N R 481088-0 1 

# syngent a

Propiconazol e Reques t fo r Waive r o f Endocrin e Disruptio n Screenin g Progra mAssay s Assessmen t

DAT A REQUIREMENT(S) : No t Applicabl e AUTHOR(S) : Richar dPeffer , Ph.D. , DAB T Jame s T . Stevens , Ph.D. , DABT , FAT S COMPLETIO N DATE : 

### May 26 , 

### 201 0 PERFORMIN G LABORATORY : Syngent aCro p Protection , Inc . 41 0 Swin g Roa d Greensboro , N C 2740 9 US A

LABORATOR Y PROJEC T ID : Report Number : TK002342 3 Tas k Number : TK002342 3 SUBMITTER/SPONSOR : Syngent aCro p Protection , Inc . 41 0 Swin g Roa d Pos t Offic e Bo x 1830 0 Greensboro , N C 27419-830 0 US A NOTE : Thi s repor t

contain s colo r

page(s) . VOLUM E J

_O F J

_O F STUD Y PAG E 1 O F 8 5 °

• • '• - - J 9 • ~ • ,

• • •STATEMENT O F DAT A CONFIDENTIALIT Y CLAIM S STATEMENT O FN O DAT A CONFIDENTIALIT Y CLAIM S UNDE RSPECIFIE

D FIFR A PROVISION S N o clai mo f

confidentialit

y i s mad e fo r

an y informatio n containe di n thi s study o n th e basi s o f it s fallin g withi n th e scop e o f FIFR A Sectio n lO(d)(l)(A ) (disclose s manufacturin g o r qualit y contro l processes) , (B ) (disclose s the detail s o f method s for

testing , detectin g or measurin g th e quantit

y o f an y deliberatel y adde diner t ingredien t

o f apesticide) , o r

(C ) (disclose s th e identit

y o r percentag e quantity o f

an y deliberatel y adde diner t ingredient

o f a pesticide) . Company Syngent aCrop Protection , Inc . Company Agent : Ruh i Rezaaiya n

U . S . Produc t

Registratio n Manager

Dat

e Syngent ai s th e owner

o f thi s study . Syngent aha s submitte dthi s material to th e Unite d State s Environmental Protectio n Agenc y specificall y unde r th e provision s containe di n FIFR A a s amende dand , hereby , consent s to us e an ddisclosur e o f thi s material b y EPA accordin g to FIFRA . Notwithstandin g th e wordin g o f our markin g TRAD E SECRET , thi s marking , b y itself

, convey s n o supplementa l claim s o f

confidentiality under

FIFR A Section s 10(a

) or 10(b ) (addressin g protectio n o f trad e secret s an dcommercial an dfinancia l information) . In submittin g thi s materia l to EPA accordin g to metho dan dformat requirement s containe di n P RNotic e 86-5 , w e d o no t waiv e any protectio n o r

right involvin g thi s material that

woul dhav e bee n claime db y th e compan y i f thi s materia l ha dno t

bee n submitte dt o th e EPA , no r d o w e waiv e an y protectio n o r right provide dunde r

FIFRA Sectio n 3 (concernin g dat aexclusivit y an ddat acompensation ) o r FIFRA Sectio n 10(g ) (prohibitin g disclosur

e to foreig n an dmultinationa l pesticid e companie s o r thei r agents) .

Report Number : TK002342 3 Pag e 2 o f 8 5GOO D LABORATORY PRACTIC E COMPLIANC E STATEMENT

A s thi s volum e contain s comment s an dassessment s o f informatio n an ddat ai n report s submitte dseparately , aGoo dLaboratory Practic e Complianc e Statement i s no t appropriate . Stud y Director : There i s n o GL P Study Director fo r thi s volume .

Dat e

. 1 presentativ e of

Submitter/Sponso r

Performin g Laboratory : Syngent aCrop Protection , Inc . 41 0 Swin g Road Greensboro , N C 2740 9 US A

Repor t Number : TK002342 3 Pag e 3 o f 8 5TABLE O FCONTENT S

STATEMEN T O F DATA CONFIDENTIALIT Y CLAIM S 2 GOO D LABORATOR Y PRACTIC E COMPLIANC E STATEMEN T 3 TABL E O FCONTENT S 4 1. 0 EXECUTIV E SUMMAR Y 6 2. 0 INTRODUCTIO N 8 3. 0 RATIONAL E FO RACCEPTANC E O F PROPICONAZOL E OSR I FO RSPECIFI C TIE R1 ASSAY S 9 3. 1 Amphibia n Metamorphosi s Assay (OPPT S 890.1100 ) 9 3. 2 Androge n Recepto r Bindin g (OPPT S 890.1150 ) 1 4 3. 3 Aromatas e -Human Recombinan t (OPPT S 890.1200 ) 1 8 3. 4 Estroge n Recepto r Bindin g Assa y Usin g Rat Uterin e Cytoso l (ER-RUC ) (OPPT S 890.1250 ) 2 3 3. 5 Estroge n Recepto r Transcriptiona l Activatio n - Huma n Cel l Lin e HeLa

990 3 (OPPT S 890.1300 ) 2 6 3. 6 Fis h Shor t Ter mReproductio n (OPPT S 890.1350 ) 3 0 3. 7 Hershberge r - Ra t (OPPT S 890.1400 ) 3 2 3. 8 Femal e Puberta l - Rat (OPPT S 890.1450 ) 3 5 3. 9 Mal e Puberta l - Rat (OPPT S 890.1500 ) 3 8 3.1 0 Steroidogenesi s - Huma n Cel l Lin e H295 R(OPPT S 890.1550 ) 4 3 3.1 1 Uterotrophi c - Rat (OPPT S 890.1600 ) 4 6 4. 0 REPRODUCTIV E AN D DEVELOPMENTA L TOXICIT Y

CHARACTERISTIC S O FPROPICONAZOL E 4 9 5. 0 CONCLUSIO N 5 1 6. 0 REFERENCE S 5 2 APPENDICE S SECTIO N 5 6 APPENDI X 1 ToxCast TM 5 7 APPENDI X 2 Assa y Descriptio n Shee t fro mCalipe r

LifeScience s -Bovin e Estroge n Recepto r Bindin g Assa y 6 5 APPENDIX 3 Assa

y Descriptio n Sheet

fro mCaliper

LifeScience s -Huma

n Estrogen Recepto r Bindin g Assa y 6 7 APPENDI X 4 Titl e Pag e an dMRI D Numbe r o f Cite dStudie s 6 9

Report Number : TK002342 3 Pag e 4 o f 8 5LIS T O F TABLE S

TABL E 1 Other

Scientificall y Relevan t

Informatio n (OSRI) : Existin g Propiconazol e Studie s Whic h Satisf y EDS P Tie r

1 Assa y Requirement s .... 7 TABL E 2 OSR I an dFIFRA 4 0 CF RPart 15 8 Studie s Whic h Includ e Examinatio n o f th e Thyroi dand/o r

Thyroi dHormone s 1 1 TABL E 3 OSRI an dFIFRA 40CF RStudie s Conducte dwith Propiconazol e whic h

Examin e Uteru s Weigh t

o r Morpholog y 4 8 TABLE 4 Submitte dReproductiv e an dDevelopmenta l Toxicity Studie s o n

Propiconazol e Technica l 5 1

Report Number : TK002342 3 Pag e 5 o f 8 5

### 1. 0 EXECUTIV E SUMMAR Y

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

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

i n human s tha t

i s similar t o a n effec t

produce db y anaturall y occurrin g estrogen , o r

othe r effect s a s [EPA ] may designate. " Sectio n 408(p)(3 ) specificall y require d that th e Administrato r

"shal l provid e fo r

th e testin g o f

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

1 Screenin g (T1S ) batter y o f eleve n separat e assay s t o identif y potentia l endocrine-modulatin g chemicals . A serie s o f guideline s fo r

th e T1 S assay s wa s published , an do n Decembe r 17 , 2009 , USEP A sen t

th e tes t order s (DC I #EDSP-122101

- 133 ) fo r th e T l S t o th e registrant s o f propiconazole . Th e technica l registrant s o f propiconazol e hav e forme daconsortiu mt o addres s th e tes t order . Th e USEP A als o indicate dtha t "othe r scientificall y relevan t

information"(OSRI ) that

i s functionall y equivalen t

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

1 assays" . Th e objectiv e o f thi s documen t

i s t o provid e th e OSRI that i s availabl e fo r

propiconazol e an ddevelo p arational e fo r justifyin g waiver s fo r

th e eleve n EDS P Tie r

1 assay s i n th e T l S battery . Th e propiconazol e consortiu midentifie daspecifi c se t o f OSR I fo r propiconazol e that establishe s tha t

mos t o f th e Tie r 1 screen s o f

th e Endocrin e Disrupte r

Screenin g Program hav e bee n previousl y conducted . A lis t o f th e 89 0 Serie s studie s an dth e correspondin g OSRI studie s ar e foun di n Tabl e 1 . Propiconazol e ha s bee n evaluate db y th e EDS P Tie r 1 assay s and/o r substantiall y similar

assays , som e o f

whic h wer e conducte dunde r th e directio n o f

th e USEP A Offic e o f

Researc h an dDevelopmen t (EPA-ORD ) a s part

o f th e ToxCas t

program (Judso n e t al. , 2010 ) o r

a s par t o f along-termEPA-OR D investigativ e progra mo f work with propiconazol e an dsevera l othe r triazol e fungicide s (Goetz e t al. , 2007 , Rockett

e t al. , 2006 ; Goetz e t al. , 2009 ; Tull y e t

al. , 2006) . Th e ToxCas t

Progra m(Phas e 1 ) wa s initiate dt o evaluat e ove r

30 0 chemicals , includin g propiconazole , i n aserie s o f hig h throughpu t

assay s t o develo p ase t

o f shorter-termassay result s an devaluat e thei r abilit y t o predic t

long-term effect s i n existin g toxicolog y testin g results . Thi s hig h throughpu t testin g include dassay s that evaluate destrogen , androge n an dthyroi drecepto r bindin g an daromatas e inhibitio n usin g th e NI H Chemica l Genomic s Center , Attagene , o r Novascree n testin g facilities . Thes e assay s fulfille dth e requiremen t t o perfor mOPPT S 890.1150 , 890.1200 , 890.1250 , 890.130 0 an d890.160 0 tests , an dpartiall y fulfille dth e nee dt o perfor m890.110 0 test s with regar dt o thyroi dresponse . Th e EPA-OR D researc h effor t o n propiconazol e an dothe r triazol e fungicide s ha s teste dpropiconazol e i n publishe dstudie s tha t includ e variant s o f

th e puberta l rat screens , both mal e an dfemale , an dsatisf y requirement s o f

OPPT S 890.145 0 an d 890.1500 . Thi s publishe dwork als o provide s dat athat

ca n b e use dt o satisf

y th e requirement s fo r

OPPT S 890.1600 , th e Uterotrophi c Assa y (Rocket t et al. , 2006) . Additionally , accordin g t o th e USEPA' s Re-registratio n Eligibility Decisio n (RED ) fo r propiconazole , ther e wa s n o evidenc e o f an y estrogen , androge n o r thyroi dmediate dtoxicit y i n th e propiconazol e toxicolog y databas e (USEPA , 2006a , b ; USEPA , 2009b) . Other Report Number : TK002342 3 Pag e 6 o f 8 5relevan t dat ai n th e publishe dliteratur e a s wel l a s th e submitte dstud y databas e fo r propiconazol e provid e OSR I fo r other EDS P Tie r

1 assay s wit h propiconazole , a s summarize di n Tabl e 1 . TABLE 1 Othe r

Scientificall y Relevan t

Informatio n (OSRI) : Existin g Propiconazol e Studie s Whic h Satisf y EDS P Tie r

1 Assay Requirement s OPPT S Guidelin e

890.110 0

890.115 0

890.120 0

890.125 0

890.130 0

890.135 0 890.140 0 Tier

I scree n

Amphibia n Metamorphosi s

Androge n Recepto r Bindin g Assay (rat ventra l prostate )

Aro mcit 3. 3 c placenta l and/o r rpf^nmhinfiti t IC^UlllUlllalll . Estroge n Recepto r bindin g Assa y (ra t uterin e cytosol ) Estroge n Recepto r Transcriptiona l Activatio n Assay

Fis h ^Jhnr t

Tpr m rioi t OHU I i i d11 1 Reproductio n Hershberge r Assa y OSR

I Reference(s ) Kjaersta de t

al.,200 7 Rocket t e t al. , 200 6 Goet z e t al. , 200 7 Taxvi g e t al. , 200 8 Judsonetal.,201 0 Sachss e e t al. , 1979 a Sachss e e t al. , 1979b Johnso n & Thompson , 198 5 Gerspach , 199

7 Hunte r e t al. , 1982 a Hunte r etal , 1982

b Baue r e t al. , 200 2 Kojim ae t al. , 200 4 Vinggaarde t

al. , 200 8 Kjaersta de t

al. , 200 7 Judsonetal.,201 0 Vinggaar de t

al. , 200 0 Henry e t al. , 200 6 Toke n e t al. , 2004 Toke n e t al. , 200 6 Kjaersta de t

al.,200 7 Ohi o e t al. , 200 4 Lavell e et al. , 200 6 Judsonetal.,201 0 Judsonetal.,201 0 Balagueretal. , 199 9 Hurs t e t al. , 200 3 Kojim ae t al. , 200 4 Kjaersta de t

al. , 200 7 Bitsc h e t al. , 200 2 Boehmle r e t al. , 200 4 Boehmle r e t al.,200 6 Judsonetal.,201 0 Breteler,198 8 Kjaersta de t

al. , 200 7 Taxvi g e t al. , 200 8 Tull y e t al. , 200 6 Goetz et al. , 200 7 Functionall y

Equivalen t Methodology ? Supplementa l Supplementa l Supplementa l Supplemental Supplementa l Higher-tie r

stud y Higher-tie r

stud y Higher-tie r

stud y Higher-tie r

stud y Higher-tie r

stud y Higher-tie r

stud y Substantiall y simila r Substantiall y simila

r Substantiall y simila r Substantiall y simila r Substantiall y simila r Substatiall y simila r Supplementa l Substantiall y similar Substantiall y simila r Substantaill y simila r Substantiall y simila r Substantiall y simila

r Substatiall y simila r Substatiall y simila r Substantiall y simila r Substantiall y simila r Substantiall y simila r Supplemental Supplementa l Supplementa l Supplementa l Substantiall y simila r Substantiall y simila r Substantiall y simila r Substantiall y simila r Higher-tie r

stud y Higher-tie r

study Comment s

N o specifi c amphibian Tie r 1 scree n available , bu t

supplementa l an dhighe r tie r mammalia n thyroi drecepto r an d hormon e informatio n satisfie s goal o f thi s screen .

hARtransfecte dto CHO hA Rtransfecte dt o aplasmi d hA Rtransfecte dto CH O hA Rtransfecte dt o CH O rat an dhuma n A Rbindin g assay s plu s huma n A Rreporte r

assay s Huma n placenta l macrodome s Trou t macrodomes(brai n & ovarian ) Huma n placenta l macrodome s Huma n placenta l macrodome s MCF- 7 Huma n Ovaria n Granulos e tumo r JEG- 3 cel l transfecte dto plasmi d Huma n aromatas e gene s Bovin e an dHuma n E Rbindin g hE Rtransfecte dt o HeL acell s hE Rtransfecte dt o yeas t hERtransfecte dto CHO MCF- 7 cell s MCF- 7 MCF- 7 MCF- 7 Huma n E Rgenes , multipl e type s Sheepshea dminnow Hershberge r

protoco l Hershberge r

protoco l EPA-OR D stud y EPA-OR D study Report Number : TK002342 3 Pag e 7 o f 8 5890.145 0 890.150 0 890.155 0 890.160 0 Femal e Puberta l

Mal e Puberta l Steroidogenesi s H295 R Uterotrophi c Assay Rockett

e t al. , 200 6 Goet z e t al. , 200 7 Goet z e t al. , 200 9 Kjsrsta de t

al. , 200 7 Sanderso n et

al. , 200 2 Rockett e t al. , 200 6 Border s e t

al. , 198 5 Sachss e e t al. , 1979 a Hunte r etal. , 1982

b Substantiall y simila r Substantiall y simila r Substantiall y simila r Substantiall y simila r Supplemental Higher-tie r

stud y Higher-tie r

stud y Higher-tie r

stud y Higher-tie r

stud y EPA-OR D stud y

EPA-OR D stud y EPA-OR D study , H295 Rcell s H295 Rcell s H295 Rcell s Uterin e weight s obtaine d Uterin e micropatholog y Uterin e micropatholog y Uterin e micropatholog y A s summarize di n th e tabl e above , propiconazol e ha s bee n teste dusin g stud y protocol s whic h ar e identica l t o o r

substantiall y equivalen t

t o th e state dpurpos e ofte n o f th e OPPT S Serie s 89 0 EDS P Tie r

1 study guidelines . Whil e n o study tha t specificall y meet s th e desig n criteri ao f th e amphibia n metamorphosi s assay i s availabl e fo r propiconazole , th e propiconazol e consortiu mrequest s awaive r fo r th e Amphibia n Metamorphosi s scree n becaus e propiconazol e ha s bee n evaluate di n multipl e mammalia n studie s fo r effect s o n th e hypothalamic-pituitary-thyroi dacces s (HPT ) an dsubsequen t thyroid-relate deffects . A s th e purpos e o f

th e Amphibia n Metamorphosi s scree n i s t o evaluat e thyroi dfunctio n i n a representativ e vertebrat e mode l (frog) , an dth e thyroi drecepto r structur e an dfunctio n ar e conserve dacros s multipl e specie s includin g mammal s an damphibians , th e dat afro m multipl e i n vitr o an di n viv o assay s i n rat an dhuma n wit

h propiconazol e provid e arobus t assessmen t

i n relevan t

vertebrat e system s tha t

g o beyon dth e Tie r

1 assay . FIFRA 4 0 CF R guidelin e studie s i n rats , mice , an ddog s als o provid e informatio n o n an y potentia l HP T axi s effect s i n multipl e specie s tha t ar e directl y relevan t

t o humans . Propiconazol e ha s bee n teste dfo r al l o f th e endpoint s covere db y Tie r 1 assay s o f

th e EDSP . Th e propiconazol e consortiu mi s therefor e requestin g waiver s fo r al l o f th e Tier 1 assay s i n th e EDS P tes t

order . 

### 2. 0 INTRODUCTIO N Th e Federa l Foo dDru g an dCosmeti c Ac t require dth e U . S . Environmenta l Protectio n Agenc y (USEPA ) t o "develo p ascreenin g program , usin g appropriat e validate dtes t system s an dother scientificall y relevan t

informatio n t o determin e whethe r certai n substance s ma y hav e an effec t

i n human s that

i s simila r t o an effec t

produce db y anaturall y occurrin g estrogen , o r

othe r effect s a s [EPA ] may designate. " Sectio n 408(p)(3 ) specificall y require d tha t th e Administrato r

"shal l provid e fo r

th e testin g o f

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

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

th e T1 S assays , an do n Decembe r 17 , 2009 , EP A sent

th e tes t order s fo r

T1 S (DC I #EDSP-122101

- 133 ) t o th e registrant s o f

propiconazole . Th e technica l registrant s o f propiconazol e hav e forme daconsortiu mt o addres s th e tes t order . EP A als o indicate dtha t

"other scientificall y relevan t

information " (OSRI ) tha t i s functionall y equivalen t

t o th e informatio n t o b e obtaine dfro mth e Tie r 1 assay s ma y b e utilize di n lie u o f Report Number : TK002342 3 Pag e 8 of8 5conductin g th e Tie r 1 assays . However , EP A indicate dth e OSR I mus t mee t specifi c qualit y an dfunctiona l equivalenc e criteri awhic h includ e th e followin g (USEPA , 2009a) : Dat aar e o f

hig h qualit y an dconducte dusin g soun dscientifi c practice s Stud y conducte di n accordanc e with accepte dscientifi c methodolog y o r protoco l Deviation s fro mTier

1 assa y protocol s identifie di n th e OSR I rational e

- Impac t

o f any deviation s fro mth e protoco l o n th e result s o f th e stud y relatin g to th e studie s valu e i n justifyin g awaive r reques t shoul db e discusse di n th e rational e Th e objectiv e o f

thi s documen t

i s t o provid e th e OSR I tha t i s availabl e fo r

propiconazol e an d t o obtai n waiver s fo r

th e eleve n EDS P Tier

1 assays :

• 890.110 0 - Amphibia n Metamorphosi s (Frog ) • 890.115 0 - Androge n Recepto r Bindin g (Ra t

Prostate ) • 890.120 0 - Aromatas e (Human Recombinant ) • 890.1250-Estroge n Recepto r Bindin g • 890.130 0 - Estrogen Recepto r Transcriptional Activatio n (Huma n Cel l Lin e HeLa-9903 ) • 890.1350

- Fis h Short-Ter mReproductio n • 890.140 0 - Hershberge r (Rat) • 890.145 0 - Femal e Puberta l (Rat ) • 890.150 0 - Mal e Puberta l (Rat ) • 890.155 0 - Steroidogenesi s (Huma n Cell Lin e - H295R) • 890.160 0 - Uterotrophi c (Rat ) Furthermore , thi s documen t review s th e endocrine-relate dendpoint s alread y routinel y assesse di n existin g EPA-require dguidelin e studie s an dothe r testin g o f

propiconazole , whic h provid e furthe r

weigh t o f evidenc e that

th e OSR I provid e "functionall y equivalent " informatio n fro mFIFRA 4 0 CF Rstudie s a s the y relat e t o th e propose dT1 S battery . 

### 3. 0 RATIONAL E FO RACCEPTANC E O FPROPICONAZOL E OSR I FO RSPECIFI C TIE R1 ASSAY S 3. 1 Amphibia n Metamorphosi s Assa y (OPPT S 890.1100 ) Purpos e o f

Assa y Th e purpos e o f

th e amphibia n metamorphosi s assa y i s t o scree n fo r

potentia l thyroi dsystem disruptin g compounds . Throug h th e hypothalamus-pituitary-thyroi d(HPT ) glan daxis , thyroi dhormon e regulate s man y processe s i n organism s includin g development , somati c growth , metabolis man dreproduction . Metamorphosi s i n anuran amphibian s i s awell

studie dmode l fo r

thyroi dhormon e function .

Report Number : TK002342 3 Pag e 9 o f 8 5

### Assa y Methodolog y Summar y

Th e OPPT S 890.110 0 protoco l involve s a21-da y exposur e o f tadpole s to th e tes t

substance , with evaluation s occurrin g o n Day 7 an dDay 21 . Th e tes t i s initiate dat

developmenta l stag e 51 , whic h can b e reache dapproximatel y 1 4 day s post-fertilization . Fou r concentration s o f th e tes t substanc e an dadilutio n wate r contro l an dfou r

replicat e tank s pe r

concentration s ar e employe di n aflow-throug h system . A t tes t initiation , atota l o f

2 0 stag e 5 1 Xenopu s laevi s tadpole s are place di n eac h o f th e fou r replicat e tank s per

treatmen t group . Environmenta

l condition s o f

22±1°C , p H o f

betwee n 6.5-8. 0 an da12:1 2 lightdark photoperio di s employed . Fiv e tadpole s fro meac h treatmen t tank ar e randoml y remove dat day 7 fo r evaluatio n o f

we t weigh t an drat e o f

metamorphosi s measure s o f developmenta l stage , whol e bod y length , hin dlimb length an dsnout-ven t length . O n Day 2 1 th e remainin g tadpole s ar e evaluate dfo r

thes e same endpoints . Histologica l evaluatio n o f

thyroi dtissu e i n asub-sampl e o f fiv e tadpole s fro meac h replicat e tan k i s als o performe di n thi s scree n i n order t o lin k an y observe dchange s i n th e rat e o f metamorphosi s t o morphologica l change s i n th e primary thyroi dsynthesi s organ . Othe r Scientificall y Relevan t

Informatio n Althoug h n o specifi c amphibia n metamorphosi s screen s with propiconazol e wer e found , th e original basi s fo r

th e Endocrin e Disrupto r

Screenin g an dTestin g Advisory Committe e (EDSTAC , 1998 ) addin g thi s scree n t o th e EDS P Tie r 1 batter y i s indicate di n th e followin g quot e fro mthei r

fina l report : ". . .ther e ar e n o clea r marker s o f

thyroi dhormon e actio n tha t coul db e use dwithi n th e contex t o f aT1 S assay . I n contrast , tai l resorptio n i n amphibia n metamorphosi s represent s a n assa y whic h utilize s specifi c thyroi dhormone-dependen t effect s a s a n endpoin t

fo r aT1 S assay. " Thi s i s re-enforce db y th e EPA' s EDS P webpag e (http://www.epa.gov/scipolv/oscpendo/pubs/amajfs.pdf : Appendi x A l - Amphibia n Metamorphosis ) whic h state s th e followin g abou t th e purpos e o f

th e Amphibia n Metamorphosi s Assay (AMA) : "The AM A i s ascreenin g assay intende dt o empiricall y identif y substance s whic h ma y interfer e with th e norma l functio n o f th e hypothalamic

pituitary-thyroi d(HPT ) axis . Th e AM A represent s ageneralize dvertebrat e mode l t o th e exten t that i t i s base do n th e conserve dstructur e an dfunction s o f thyroi dsystems . I t

i s no t intende dt o quantif

y o r confir mendocrin e disruption , o r t o provid e aquantitativ e assessmen t o f risk , bu t onl y provid e evidenc e tha t thyroi dregulate dprocesse s ma y b e sufficientl y perturbe dt o warran t

mor e definitiv e testing. " Therefore , th e amphibia n metamorphosi s stud y wa s originall y include di n th e batter y a s ascree n t o loo k at thyroi deffect . Numerou s publishe dstudie s a s wel l a s USEP A FIFRA 40CF RPar t 15 8 guidelin e studie s wit h propiconazol e examine dth e thyroi da s apotential targe t organ . Thes e studie s wer e no t necessaril y limite dt o checkin g fo r change s i n thyroi dhormones , bu t als o mor e sensitiv e marker s suc h a s thyroi drecepto r binding , weigh t

an dhistopathology . Thes e ar e summarize d i n Tabl e 2 .

Report Number : TK002342 3 Pag e 1 0 o f 8 5TABL E 2 OSR I an dFIFRA 4 0 CF RPar t 15 8 Studie s Whic h Includ e Examinatio n o f

th e Thyroi dand/o r

Thyroi dHormone s Thyroi dendpoint s Publishe dStudies : Thyroi ddependen t

cel l growth o f ara t pituitar y cel l lin e GH 3 (T-screen) , T 3 an dT 4 level s at GD21and/orPND1 6 Thyroi dweigh t

an d histopatholog y i n femal e rat s T 3 , T 4 an dTS H levels , thyroi d weigh t an dhistopatholog y i n mal e rat s T 4 level s Huma n thyroi drecepto r binding , reporte r

gen e assay s fo r thyroi daan dP receptor s ( P agonist s an dantagonists ) 4 0 CF Rpar t

15 8 Guidelin e Studies : Thyroi dhistopatholog y Thyroi dweigh t

an d histopatholog y Thyroi dweigh t

an d histopatholog y Thyroi dweigh t

an d histopatholog y Thyroi dhistopatholog y Thyroi dweigh t

an d histopatholog y OSR I Reference s

Kjaersta de t

al. , 200 7

Rockett e t al. , 200 6

Goet z e t al. , 200 7 Taxvi g e t al. , 200 8 Judsonetal.,201 0

Sachss e e t al. , 1979 a Sachss e e t al. , 1979 b Johnso n & Thompson , 198 5 Gerspach,199 7 Hunte r etal , 1982 a Hunte r e t al. , 1982 b Comment s

I n vitr o an di n viv o studie s Thes e wer e jus t

som e o f EDS P apica l endoint s evaluate di n thi s puberta l femal e stud y (Complet e HP T Axis ) Thes e wer e jus t

some o f EDS P apica l endoint s evaluate di n thes e puberta l mal e stud y (Complet e HP T Axis ) Hershberge r

scree n (Complet e HP T Axis )
EP A ToxCas t

assay s

90-da y feedin g i n rat s (Complet e HP T Axis ) 90-da y feedin g i n dog s (Complet e HP T Axis ) 1-yea r feedin g i n dog s (Complet e HP T Axis ) 1 8-month oncogenicity stud y i n mic e (Complet e HPT Axis ) 2 -yea r oncogenicit y stud y i n mic e (Complet e HP T Axis ) 2 -yea r chroni c toxicity/oncogenicit y stud y i n rat s (Complet

e HPT Axis ) Kjaersta de t

al. , 200 7 Fo r th e i n vitr o detectio n o f

agonisti c an dantagonisti c propertie s o f th e azol e fungicide s toward s th e thyroi drecepto r (TR) , th e relativel y ne w tes t method , calle dth e Tscreen , wa s used . Th e assay i s base do n th e thyroi dhormon e dependen t cel l growt

h o f arat pituitar y tumo r cel l lin e (GH3) . Thi s cel l lin e expresse s intracellular T Ri n very hig h amounts , an dth e assay ca n b e use dt o study interferenc e o f compound s with thyroi dhormon e at th e cellular Report Number : TK002342 3 Pag e 1 1 o f 8 5

### level . Th e GH 3 cell s wer e culture dat 37° C humidifie datmospher e o f 5 % C02/95 % ai r

i n DMEM/F1 2 supplemente dwith 10 0 U/m l penicillin , 10 0 ^g/m l streptomyci n an d0.2 5 ug/m l amphoterici n B an d1 0 % feta l cal f serum . Forty eight

hour s prio r

t o platin g th e cell s int o 96-wel l microplate s fo r

th e experiment , standar dcultur e mediu mwa s change dt o tes t mediu mcontainin g 10 % (v/v ) T3

- an dT4-deplete ddextra n charcoa l treate dPC S (DC-FCS) . Afte r 4 8 h i n DC-FCS , whic h wa s change donc e afte r 2 4 h , th e GH 3 cell s wer e release d usin g acellscraper

an dseededi n 96-wel l black , clea r botto mmicroplates , 5 0 u l cel l suspensio n an d5 0 u l tes t

compoun dpe r

well , at adensit y o f

250 0 cells/well . Propiconazol e wa s teste dat

concentration s o f

0 , 0.01 , 0.375,1,3 , 1 0 an d3 0 uM , an di n th e absenc e an d presenc e o f

0.2 5 n M T3 , t o tes t

fo r agonisti c an dantagonisti c potency . Th e plate s wer e incubate dfo r

9 6 h , an dcel l growt

h wa s analyze dafte r

additio n o f

AlamarBlue TM b y measurin g fluorescence . Rocket t et al. , 200 6 Thi s specialize done-generatio n reproductiv e toxicit y stud y i n rats , wit h aparticular emphasi s o n response s i n F l femal e offspring , evaluate dth e respons e t o thre e differen t tes t substance s includin g propiconazole . Propiconazol e wa s administere dt o timed-pregnan t Wistar-Han rat s (Charle s Rive r

Laboratory , Raleigh , NC ) a t 0,100 , 50 0 an d250 0 pp mvi ath e fee dfro m gravi dday (GD ) 6 t o postnata l day (PND ) 98 . Th e hig h dos e o f 250 0 pp mwa s considere dt o b e at o r abov e th e MTD . Eac h treatmen t grou p consiste do f

approximatel y 3 5 dams . Dam s were allowe dt o deliver

naturally , with th e day o f deliver y designate da s PN D 0 fo r th e F l offspring . Pup s wer e weighe do n PN D 0 . Th e siz e an dgende r rati o o f eac h litte r

wa s noted . Litter s wer e culle dt o eigh t pup s o n PN D 8 usin g blinde drando mselection , an dth e pup s weane do n PN D 22 . Femal e weanling s wer e continue do n th e sam e die t a s thei r dam s ha d bee n expose dt o unti l terminatio n o n PN D 99 . Amon g othe r things , bod y an dorgan weight s (brain , hippocampus , hypothalamus , liver , pituitary , thyroi dan duterus ) wer e measure dat necropsy . Th e liver , thyroi d(score dfo r colloi ddepletion , follicula r cel l hypertroph y and/o r follicula r cel l hyperplasia ) an dovarie s (ever y twentiet h sectio n wa s staine dwit

h H an dE an d examine dfo r

edema , cysti c follicle s an dretaine doocytes ) wer e rea dfo r histopathology . Goetz e t al. , 200 7 Thi s specialize done-generatio n reproductiv e toxicity stud y i n rats , wit h aparticular emphasi s o n response s i n F l mal e offspring , evaluate dth e respons e t o fou r differen t tes t substance s includin g propiconazole . Time dpregnan t Wistar Ha n IG S rat s wer e receive dfro mCharle s Rive r Laboratorie s (Raleigh , NC ) o n gestatio n da y (GD ) 1- 3 an dassigne dto treatmen t group s s o a s t o achiev e equivalen t weigh t mean s acros s treatmen t

groups . Propiconazol e wa s administere di n th e die t

at concentration s o f

0 , 100 , 50 0 an d250 0 pp mfro mGD 6 throug h PND92 . Th e hig h dos e o f 250 0 pp mwa s considere dt o b e at o r abov e th e MTD . Dam s wer e allowe dt o delive r

naturally , with day o f

delivery designate da s postnata l day (PND ) 0 fo r th e F l offspring . O n PN D 8 , litter s wer e weighe dan dthe n culle dt o eigh t pup s pe r dam , retainin g male s preferentially , t o maximiz e uniformit y i n growt h rates . Th e F l generatio n continue do n th e sam e treate dfee ddiet s upo n weanin g at PND23 . F l offsprin g fee dintak e an dbod y weight s were measure dweekl y unti l necropsy . On e mal e fro meac h litte r wa s take n t o necrops y at

PND1 , 22 , 5 0 o r

9 2 t o asses s affect s o n selec t organ weights , histolog y an dhormon e measures . Whole-bod y weight s wer e measured , an dthe n brain , hypothalamus , hippocampus , pituitary , thyroid , liver , testis , epididymis , ventral prostate an dsemina l Repor t Number : TK002342 3 Pag e 1 2 o f 8 5

### vesicle s wer e removed , weighe dan dfixe di n 10 % neutra l buffere dformali n fo r histology . Seru mlevel s o f

th e thyroi dhormone s T 3 an dT 4 a s wel l a s estradio l an dtestosteron e wer e measure dwith acommercia l radioimmunoassa y kits . Thyroi dstimulatin g hormon e (TSH ) wa s measure dusin g aspecialize dradioimmunoassa y method . Taxvi g e t al. , 200 8 I n aHershberge r

assay , propiconazol e wa s administere dorall y at dose s o f 0 , 50,10 0 o r

15 0 mg/k g body weight/day fo r seven day s t o castrate dmal e rat s treate dconcurrentl y with testosterone . On e grou p o f castrate drat s serve da s negativ e control s an dwer e give n peanu t oi l only . Th e rat s i n th e remainin g group s were al l dose dwit h testosteron e propionat e (0. 5 mg/kg/da y s.c. ) fo r

seve n days . On e group wa s onl y administere dtestosteron e propionat e an dserve da s th e contro l grou p t o whic h al l propiconazole-treate dgroup s wer e compared . Effect s wer e examine di n castrate dtestosterone-treate dmale s b y analyzin g weight s o f reproductiv e organs , seru mhormon e level s an dprostat e gen e expression . Fo r seru m hormones , rat

luteinizin g hormon e (LH) , rat follicle-stimulatin g hormon e (FSH ) an dT 4 hormon e level s wer e analyzed . Judso n e t

al. , 201 0 Th e ToxCas t

group identifie s an dprioritize s reliabl e hig h throughpu t scree n (HTS ) assay s t o assess , amon g othe r

things , agonist/antagonis t activit y fo r

anumbe r o f nuclear receptor s an d gene s involve di n steroi dmetabolism . Fo r th e thyroid , Attagene' s THRal_TRANS ; Novascreen' s NR_hTRa ; an dNCGC' s TRbeta_Agonis t an dTRbeta_Antagonis t

gene s wer e evaluate di n th e presenc e o f propiconazole . Se e Appendi x 1 an dJudso n e t al. , 200 9 fo r

mor e detail s o n th e methodolog y use di n thes e screen s an dacces s t o th e ToxCas t result s vi athei r websit e links . Sachss e et

al. , 1979 a I n thi s Part

15 8 90-da y rat

stud y (MRID Numbe r

0058606) , th e thyroi dgland s o f bot h female s an dmale s wer e evaluate db y histopatholog y at th e en do f

th e study . Sachss e et

al. , 1979b I n thi s Part

15 8 90-da y do g stud y (MRID Numbe r 00058607) , th e thyroi dgland s o f both female s an dmale s wer e weighe dan devaluate db y histopatholog y at th e en do f

th e study . Johnso n an dThompson , 198 5 I n thi s Par t 15 8 1-yea

r do g stud y (MRID Numbe r 00151515) , th e thyroi dgland s o f bot h female s an dmale s wer e weighe dan devaluate db y histopatholog y at th e en do f

th e study Gerspach , 1997 I n thi s Part

15 8 18-month mous e oncogenicity stud y (MRI D Numbe r 44381401) , th e thyroi d gland s were weighe dan devaluate db y histopatholog y at th e en do f

th e stud y Hunte r e t al. , 1982 a I n thi s Part

15 8 2-yea

r mous e oncogenicity stud y (MRI D Numbe r 00129570) , th e thyroi d gland s o f mal e an dfemal e mic e wer e evaluate db y histopatholog y at th e en do f

th e stud y Repor t Number : TK002342 3 Pag e 1 3 o f 8 5

### Hunte r e t al. , 1982 b I n thi s Part

15 8 2-year

rat combine dchroni c toxicity/oncogenicit y study (MRID Numbe r 00129918) , th e thyroi dgland s wer e weighe dan devaluate db y histopatholog y at 5 2 week s an dat th e terminatio n o f

th e study . Conclusion : Th e propiconazol e consortiu mrequest s awaive r fo r th e Amphibia n Metamorphosi s scree n becaus e propiconazol e ha s bee n evaluate di n multipl e mammalia n studie s fo r effect s o n th e hypothalamic-pituitary-thyroi daxi s (HPT ) an dsubsequen t thyroid

relate deffects . Publishe dwork ha s bee n conducte db y anumbe r o f differen t researc h team s wit h experienc e i n thes e type s o f assays , includin g th e EPA-ORD , whos e work include d evaluation s i n both rat

an dhuma n assays . Th e wid e rang e o f differen t thyroid-mediate d endpoint s examine dinclude dexamination s fo r effect s o n th e thyroi dreceptor s i n vitro , an d effect s o n th e HP T axi s i n viv o throug h thyroi dweigh t and/o r histolog y and/o r

thyroi d hormones . A s th e purpos e o f th e Amphibia n Metamorphosi s scree n i s t o evaluat e thyroi d functio n i n arepresentativ e vertebrat e mode l (frog) , base do n th e conserve dstructur e an d function s o f

thyroi dsystems , th e dat afro mrat an dhuma n with propiconazol e provid e a robus t assessmen t

i n relevan t

vertebrat e system s tha t

g o beyon dth e Tie r

1 assay . FIFR A 4 0 CF RPart 15 8 guidelin e studie s i n rats , mice , an ddog s als o provid e informatio n o n an y potentia l HP T axi s effect s i n multipl e specie s that ar e directl y relevan t

t o humans . 3. 2 Androge n Recepto r Bindin g (OPPT S 890.1150 ) Purpos e o f

Assay Th e androge n receptor

(AR ) bindin g assa

y i s a n i n vitr o tes t

to detec t chemical s that

ma y affec t th e endocrin e syste mb y bindin g t o th e androge n receptor . Th e tes t measure s th e abilit y o f acompoun dt o compet e with a[ 3 

### H]-ligan dfo r bindin g i n acytoso l homogenat e o f rat ventra l prostat e (VP ) tissue , whic h i s replet e wit h androge n receptors . Th e A Rbindin g assay doe s no t

asses s functiona l o r

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

antagonisti c resul t

i n th e Hershberge r

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

eac h contain s an aliquo t o f V P cytosol . On e se t

o f tube s receive s variou s dilution s o f th e tes t substance , a secon dse t receive s dilution s o f

aknow n hig h affinit y androge n ligan dan dathir dse t receive s dilution s o f

aknow n lo w affinit y ligand . A precis e amoun t o f [ 3 

### H]-R-1881 , th e hig h affinit y ligand , i s the n dispense dt o al l tubes , whic h ar e the n incubate dovernigh t at 4°C . O n th e followin g day aseparatio n ste p isolate s radioactivit y boun dt o A Ri n eac h tube , whic h i s counte db y liqui dscintillation . Th e principl e o f th e assay i s tha t

acompetin g ligan dabl e t o bin dt o A Rwil l displac e [ 3 

### H]-labele dligan dan dproduc e anegativ e sigmoi ddose-respons e plo t o f boun dradioactivity . A curve-fittin g compute r programca n determin e th e relativ e affinit y o f th e tes t substanc e b y comparin g it s plo t parameter s versu s thos e o f th e know n binders . Th e assay principl e furthe r hold s that

acompoun dwhic h displace s n o radiolabele d ligan di s unabl e t o bin dt o AR ; it s dose-respons e plo t wil l res t at 100 % maximu mbindin g an dhav e n o downwar dslope . Report Number : TK002342 3 Pag e 1 4 o f 8 5Othe r Scientificall y Relevan t

Informatio n OSR I fo r propiconazol e tha t

i s substantiall y simila r

t o th e androge n recepto r

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

Tie r 1 scree n Recombinan t

huma n A R Human A Ri n CH O cell s Huma n A Ri n CH O cell s Huma n A Ri n CH O cell s Human A Ran drat

AR , agonis t an dantagonis t

assay s OSR I Reference s Baue r etal. , 200 2 Kojim ae t al. , 200 4 Vinggaar de t

al. , 200 8 Kjaersta de t

al. , 200 7 Judso n etal. , 201 0 Comment s

Bindin g affinit y i n recombinan t huma n A R Agonis t an dantagonis t

activit y i n hA R expresse di n cell s wit h areporte r gene . Antatgonis t

activit y i n hA Rexpresse di n cell s wit h areporte r

gene . Antatgonis t

activit y i n hA Rexpresse di n cell s with areporter

gene . EPA' s HT S fo r

examinin g EDSP-selecte d chemical s Baue r e t al. , 200 2 Thi s stud y assesse dcompetitiv e bindin g o f 2 9 compounds , includin g propiconazole , t o a recombinan t

huma n androge n recepto r (rhAR ) i n 96-wel l plates . Briefly , rhA Rwa s expresse db y avira l vecto r withi n insect

cells , an dth e recombinan t A Rprotei n recepto r

wa s isolated . Th e radiolabele dtrace r wa s adifferen t

high-affinit y ligan dfro mR-188 1 use di n th e EDS P Guidelin e protocol , namel y 3 H-dihydroxytestosteron e ( 3 H-DHT) . Th e recepto r preparatio n wa s dilute di n assa y buffe r an da10 0 u l aliquo t

pe r wel l wa s incubate d1 6 h at 4° C wit h 0.4 4 n M 3 H-DH T i n th e presenc e o r absenc e o f

increasin g concentration s o f propiconazol e o r

DHT . Th e recepto r

wa s fixe do n th e microtite r plat e wit h a n antibod y specifi c t o th e rhAR . Afte r washin g th e plate s tw o time s wit h washin g buffer , th e microtite r well s wer e separate db y breaking , transferre dint o scintillatio n vial s an dcounte di n 3 m l scintillatio n fluid . Specifi c A Rbindin g i n term s o f IC5 0 an dKi value s wer e calculated , base do n th e differenc e o f tota l bindin g compare dt o non-specifi c bindin g i n th e presenc e o f a 200-fol dsurplu s o f

unlabele dDHT . Therefore , th e bindin g assay desig n wa s fundamentall y simila r t o th e EDS P Guidelin e assay , bu t adifferen t

recepto r typ e (human ) an dmetho do f separatio n wa s utilized . Prio r wor k cite db y th e author s indicate dthat thi s recombinan t huma n A Rexhibit s th e same bindin g characteristic s a s th e nativ e hAR . Th e stud y wa s conducte da t

aUniversity i n Germany . Kojim ae t

al. , 200 4 Thi s study examine dth e abilit y o f 20 0 chemical s t o induc e a n agonis t o r antagonis t

respons e i n a n AR-linke dreporte r

system . Huma n A Rwa s transfecte dint o Chines e hamste r ovary (CHO ) cell s wit h avecto r an daluciferas e reporte r

gen e wa s linke ds o tha t

bindin g o f

a ligan dt o A Rwoul dproduc e aluminescen t signal . I n th e antagonis t

assay , varyin g concentration s o f

th e tes t substanc e wer e teste di n th e presenc e o f th e mode l A Ragonis t DHT . Althoug h a n AR-linke dreporter syste mha s no t

bee n validate dfo r

inclusio n i n th e EDS P Tie r

1 scree n battery , th e principl e i s presentl y bein g evaluate dan dwil l likel y b e Report Number : TK002342 3 Pag e 1 5 o f 8 5adopte di n th e future . I t i s highl y specifi c fo r

A Rbinders . Propiconazol e wa s on e o f th e 20 0 teste dchemicals . Vinggaar de t

al. , 200 8 Thi s stud y examine dth e ability o f 39 7 chemical s t o induc e an anti-androgeni c respons e i n an A Rreporte r

assay system , i n th e presenc e o f th e mode l A Ragonis t

R-1881 . Human A Rwa s transfecte dint o Chines e hamste r ovary (CHO ) cell s wit h avecto r an daluciferas e reporte r gene , whic h wa s linke ds o tha t bindin g o f

aligan dt o A Rwoul dproduc e aluminescen t signal . Th e cell s wer e incubate dwith varyin g concentration s o f th e propiconazol e i n th e presenc e o f

th e A Ragonis t

R-1881 , t o examin e antagonis mo f th e AR-induce dreporte r response . Despit e th e fac t that th e AR-linke dreporte r

syste mha s no t

bee n validate dfo r inclusio n i n th e EDS P Tier 1 scree n battery , i t

i s widel y used , th e principl e i s presentl y bein g evaluate dan di t

wil l likel y b e adopte di n th e future . I t i s highl y specifi c fo r

A Rbinders . Propiconazol e wa s on e o f th e 39 7 teste dchemicals . Thi s study wa s conducte dat Nationa l Foo dInstitute , Departmen t o f Toxicolog y an dRis k

Assessment , Technica l Universit y o f Denmark , S0borg , Denmark , Kjaersta de t

al. , 200 7 Thi s study examine dth e abilit y o f 4 fungicide s t o induc e an anti-androgeni c respons e i n an A Rreporte r

assa y system , i n th e presenc e o f th e mode l A Ragonis t

R-1881 . Huma n A Rwa s transfecte dint o Chines e hamste r ovary (CHO ) cell s with avecto r an daluciferas e reporte r gene , whic h wa s linke ds o that bindin g o f

aligan dt o A Rwoul dproduc e aluminescen t signal . Th e cell s wer e incubate dwith twelv e concentration s o f propiconazol e rangin g fro m 0.02 5 t o 5 0 uM , combine dwit h 0. 1 n M o f th e A Ragonis t

R1881 , t o examin e antagonis mo f th e AR-induce dreporte r

response . Despit e th e fac t tha t th e AR-linke dreporte r

syste mha s no t bee n validate dfo r

inclusio n i n th e EDS P Tie r 1 scree n battery , i t

i s widel y used , th e principl e i s presentl y bein g evaluate dan di t wil l likel y b e adopte di n th e future . I t i s highl y specifi c fo r

A Rbinders . Judso n e t

al. , 201 0 Th e ToxCas t

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

activit y fo r

anumbe r

o f nuclear receptor s an dgene s involve di n steroi dmetabolism . T o examin e androge n an danti-androgeni c potential , propiconazol e wa s evaluate di n th e followin g assays : Attagene' s ARJTRANS ; Novascreen' s NR_rA Ran d NR_hAR ; an dNI H Chemica l Genomic s Center' s AR_Agonis t an dAR_Antagonist . Se e Appendi x 1 an dJudso n e t al. , 200 9 fo r

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

websit e links .

Report Number : TK002342 3 Pag e 1 6 o f 8 5Deviation s to OPPT S 890.115 0 Protoco l OPPT S 890. 1 IS O Requirement s Criteri a Baue r etal. , 200 2 Deviation s t o OPPT S 890 . 1 1 5 0 Kojim ae t al. , 200 4 Deviation s t o OPPT S 890 . 1 1 5 0 Vinggaar de t

al. , 200 8 Deviation s t o OPPT S 890 . 1 1 5 0 Kjaersta de t

al. , 200 7 Deviation s t o OPPT S 890 . 1 1 5 0 Judso n etal. , 201 0 Deviation s t o OPPT S 890 . 1 1 5 0

Justificatio n fo r OSRI A Ri n Ra t V P Cytoso l

Recombinan t

huma n A R

Huma n A Ri n CH O cell s

Huma n A Ri n CH O cell s

Human A Ri n CH O cell s

Huma n an drat

A R bindin g assay s (Novascreen ) Huma n A Rreporte r assay s Binder s reac t

sam e wit h bot h huma n an d ratAR . Both hav e bee n teste dwith propiconazole . 3 H-competito r i s R

188 1

3 H-labele dcompetito r isDH T

N o radiolabel ; bindin g respons e measure d wit h linke dreporte r (Agonis t + antagonis t experiments ) N o radiolabel ; bindin g respons e measure d wit h linke dreporte r (Antagonis t experiments ) N o radiolabel ; bindin g respons e measure d with linke dreporte r (Antagonis t experiments ) 3 H-methy l trienolon e N o radiolabel ; bindin g respons e measure d with linke dreporte r (Agonis t + antagonis t experiments ) Know n androge n ligand s respon di n a n identica l manne r Separatio n o f boun d an dfre e trace r

usin g hydroxyapetit e ge l Separatio n o f

boun d an dfre e b y fixin g th e boun drecepto r comple x t o microtite r well s with hA R antibody . N o separation step ; A R bindin g i s linke dt o a reporte r N o separatio n step ; A R bindin g i s linke dt o a reporte r N o separation step ; A R bindin g i s linke dt o a reporte r No t stated , bu t availabl e fro mCalipe r LifeScience . N o separatio n step ; A R bindin g i s linke dt o a reporte r Method s ar e equall y vali d

Conclusions : Th e propiconazol e consortiu mrequest s awaive r fo r th e A Rbindin g assay becaus e propiconazol e ha s bee n evaluate db y anumbe r o f othe r researcher s usin g substantiall y similar

methodology , includin g assay s performe db y th e EPA-OR D i n it s ToxCas t screens . Th e abilit y o f

propiconazol e t o bin do r displac e arecepto r

ligan dha s bee n evaluate di n at

leas t 5 separat e studie s o f

A Rbindin g potential . Methodologica l deviation s shoul dhav e n o impac t

o n justificatio n fo r

usin g OSRI i n consideratio n o f propiconazol e interactio n with androge n receptors .

Report Number : TK002342 3 Pag e 1 7 o f 8 5

### 3. 3 Aromatas e - Huma n Recombinan t (OPPT S 890.1200 ) Purpos e o f

Assa y Th e Aromatas e (Huma n Recombinant ) Assay i s ascree n intende dt o identif y chemical s tha t may affec t th e endocrin e syste m(e.g. , steroidogenesis ) b y inhibitin g catalyti c activit y o f aromatase , th e enzyme responsibl e fo r th e conversio n o f

androgen s t o estrogens . Aromatas e (ofte n calle dCYP19 ) i s acytochrom e P45 0 enzym e comple x tha t produce s estrogen s fro m an androge n substrate . Alteration s i n th e amoun t o f aromatase , o r

o f th e catalyti c activity , can alte r th e level s o f

estrogen s i n tissue s an dpotentiall y disrup t estroge n hormon e action . Inhibitio n o f

aromatas e can als o resul t

i n diminishe dbloo dlevel s o f estrogens . Assa y Methodolog y Summar y Th e assay selecte db y th e EP A fo r validatio n use s ahuma n placenta l microsoma l preparatio n tha t contain s aromatase . Th e assay measure s th e conversio n o f androgeni c t o estrogeni c structure s i n microsome s isolate dfro mvariou s targe t tissue s o r cel l line s containin g aromatase , o r

recombinan t

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

an NADPH-generatin g system . Th e describe dassay ha s bee n optimize dfo r us e o f recombinan t

microsom e preparations , whic h can b e obtaine dfro mcommercial sources . On e cite dsourc e i s manufacture db y Gentes t

(Woburn , MA ) tha t

include s huma n recombinan t aromatas e an d P45 0 reductas e (SUPERSOMES®) . Enzym e activity i s measure db y quantifyin g [ 3 

### H]

- labele dwater . Method s usin g both recombinan t an dplacenta l extrac t

were carrie dthroug h th e EDS P validatio n stage s t o demonstrat e thei r equivalence . Th e assay i s conducte db y incubatin g aradioactiv e substrat e ( 3 

### H-androstenedione ) an d NADP H with th e microsom e preparation . 3 

### H 2 

### O i s release ddurin g th e conversio n o f androstenedion e t o estrone , an dresult s ar e reporte da s adirec t measuremen t

o f aromatas e activit y pe r

uni t reactio n time . Competitiv e inhibitio n o f aromatas e activit y ca n b e detecte d b y seria l reactio n tube s containin g increasin g concentration s o f th e competito r

o f interest .

Report Number : TK002342 3 Pag e 1 8 o f 8 5Othe r Scientificall y Relevan t

Informatio n Propiconazol e ha s bee n evaluate di n at leas t 4 assay s wit h th e sam e objectiv e a s OPPT S 890.1200 . On e o f

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

battery , th e 3 othe r studies , publishe di n peer-reviewe djournals , als o reporte dresult s o f propiconazol e activit y o n aromatas e with tissu e preparation s containin g th e CYP1 9 complex .

Tie r I scree n Huma n placenta l microsome s Microsome s extracte dfro mtrou t brai n & ovary Recombinan t

huma n microsome s Recombinan t

huma n microsome s Huma n placenta l microsome s Huma n placenta l microsome s Huma n placenta l microsome s Huma n CYP C 1 9 gen e OSR 1 Reference(s ) Vinggaar de t

al , 200 0 Hinfra y e t

al. , 200 6 Troske n e t

al. , 200 4 Troske n e t

al. , 200 6 Kjsersta de t

al. , 200 7 Ohn o e t al. , 200 4 Lavill e e t al. , 200 6 Judsonetal. , 201 0 Comment s Huma n placenta l microsome s an d3H-androstendion e wa s th e substrat e Teleos t fis h aromatase , [ 3 H]-labele dsubstrat e Recombinan t

huma n microsomes , fluorescen t substrat e SUPERSOME S wer e use d MCF- 7 cell s KG N cell s wit h androstendion e a s th e substrat e an d estrone a s th e produc t 3 H-androstendion e wa s th e substrate ; JEG- 3 huma n choriocarcinom acell s wer e use da s amode l Novascree n cell-fre e hCYPC1 9 enzym e inhibitio n assa y Vinggaar de t

al. , 200 0 Thi s study wa s conducte dat th e Institut e o f

Foo dSafety Laboratory i n Denmark an duse d huma n placenta l microsome s a s th e mode l tes t syste mwith 2 2 differen t

chemicals . 3 H

androstendion e wa s use da s substrate . 4-Androsten-4-ol-3,17-dion e wa s use da s positiv e aromatas e inhibitor . Cytotoxicit y wa s evaluate db y measurin g th e reductio n o f AlamarBlueTM . Propiconazol e wa s evaluate dat aconcentratio n o f

50um . Hinfra y e t

al. , 200 6 Thi s study , conducte di n aFrenc h governmen t environmenta l researc h laboratory , examine d th e abilit y o f

4 3 divers e chemical s t o affec t aromatas e activity . Th e methodolog y use d microsome s prepare dfro mrainbo w trou t ovary an dbrai n tissue . [ 3 H]-androstenedion e wa s incubate dfo r

3 0 minute s i n vitro , ± variou s tes t substances , an dradiolabele destron e wa s extracte dan dcounted . Th e manuscrip t include ddetail s o f

an extensiv e validatio n o f th e protocol . A wid e variety o f chemicals , includin g some heav y metals , wer e evaluated . Propiconazol e wa s incubate dat on e concentratio n (1 0 [iMolar) , usin g brai n an dovaria n microsom e preparations .

Repor t Number : TK002342 3 Pag e 1 9 o f 8 5Troske n e t al. , 200 4 Thi s study wa s conducte di n universitie s i n Wuerzberg , German y an dZuerich , Switzerland . A fluorimetri c assa y base do n huma n recombinan t CYP1 9 enzym e with dibenzylfluorescei n (DBF ) a s asubstrat e wa s use dt o compar e th e inhibitor y potenc y o f 2 2 azol e compounds , includin g propiconazole . Oxidize dDBFforme dby aromatas e was cleave dby 2 M NaO H to generat e afluorescen t

produc t fo r measuremen t

i n thi s assay . Dos e response s wer e establishe dan dduplicat e dataset s wer e analyze dstatisticall y t o deriv e IC5 0 value s (50 % inhibitory concentration ) fo r aromatas e inhibition . Troske n e t

al. , 200 6 Thi s study , conducte di n auniversity i n Wuerzberg , Germany , examine dth e abilit y o f 2 1 azol e compounds , includin g propiconazole , t o inhibi t aromatase . Microsome s ("Supersomes" ) containin g huma n aromatas e (CYP19 ) coexpresse dwit h huma n cytochrom e P45 0 reductas e (baculovirus/insec t cel l expressed ) wer e fro mB D Gentest . Testosteron e wa s utilize da s th e substrat e i n th e presenc e o f varyin g concentration s o f eac h tes t substance . Estradio l produc t

formatio n wa s measure db y anewl y develope dan dfull y validate d analytica l metho dbase do n liqui dchromatography-tande mmas s spectrometry utilizin g photospra y ionizatio n (APPI) . Potenc y o f enzym e inhibitio n wa s expresse di n term s o f IC5 0 concentrations . Kjaersta de t

al. , 200 7 A huma n breas t

cance r cel l lin e (MCF- 7 cells) , whic h expres s th e enzym e aromatas e naturally , wer e utilize di n thi s study . Th e respons e obtaine di n thi s assa y i s th e combine d effec t o f aromatas e inhibitio n an danti-estrogenicity , a s aromatas e convert s testosteron e t o estrogen , whic h the n induce s proliferatio n o f th e cells . B y co-treatin g th e cell s wit h testosteron e an dfungicide s a n inhibitio n o r stimulatio n o f

th e enzym e activit y wa s measure d b y determinin g th e relativ e proliferativ e effec t (RPE) . Prochlora z ha s bee n demonstrate dt o inhibit aromatas e activit y i n differen t i n vitr o assay s an dwa s include da s positiv e control . Fou r azol e fungicide s includin g propiconazol e wer e teste di n thre e independen t experiments at sixtee n differen t

concentrations , an dIC5 0 value s were determined . Ohn o e t al. , 200 4 Thi s study utilize danon-radioactiv e cell-base dassay tha t coul ddetec t

bot h inhibitio n an d inductio n o f

aromatase . Th e assay wa s performe dusin g KG N cells , whic h ar e a steroidogeni c huma n ovaria n granulosa-lik e tumo r cel l lin e tha t

possesse s hig h aromatas e activity that

i s stimulate db y follicl e stimulatin g hormon e o r cAMP . I n addition , th e KG N cell s canno t

synthesiz e androge n o r

estroge n b y themselve s du e t o th e absenc e o r lo w leve l o f 17a-hydroxylase . Therefore , th e aromatas e activit y wa s evaluate dsimpl y b y culturin g th e cell s with androstenedion e an dmeasurin g th e estron e leve l i n th e cultur e mediu mwit h a specifi c enzyme-linke dimmunosorben t assay (ELISA) . Effect s o f treatmen t

wit h propiconazol e a t

si x differen t

concentration s wer e evaluated , alon g wit h 2 2 othe r compounds . Appropriat e controlle dexperiment s demonstratin g th e specificit y o f th e estron e ELIS A assay , an examinatio n o f cytotoxicity , reproducibilit y o f th e assay an dcompariso n t o other reporte daromatas e inhibitio n o r activatio n assay s wer e provided .

Repor t Number : TK002342 3 Pag e 2 0 o f 8 5Lavill e e t al. , 200 6 I n thi s study , abilit y o f

3 0 compound s t o modulat e aromatas e activit y wa s studie s i n ahuma n choriocarcinom aJEG- 3 cel l line . 3 H-androstendion e wa s use da s substrate , an dreleas e o f tritiate dwate r

wa s evaluate dafte r

bot h shor t (2h ) an dlon g (2 4 h ) time s o f exposure , t o identif y eithe r inhibitio n o r

inductio n o f

aromatase , respectively . Propiconazol e wa s evaluate dat

1, 3 an d1 0 uM ; mor e extensiv e follow-u p assay s includin g measuremen t o f mRN A level s o f

CYP1 9 wer e conducte do n certai n tes t substances , bu t

no t fo r propiconazole . Judso n e t

al . 201 0 Th e ToxCas t

grou p identif

y an dprioritiz e reliabl e HT S assay s t o asses s amon g othe r thing s agonist/antagonis t

activity fo r

anumber o f nuclea r receptor s an dgene s involve di n steroi d metabolism . T o examin e aromatas e inhibition , propiconazol e wa s evaluate dNovascreen' s ADME_hCYPC1 9 assay . Se e Appendi x 1 an dJudso n e t al. , 200 9 fo r

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

websit e links .

Repor t Number : TK002342 3 Pag e 2 1 o f 8 5Deviation s t o OPPT S 890.1200 Protoco l OPPT S 890.120 0 Requirement s Criteri a Vinggaar de t

al. , 200 0 Deviation s to OPPT S 890.120 0 Hinfra y e t al. , 200 6 Deviation s t

o OPPT S 890.120 0 Troske n e t al.,200 4 Deviation s t o OPPT S 890.120 0 Troske n e t al.,200 6 Deviation s t o OPPT S 890.120 0 Kjaersta de t

al.,200 6 Deviation s t o OPPT S 890.120 0 Ohn o e t al. , 200 4 Deviation s t o OPPT S 890.1200

Lavill e e t al. , 200 6 Deviation s t o OPPT S 890.120 0 Judsonetal.,201 0 Deviation s t o OPPT S 890.1200

Justificatio n fo r OSR I Recombinan t huma n microsomc s a s CYP1 9 sourc e Huma n placenta l extrac t Aromatas e i n microsome s extracte d fro mfis h brai n an d ovary Non e (Huma n recombinant ) Non e (Huma n recombinant )

Aromatas e naturall y expresse denzym e i n MCF 7 cell s KG N cell s

Aromatas e naturall y expresse denzym e i n JEG- 3 cell s

Non e (huma n recombinant , Novascree n hCYPC19 ) Aromatas e i s bioidentica l i n numerou s anima l specie s Us e [ 3 H]

-

androstenedion e a s enzym e substrat e Non e

Non e

Use d dibenzylfluorescei n a s asubstrat e Use dtestosteron e a s a substrat e

Use dtestosteron e a s a substrat e Non e

Non e

Use d dibenzylfluorescei n a s asubstrate

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

CY P 1 9 Enzym e activity

measure da s [ 3 H]

- water , produce dfro m substrat e b y enzym e actio n Non e Enzym e activit y measure da s [ 3 H]

- estrone , th e steroi d produc t o f th e enzym e actio n Use dth e fluorescen t produc t liberate di n th e cleavag e o f

oxidize d DB F Enzym e activit y measure destradio l produc t o f th e enzym e actio n usin g LC

MS/M S Produc t formatio n Enzym e activit y measure da s [ 3 H]

- estrone , th e steroi d produc t o f th e enzym e actio n Non e (Aromatas e activit y wa s determine db y usin g th e tritiu m releas e assay ) Use dth e fluorescen t produc t liberate di n th e cleavag e o f

oxidize d DB F

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

enzym e i n th e method s use d Conclusions : Th e propiconazol e consortiu mrequest s awaive r fo r th e aromatas e assay becaus e propiconazol e ha s bee n evaluate db y anumbe r o f other researcher s usin g substantiall y similar

methodology , including assay s performe db y th e EPA-OR D i n it s Repor t Number : TK002342 3 Pag e 2 2 o f 8 5ToxCas t screens . Methodologica l deviation s shoul dhav e n o impac t o n justificatio n fo r

usin g OSRI i n consideratio n o f propiconazol e interactio n wit h th e aromatas e complex . 

### 3.4 Estroge n Recepto r Bindin g Assa y Usin g Rat Uterin e Cytoso l (ER-RUC ) (OPPT S 890.1250) Purpos e o f

Assay Thi s assa y wil l b e use dto provid e informatio n o n th e ability o f acompoun dto interac t

wit h th e estroge n receptor s (ERs ) isolate dfro mth e rat uterus . Th e assay i s no t intende dt o b e use dt o sho w tha t

th e interactio n is , specifically , one-sit e competitiv e binding , o r t o characteriz e precisel y th e strengt h o f th e binding . Th e expressio n o f

ahormone' s activit

y begin s whe n i t

bind s to it s specifi c recepto r an d initiate s acascad e o f

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

cells , i n o r

near th e nucleus , an dhormone-boun dE Rinteract s with specifi c site s i n th e genom e o f th e cell t o contro l productio n o f mRNA . Th e hormone

bindin g domai n (HBD ) o f th e estroge n recepto r

i s highl y specifi c compare dt o receptor s fo r other steroi dclasses . It

i s highl y conserve dacros s species . Environmental chemical s that compet e with endogenou s estrogen s hav e th e potentia l t o eithe r induc e (agonist ) hormone

dependen t

transcriptiona l activity , o r t o bloc k (antagonist ) normal hormon e functio n b y preventin g th e endogenou s hormon e fro mbindin g t o th e receptor . Thu s ates t o f a compound' s ability t o bin dt o E Rconstitute s adirect , simpl e evaluatio n o f it s estrogeni c potentia l i n vertebrat e species . Assa y Methodolog y Summar y Th e assay describe di n th e OPPT S protoco l (890.125 0 - Estroge n Recepto r Binding ) measure s th e abilit y o f

aradiolabele dligan d(17p-estradiol ) t o interac t wit h th e E Ri n th e presenc e o f

increasin g concentration s o f ates t chemical . Rat

uterin e cytoso l (RUC ) containin g E Ri s incubate di n tes t tube s with concentration s o f ates t substanc e an dan aliquo t o f radiolabele d[ 3 H]-estradiol . I f th e tes t substanc e interact s wit h th e receptor' s HBD , les s radioligan dca

n bin dan da

n activ e competito r

produce s adescendin g dose-respons e plot . Similarly , compound s tha t d o no t displac e radiolabele destradio l fro mE Rar e presume dt o b e devoi do f receptor-bindin g activity . Th e assay ha s alon g histor y o f us e withi n th e researc h community fo r

rapi dan drelativel y inexpensiv e detectio n o f chemical s wit h E Rbindin g capability . Th e Tie r 1 scree n E Rbindin g assay (ER-RUC ) consist s o f asaturatio n bindin g experimen t t o optimiz e th e tes t

concentratio n o f

estradio l unde r

th e relevan t

assay conditions . Thi s i s achieve dwhe n result s produc e aplatea u i n th e specifi c bindin g curve , alinear Scatchardplot , an daK di n th e rang e o f

0.0 3 t o 1. 5 nM . Afte r

optimizin g condition s i n tha t assay o r laboratory , acompetitiv e bindin g experiment with th e tes t

chemica l i s performed . At

eac h o f 8 concentration s (o f

radiolabele destradio l i n th e saturatio n bindin g assay ; o f tes t chemica l i n th e competitiv e bindin g assay) , thre e simultaneou s replicate s ar e prepared . Eac h se t o f 8 concentration s constitute s arun . Thre e adequat e independen t run s constitut e a n experiment . Repor t Number : TK002342 3 Pag e 2 3 o f 8 5Othe r Scientificall y Relevan t

Informatio n Propiconazol e wa s evaluate db y th e EP A ToxCas t Grou p i n several estroge n recepto r bindin g assays , usin g system s tha t ar e substantiall y similar

t o th e OPPT S 890.125 0 guidelin e E R bindin g assay . Method s an dresult s o f thes e assay s ar e summarize di n Judso n e t al. , 2010 : Judso n et

al. , 201 0 Th e ToxCas t

grou p ha s examine dth e competitiv e bindin g profil e o f 32 0 chemicals , includin g propiconazole , i n th e Novascree n assay s tha t ar e abbreviate dNVS_NR_bE Ran d NVS_NR_hE Rwithi n thei r databas e o f

man y high-throughpu t

screens . Mor

e detail s o n th e methodolog y use di n thes e studie s a s part o f ToxCas t can b e foun di n Appendi x 1 an dJudso n e t al. , 2009 . Specifi c description s o f thes e estroge n bindin g assay s wer e obtaine dfro m Calipe r Lif e Science s an dar e provide di n Appendice s 2 an d3 . NVS_NR_bE Rteste dth e competitiv e bindin g o f propiconazol e i n estroge n recepto r preparation s derive dfro mbovin e uterin e membranes . 3 H-Estradio l wa s use da s th e radiolabelle dligand . IVp-Estradio l wa s use da s apositiv e control . Reaction s wer e carrie d ou t i n an appropriat e buffe r

solutio n at

0-4° C fo r

1 8 hours . Th e reactio n wa s terminate db y th e additio n o f

dextran coate dcharcoa l an dincubate dfo r 2 0 minutes at

0-4°C . Th e reactio n mixture s wer e centrifuge dan dth e radioactivit y boun di n th e supernatan t wa s assesse dan d compare dt o contro l value s i n orde r t o ascertai n an y interaction s o f tes t compoun dwith th e estradio l bindin g site . NVS_NR_hE Rteste dth e competitiv e bindin g o f propiconazol e i n estroge n recepto r preparation s derive dfro mhuma n primary breas t cance r cel l lin e (MCF- 7 cells) . 3 H-Estradio l wa s use da s th e radiolabelle dligand . l?p-Estradio l wa s use da s apositiv e contro l an da s a mode l substrat e fo r

compariso n t o propiconazole . Reaction s wer e carrie dou t i n an appropriate buffe r

solutio n at

0-4° C fo r

1 8 hours . Th e reactio n wa s terminate db y th e additio n o f

dextran coate dcharcoa l an dincubate dfo r 2 0 minutes at

0-4°C . Th e reactio n mixture s wer e centrifuge dan dth e radioactivit y boun di n th e supernatan t wa s assesse dan d compare dt o contro l value s i n orde r t o ascertai n an y interaction s o f tes t compoun dwit h th e estradio l bindin g site . Fo r th e saturatio n bindin g experiments , reproducibl e K dvalue s an dBmax value s fro mprio r run s at Calipe r Lif e Science s ar e show n i n Appendice s 2 an d3 , an dfal l withi n th e acceptabl e rang e at th e typica l substrat e concentratio n tha t i s used . Chemical s i n ToxCas t wer e initiall y screene dat

asingl e concentratio n i n duplicat e well s at aconcentratio n o f

2 5 u M fo r

th e E R bindin g assays . Chemicals-assay pair s tha t showe dsignifican t

activit y wer e the n ru n i n concentratio n respons e format , fro mwhic h a n AC5 0 valu e wa s extracted . Fo r concentratio n response , 8 concentration s wer e teste di n th e range s 0.0229-5 0 u M fo r non-CY P assays . Thes e assay s wer e ru n b y Novascree n ( apart o f Calipe r Lif e Sciences , Hanover , MD ) unde r contrac t t o EPA .

Report Number : TK002342 3 Pag e 2 4 o f 8 5Result s o f thes e assay s ar e publicl y availabl e o n th e ToxCas t websit e usin g th e followin g information : http://www.epa.gov/ncct/toxcas t #Dat aSe t

Name : Novascree n #Fil e Name : /share/server_data/data/ACToR/ToxCast/data/ToxMinerExport/ToxCast_Novascree n _20090807.tx t Deviation s t o OPPT S 890.125 0 Protoco l OPPT S 890.125 0 Requirement s Criteri a Judsonetal . 201 0 Bovin e E RAssay NVS_NR_bE R Deviation s t o OPPT S 890.125 0 Judsonetal . 201 0 Huma n E RAssay NVS_NR_hE R Deviation s t o OPPT S 890.125 0 Justificatio n fo r OSR I E Rfro mra t uterin e cytoso l Mino r (bovin e uteru s preparation )

Huma n primar y cel l lin e a s sourc e

Mino r differences ; eac h represent s a conserve dmammalia n E Rrecepto r

source . Us e [ 3 H]-estradio l a s

enzym e substrat e Non e

Non e

N o deviatio n Hydroxyapetit e ge l

fo r separatio n o f boun dan dfre e Dextran-coate d charcoa l fo r

separatio n

Dextran-coate d charcoa l fo r

separatio n

Mino r difference . Eithe r separatio n metho dha s bee n demonstrate dt o b e effectiv e i n thes e bindin g assays . I n addition , numerou s studie s testin g propiconazol e i n estroge n recepto r reporte r assay s ar e available , a s describe di n Sectio n 3.4 . Thes e provid e very similar evaluation s o f

estrogeni c potentia l t o th e direc t

bindin g assay an dfurthe r suppor t tha t OSRI ar e availabl e fo r propiconazole . Conclusions : Th e propiconazol e consortiu mrequest s awaive r fo r th e conduc t

o f th e Estrogen Receptor

Bindin g (OPPT S 890.1250 ) i n ra t uterin e cytoso l becaus e propiconazol e ha s bee n evaluate di n th e EPA' s ToxCas t progra mwith bot

h huma n an dbovin e estroge n recepto r bindin g assay s that

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

usin g OSR I i n consideratio n o f propiconazole' s interactio n with th e estroge n receptor .

Repor t Number : TK002342 3 Pag e 2 5 o f 8 5

### 3. 5 Estroge n Recepto r Transcriptiona l Activatio n - Huma n Cel l Lin e HeLa

990 3 (OPPT S 890.1300) Purpos e o f

Assay Th e purpos e o f

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

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

i n away tha t

promote s subsequen t

downstreamtranscriptiona l activation . T A assay s hav e lon g bee n use da s screen s t o evaluat e th e specifi c gen e expressio n regulate db y specifi c nuclear receptors , suc h a s th e ERs , an dhav e bee n propose d b y th e EDS P fo r

th e detectio n o f

estrogen-mediate dtransactivation . Nuclear ER s exis t a s at leas t tw o subtypes , terme daan dp . The y are encode db y distinc t genes , an dhav e differen t tissu e distribution , relativ e ligan dbindin g affinitie s an dbiologica l functions . Nuclear ER a mediate s th e classi c estrogeni c responses , an dtherefor e model s currentl y bein g develope dt o measur e E Ractivatio n mainl y us e ERa-linke dreporters . Thi s OPPT S Tes t

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

b e directl y extrapolate dt o th e mor e comple x i n viv o situatio n o f al l estroge n mode s o f

regulatio n o f

cellular processes . Furthermore , thi s Tes t

Guidelin e doe s no t

addres s antagonis t

interactio n wit h th e hERaan d it s subsequen t

effec t o n transcription . Assay Methodolog y Summar y Th e TA assa

y describe di n OPPT S 890.1300 , th e Estrogen Receptor Transcriptional Activatio n Assay (Human Cel l Lin e HeLa-9903) , use s areporte r gen e technique , an di t

i s an i n vitr o too l tha t

provide s mechanisti c data . Th e assay i s use dt o signa l bindin g o f th e estroge n recepto r

with aligand . Th e receptor-ligan dcomple x the n translocate s t o th e nucleus , bind s specifi c DN A respons e elements , transactivate s a n associate dfirefl y luciferas e reporter gen e an dthereb y increase s cellular expressio n o f

luciferas e enzyme . Luciferi n i s a substrat e tha t

i s transforme db y th e enzym e t o abioluminescen t produc t tha t ca n b e simpl y an dquantitativel y measure dwith aluminometer . Th e tes t syste mprovide di n thi s guidelin e utilize s th e hERa-HeLa-990 3 cel l line , whic h i s derive dfro mahuma n cervical tumor . Th e cell s contai n tw o stabl y inserte dconstructs : Th e hER aexpressio n construc t (encodin g th e full-lengt h huma n receptor) . A firefl y luciferas e reporter construc t bearin g fiv e tande mrepeat s o f avitellogeni n Estrogen-Responsiv e Elemen t (ERE ) drive n b y amous e metallothionei n (MT ) promote r TAT A element . Consequently , thi s hERa-HeLa-990 3 cel l lin e can measur e th e ability o f ates t chemica l t o induc e hERa-mediatedtransactivatio n o f luciferas e gen e expression . Dat ainterpretatio n fo r

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

chemica l equal s o r

exceed s an agonis t

respons e equa l t o 10 % o f

tha t induce db y amaximall y inducin g ( 1 nM ) concentratio n o f th e positiv e contro l 17( 3 estradio l (EZ)(i.e. , thePCIO) . Report Number : TK002342 3 Pag e 2 6 o f 8 5Othe r Scientificall y Relevan t

Informatio n Propiconazol e ha s bee n adequatel y teste dregardin g th e estroge n recepto r transcriptiona l activatio n assay . Th e followin g studie s com e fro mreputabl e an dexperience dlaboratorie s tha t hav e an extensiv e publishe dliteratur e i n us e o f transfecte dsteroi dreceptor-linke dcel l reporte r systems . Al l use dwidel y accepte dtechnolog y i n cel l line s tha t hav e bee n use dfo r man y years . Methodologica l deviation s shoul dhav e n o impac t o n justificatio n fo r

usin g OSR I i n consideratio n o f propiconazol e interactio n with estroge n recepto r transactivatio n systems . Tie r I scree n

E Rtransfecte dwit h reporte r Reference(s ) Balague r e t al. , 199 9 Hurs t an dSheahan , 200 3 Kojim ae t al. , 200 4 Kja?rstadetal.,200 7 Bitsc h e t al. , 200 2 Boehmle r e t al. , 200 4 Boehmle r e t al. , 200 6 Judsonetal. , 201 0 Comment s hER dtransfecte dint o HeL acell s hER atansfecte dint o yeas t cell s hER atransfecte dint o CH O hER atransfecte dint o MCF- 7 cell s MCF- 7 cell s proliferatio n MCF- 7 cell s proliferatio n MCF- 7 cell s proliferatio n Multipl e huma n E Rgen e transactivatio n reporte r assay s Balague r et al. , 199 9 Thi s stud y wa s th e closes t t o th e EDS P validate dprotocol . HeL acell s wer e stabl y transfecte dwit h achimeri c recepto r expressio n syste mcontainin g ER aan dluciferase . Abou t 8 putativ e xenoestrogen s wer e incubate dat concentratio n range s o f

0. 1 nMola

r t o 1 0 |o.Molar , an dcompare dt o estradio l at 0. 1 pMolar

t o 1 0 nMolar . Experiment s wer e conducte d a t aFrenc h university . Hurs t an dSheahan , 200 3 Thi s study , conducte dat

th e Centr e fo r

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

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

genome , togethe r

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

gen e Lac-Z . I n th e presenc e o f

estrogen s o r

chemical s wit h estrogeni c activit y (whic h bin dt o an dactivat e th e receptor) , p-galactosidas e i s synthesize dan dsecrete dint o th e assay mediu mi n whic h th e yeas t i s grown . Th e P-galactosidas e the n break s dow n th e chromogeni c substrat e chlorophenol . Twelv e differen t concentration s o f

propiconazol e a s wel l a s 2 5 othe r pesticide s wer e teste di n thi s model . Report Number : TK002342 3 Pag e 2 7 o f 8 5Kjaersta de t al. , 200 7 Thi s stud y use dMCF- 7 cells , ahuma n breas t cancer-derive dcel l line , transientl y transfecte d with a n E Rchimer aan daluciferas e reporte r gene . Cell s wer e plate dan dincubate dwit h medi acontainin g propiconazol e ( 1 nMolar t o 15 0 uMolar ) o r

estradiol-17 p (positiv e control , 1 pMolar t o 1 0 nMolar ) fo r

2 4 h r befor e luciferas e activit y wa s measured . Eac h azol e fungicid e wa s teste di n a t leas t thre e independen t

experiment s an di n twenty-tw o differen t concentrations . Kojima , 200 4 Thi s stud y examine dth e abilit y o f 20 0 chemical s t o induc e arespons e i n an ER-linke d reporte r system . Huma n ER awa s transfecte dint o Chines e hamste r ovary (CHO ) cell s wit h a vector , an daluciferas e reporte r gen e wa s linke ds o tha t

ligan dbindin g t o th e recepto r woul d produc e aluminescen t

signal . Propiconazol e (1 0 nMolar t o 1 0 uMolar ) wa s on e o f th e 20 0 teste dchemical s i n compariso n t o estradiol-17 p (at 0.0 1 pMolar

t o 10 0 nMolar) . Bitsc h e t al. , 200 2 I n thi s study , at

leas t 5 7 activ e ingredient s o f

pesticides , includin g propiconazole , an dgrowt h regulator s wer e examine dfo r thei r possibl e estrogeni c activity . Th e E Screen-Assay base d o n th e huma n breas t

cance r cel l lin e MCF- 7 wa s use da s th e suitabl e tes t system . MCF- 7 cell s wer e seede di n microtite r plate s an dafte r

2 4 h , concentration s (1.4 5 x 10" 1 0 t o 1 x 10" 4 molar ) o f tes t chemical s wer

e adde dan dincubate dfo r si x days . Cell s were fixe dan d stained . Th e numbe r

o f livin g cell s wa s measure dusin g an ELISA-reader . Thi s stud y wa s performe dat

th e Jusrus-Liebi g University i n Giessen , Germany . Boehmle r e t al. , 200 4 I n thi s study , conducte dat Germa n Consume r

Protectio n an dFoo dSafet y Bureau , propiconazol e alon g wit h 1 6 othe r pesticide s wer e evaluate dfo r thei r possibl e estrogeni c activity . Th e E Screen-Assay base do n th e huma n breas t cance r cel l lin e MCF- 7 wa s use da s th e suitabl e tes t

system . Propiconazol e wa s evaluate dat concentration s rang e fro m10" 8 t o 10" 5 molar an dsolubilize di n 0.0 1 molar ethanol . MCF- 7 cell s wer e seede di n microtite r plate s an dafte r

2 4 h , concentration s (1.4 5 x 10"' ° t o 1 x 10" 4 molar ) o f tes t chemical s wer e adde dan dincubate dfo r

si x days . Cell s wer e fixe dan dstained . Th e numbe r o f livin g cell s wa s measure dusin g an ELISA-reader . Boehmle r

e t al. , 200 6 Thi s secon dstudy , conducte dat German Consume r

Protectio n an dFoo dSafet y Bureau , agai n looke dat

propiconazol e alon g with 1 6 other pesticide s t o evaluat e thei r possibl e estrogeni c activity . Th e experimental desig n wa s identica l t o th e study publishe db y Boehmle r e t al. , 2004 . Propiconazol e wa s evaluate dat concentration s rang e fro m10" 8 t o 10" 5 molar an dsolubilize d i n 0.0 1 molar

ethanol . MCF- 7 cell s wer e seede di n microtite r plate s an dafte r

2 4 h , concentration s (1.4 5 x 10" 1 0 t o 1 x 10" 4 molar ) o f tes t chemical s wer e adde dan dincubate dfo r si x days . Cell s wer e fixe dan dstained . Th e numbe r o f livin g cell s wa s measure dusin g an ELISA-reader . Report Number : TK002342 3 Pag e 2 8 o f 8 5Judso n e t al. , 201 0 Th e ToxCas t

grou p utilize dreporte r

gen e assay s o f

th e followin g typ e relate dt o estroge n receptors : • NI H Chemica l Genomic s Cente r (NCGC ) assay , fo r

hER aagonis t

an dfo r hER a antagonis t functio n i n HEK293 H cells , utilizin g an Invitroge n GeneBLaze r reporte r assay system . • Attagen e reporte r

assays , fo r

huma n hERa , huma n estroge n respons e element , huma n estrogen-relate drecepto r

a , an dhuma n estrogen-relate drecepto r y , i n multiple x transcriptio n reporte r

assay s i n HepG 2 cells . Propiconazol e wa s evaluate db y th e EP A i n thes e screens . Se e Appendi x 1 an dJudso n e t al. , 200 9 fo r mor e detail s o n th e methodolog y use di n thes e screen s an dacces s t o th e ToxCas t result s vi athei r

websit e links . Deviation s t o OPPT S 890.1300 Protoco l OPPT S 890.130

0 Requirement s Criteri a Balague r e t al. , 199 9 Deviation s t o OPPT S 890.130 0 Hurs t e t al. , 200 3 Deviation s t o OPPT S 890.130 0 Kojim ae t al. , 200 4 Deviation s t o OPPT S 890.130 0 Bitsc h e t al. , 200 2 Deviation s t o OPPT S 890.130 0 Boehmle r e t al. , 200 4 Deviation s t o OPPT S 890.130 0 Boehmle r e t al. , 200 6 Deviation s t o OPPT S 890.130 0 Kjaersta de t

al. , 200 7 Deviation s t o OPPT S 890.130 0 Judso n etal. , 201 0 Deviation s t o OPPT S 890.130 0

Justificatio n fo r

OSR I HeL acel l lin e Non e Modifie dyeas t

cell s

- develope db y Glax o Wellcome . Chines e Hamste r

Ovar y Cell s MCF- 7 cel l lin e

MCF- 7 cel l lin e

MCF- 7 cel l lin e

MCF- 7 cel l lin e

HEK293 H cell s an dHepG 2

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

Non e No t transfecte dt o ER ; relativ e cel l proliferatio n rat e measure d No t transfecte dt o ER ; relativ e cel l proliferatio n rate measure d No t transfecte dt o ER ; relativ e cel l proliferatio n rat e measure d No t transfecte dt o ER ; relativ e cel l proliferatio n rat e measure d Non e ER alinke dt o reporte r Al l construct s and/o r

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

estroge n mediate d growt h Report Number : TK002342 3 Pag e 2 9 o f 8 5Conclusions : Th e propiconazol e consortiu mrequest s awaive r fo r th e estroge n recepto r transcriptiona l activatio n assay becaus e propiconazol e ha s alread y bee n thoroughl y teste dfo r estroge n recepto r

transcriptiona l activatio n i n existin g studies , usin g both substantiall y similar reporte r gen e methodologie s an dth e E-scree n i n whic h estroge n recepto r activatio n i s measure db y acel l proliferatio n response . Al l report s com e fro mreputabl e an dexperience d laboratorie s that

hav e an expans e o f

publishe dliteratur e i n th e us e o f transfecte dsteroi d receptor-linke dcel l reporte r systems . Al l use dwidel y accepte dtechnolog y i n cel l line s tha t hav e bee n use dfo r

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

usin g OSRI i n consideratio n o f propiconazol e interactio n with estroge n recepto r transactivatio n systems . 3. 6 Fis h Shor t

Ter mReproductio n (OPPT S 890.1350) Purpos e o f

Assa y Th e fis h short-termreproductio n assay wa s develope di n a n effor t t o identif y change s i n morpholog y (secondar

y sexua l characteristics) , histopathology , spawnin g an dspecifi c biochemica l endpoint s whic h migh t reflec t interferenc e wit h th e norma l functionin g o f th e hypothalamic-pituitary-gonada l (HPG ) axis . Assa y Methodolog y Summar y Th e Fis h Short-TermReproductio n assay utilize s matur e fathea dminnow s (Pimephale s promelas ) tha t

ar e expose dt o aminimu mo f thre e concentration s o f

tes t chemica l an d appropriat e control(s ) fo r 2 1 days . Spawnin g i s establishe di n apre-exposur e perio do f at leas t 1 4 days . I n eac h o f

fou r replicat e tank s pe r

concentratio n ther e ar e fou r female s an d tw o males . Th e endpoint s assesse dinclud e survival , behavior , fecundit y measure da s numbe r o f spawn s an dnumbe r

o f egg s pe r femal e reproductiv e day , fertilizatio n succes s measure da s numbe r

o f fertil e egg s pe r

femal e reproductiv e day an dpercen t fertil e eggs , gonada l histopathology , gonadosomati c index , secondar y se x characteristic s (measure d primaril y a s tubercl e number , bu t coul dals o includ e bod y coloration , fatpa dinde x an d oviposito r size ) an dbiochemica l measure s o f vitellogenin , estradio l an dtestosterone . Othe r Scientificall y Relevan t

Informatio n Propiconazol e ha s bee n teste di n asheepshea dminno w ful l lifecycl e stud y an dtw o fathea d minno w earl y lifestag e test s (Iley , 198 1 an dSuprenant , 1987 : no t presente dbu t

availabl e a s supplementa l information) . Th e sheepshea dminno w ful l lif e stud y i s mos t

relevan t (include dfecundity ) an di s describe dbelow :

Stud y Fis h foi l lifecycl e stud y i n sheepshea dminno w Reference(s ) Bretele r 198 8 Comment s

Sheepshea dminno w foi l lifecycl e stud y (100-day exposure) . Assessmen t

o f effect s o n th e F O generatio n fro meg g t o eg g an dF l generatio n t o 2 1 -day s pos t hatc h

Repor t Number : TK002342 3 Pag e 3 0 o f 8 5

### Breteler , 198 8

I n thi s Part

15 8 guidelin e stud y (MRI D 40882001) , sheepshea dminno w (Cyprinodo n variegatus ) wer e continuousl y expose dt o si x concentration s o f propiconazol e an daseawate r contro l throughou t

alif e cycl e (eg g t o egg) . I n addition , thei r progen y wer e reare dfo r

2 8 day s post-hatch . Th e effect s o f exposure o n hatchin g success , survival , growt h an d reproductiv e succes s o f

firs t generatio n (FO ) fis h an dth e hatchin g success , survival an d growt h o f thei r progen y (Fl ) wer e assessed . Thi s study i s robus t relativ e t o assessmen t

o f th e chroni c effect s o f

propiconazol e o n fish , includin g effect s tha t mayb e mediatedvi a endocrin e disruption . Deviation s to OPPT S 890.135 0 Protoco l Compare dt o th e OPPT S 890.135 0 protocol , th e fis h ful l lifcycl e stud y i s ahighe r tie r study . Th e ful l lifecycl e tes t

addresse s al l relevan t

lifestage s no t

jus t reproductio n a s i s th e cas e fo r th e fis h short

termreproductio n assay . I t i s als o importan t

t o not e tha t

ther e i s alon g preceden t fo r sheepshea dminno w studie s o f thi s typ e a s chroni c reproductio n tes t method s includin g th e assessmen t

o f endocrin e disruptio n (Zilliou x e t al. , 2001) . Th e ful l lifecycl e tes t doe s no t includ e vitellogeni n o r

gonada l histopatholog y (require dendpoints ) o r bloo d plasm aestroge n o r

testosteron e titre s (optiona l endpoints) . Th e uncertaint y o f no t includin g thes e specifi c endocrin e biomarker s can b e addresse db y aweigh t o f evidenc e evaluatio n o f al l existin g toxicologica l data . Deviation s t o OPPT S 890.135 0 Protoco l OPPT S 890.135 0 Requirement s Criteri a Breteler , 198 8 Deviation s t o OPPT S 890.135 0
Justificatio n fo r OSRI Tes t

Specie s Fathea d minno w Sheepshea d minno w

Mino r differenc e i n species , bu t sheepshea d minno w ar e a standar dspecie s fo r chroni c reproductio n tes t method s includin g endocrin e disruption . Thre e chemica l

treatmen t

level s require d N o deviatio n 6 chemica l treatmen t level s use d N o deviatio n Flo w throug h

exposure s N o deviatio n Flo w throug h

N o deviatio n Measur e

vitellogeni n an d gonada l histopatholog y No t conducted .

Th e uncertaint y o f no t includin g thes e specifi c endocrin e biomarker s ca n b e addresse db y aweigh t o f evidenc e evaluatio n o f

al l existin g toxicologica l data . Secondar y

sexua l character s N o deviatio n Evaluate d

N o deviatio n



### Conclusions : Th e propiconazol e consortiu mrequest s awaive r fo r th e fis h short

term reproductio n assay a s dat ao n th e lon g termimpac t o f propiconazol e o n fis h reproductio n an d developmen t

hav e bee n provided . Th e dat aprovide dar e fro mahighe r tie r stud y that measure s mor e endpoint s includin g effect s tha t ma y b e mediate dvi aendocrin e disruption . Additiona l fis h studie s fro mshor t t o long-termar e als o available . Report Number : TK002342 3 Pag e 3 1 o f 8 53. 7 Hershberge r - Ra t (OPPT S 890.1400 ) Purpos e o f

Assa y Th e Hershberge r

Bioassa y i s ashort-ter mi n viv o screenin g tes t usin g accessory tissue s o f th e mal e reproductiv e tract . Th e bioassa y serve s a s amechanisti c i n viv o screenin g assay fo r androge n agonists , androge n antagonist s an d5a-reductas e inhibitors . I t i s intende dt o b e include di n abattery o f

i n vitr o an di n viv o test s t o identif y substance s wit h potentia l t o interac t wit h th e endocrin e system . Assa y Methodolog y Summar y Th e Hershberge r

assay i s designe dt o identif y change s i n weigh t

o f fiv e androgen-dependen t tissue s i n th e castrate-peripuberta l mal e rat . I t evaluate s th e abilit y o f

achemica l t o elici t biologica l activitie s consisten t wit h androge n agonists , androge n antagonist s o r 5a-reductas e inhibitors . Th e fiv e androgen-dependen t tissue s include di n thi s Tes t Guidelin e ar e th e ventra l prostat e (VP) , seminal vesicl e (SV ) (plu s fluid s an dcoagulatin g glands) , levato r ani

bulbocavernosu s (LABC ) muscle , paire dCowper' s gland s (COW ) an dth e glan s peni s (GP) . I n th e castrate-peripuberta l mal e rat , thes e fiv e tissue s al l respon dt o androgen s with a n increas e i n absolut e weight . Whe n thes e sam e tissue s ar e stimulate db y administratio n o f a poten t referenc e androgen , the y al l respon dt o antiandrogen s with adecreas e i n absolut e weight . Th e scree n i s conducte db y dail y administratio n (gavag e o r subcutaneou s injection ) fo r a perio dofte n consecutiv e days , t o aminimu mo f tw o treatmen t

group s o f

experimenta l animals , at

on e dos e leve l pe r

group . Th e animal s ar e necropsie dapproximatel y 2 4 hour s afte r th e las t administration . A significan t increas e i n tw o o r

mor e targe t

organ weight s o f th e tes t substanc e groups , compare dt o th e vehicl e contro l group , indicate s tha t th e tes t substanc e i s positiv e fo r

androgeni c activity . Certai n androgens , suc h a s trenbolone , tha t canno t b e Sa-reduced , hav e amor e pronounce deffec t o n th e LAB C an dG P versu s testosteron e propionat e (TP) , bu t al l responsiv e tissue s ar e expecte dt o displa y increase d growt h i n th e presenc e o f

an androgen . Whe n screenin g fo r

potentia l antiandrogeni c activity , th e tes t substanc e i s administere ddail y b y oral gavag e o r

subcutaneou s injectio n fo r aperio dofte n consecutiv e days , i n concer t wit h dail y s c (subcutaneous ) injection s o f testosteron e propionat e (TP , 0. 2 o r 0. 4 mg/kg/d) . Th e validatio n progra mdetermine dthat eithe r dos e o f T P coul db e used . Graduate ddose s o f th e tes t substanc e ar e administere dt o aminimu mo f thre e treatmen t

group s o f

animals . Th e animal s ar e necropsie dapproximatel y 2 4 hour s afte r th e las t dose . A significan t

decreas e i n tw o o r mor e targe t

organ weight s o f

th e tes t substanc e plu s T P groups , compare dt o th e T P onl y contro l group , suggest s that th e substanc e i s positiv e fo r antiandrogeni c activity . Othe r Scientificall y Relevan t

Informatio n Propiconazol e ha s bee n previousl y teste di n aHershberge r assay fo r anti-androgeni c effect s usin g substantiall y similar methodolog y a s th e EDS P Tie r 1 scree n guidelines , an di n mor e extensiv e studie s o f

androgen-sensitiv e endpoint s i n mal e rats .
Report Number : TK002342 3 Pag e 3 2 o f 8 5Tie r I scree n Hershberge r Mal e reproductio n studie s OSR I Reference(s ) Kjaersta de t

al. , 200 7 Taxvi g e t al. , 200 8 Tullyetal.,200 6 Goet z e t al. , 200 7 Comment s

Hershberge r

protocol , fo r

anti-androgeni c effect s Hershberge r

protocol , fo r

anti-androgeni c effect s Mal e ra t stud y wit h androgen-responsiv e endpoint s measure dafte r

14-day treatment . Reproductio n stud y i n rats , wit h specia l emphasi s o n mal e ra t

effect s includin g multipl e androgen-responsiv e endpoint s Kjaersta de t

al. , 200 7 (als o publishe db y Taxvi g e t al. , 2008 ) I n thi s stud y conducte db y researcher s at Departmen t

o f Toxicolog y an dRis k

Assessment , Nationa l Foo dInstitute , Technica l Universit y o f Denmark , S0borg , Denmark , fungicide s includin g propiconazol e wer e administere dt o castrate dmal e rat s treate dwit h testosteron e an dth e effect s wer e examine di n bot h intac t an dcastrate dtestosterone-treate dmale s b y analyzin g weight s o f

reproductiv e organs , seru mhormon e level s an dprostat e gen e expression . On e grou p o f intac t animal s an dfurthe r

group s o f

castrate dmal e Wistar

rat s wer e include di n th e study ( n = 6 pe r group) . Th e intac t

rat s an don e grou p o f

castrate drat s serve da s negativ e control s an dwere give n peanu t oi l only . Th e rat s i n th e remainin g castrate dgroup s wer e al l dose dwit h testosteron e propionat e (0. 5 mg/kg/da y s.c.) . On e group wa s onl y administere dtestosteron e propionat e an dserve da s th e contro l grou p t o whic h al l othe r

group s wer e compared . Th e positiv e contro l group receivedflutamid e (1 0 mg/k g bod y weight/da y orally) . Th e las t thre e group s o f

castrate drat s received propiconazol e orall y at

dose s o f 50,10 0 o r

15 0 mg/k g bod y weight/da y fo r seve n days . Afte r sacrifice , secondary se x organ s wer e remove dan dweighed . I n addition , seru mwa s collecte dan dluteinizin g hormone , rat follicle-stimulatin g hormon e an dT 4 hormon e level s wer e analyzed . Tull y e t al. , 200 6 Fou r triazol e fungicide s includin g propiconazol e wer e studie dusin g both traditiona l toxicity endpoint s an dtoxicogenomi c techniques , an dtherefor e thi s stud y provide dsupplementa l measurement s o f

possibl e androgeni c effect s i n mal e rats . Adul t mal e Sprague-Dawle y rat s wer e dose dfo r

1 4 day s b y gavag e (PND 6 0 t o PN D 74) . Propiconazol e wa s administere dat concentration s o f

0 , 10 , 7 5 an d15 0 mg/kg/day . Followin g exposure , seru mwa s collecte dfo r hormon e measurement s (testosterone , LH , FSH , an destradiol) , an dlive r an dteste s wer e collecte dfo r

histology , enzym e biochemistry o r gen e expressio n profiling . Thi s stud y measure dorga n weight s o f androgen-sensitiv e organ s including lef t testi s an dlef

t epididymis , a s wel l a s spermmorpholog y followin g a14-day propiconazol e treatment . Goet z e t al. , 200 7 Thi s specialize done-generatio n reproductiv e toxicit y stud y i n rats , wit h aparticular emphasi s o n response s i n F l mal e offspring , evaluate dth e respons e t o fou r differen t tes t substance s includin g propiconazole . A mor e complet e descriptio n o f th e publishe dstud y i s presente di n Sectio n 3. 8 Mal e Puberta l Rat Assay ; th e endpoint s evaluate di n thi s stud y giv e higher-tie r evaluation s o f

potentia l androgeni c an danti-androgeni c effect s an dar e relevan t t o OSR I fo r th e Hershberge r

Assay . Time dpregnan t

Wistar Ha n IG S rat s wer e receive dfro mCharle s Rive r Laboratorie s (Raleigh , NC ) o n gestatio n day (GD ) 1- 3 an dassigne dt o treatmen t Report Number : TK002342 3 Pag e 3 3 o f 8 5group s s o a s t o achiev e equivalen t weigh t mean s acros s treatmen t

groups . Propiconazol e wa s administere di n th e die t

at concentration s o f

0 , 100 , 50 0 an d250 0 pp mfro mGD 6 throug h PND92 . Th e hig h dos e o f 250 0 pp mwa s considere dt o b e at o r abov e th e MTD . Dam s wer e allowe dt o delive r

naturally , with day o f

deliver y designate da s postnatal day (PND ) 0 fo r th e F l offspring . O n PN D 8 , litter s wer e weighe dan dthe n culle dt o eigh t pup s pe r dam , retainin g male s preferentially , t o maximiz e uniformit y i n growt h rates . Th e F l generatio n continue do n th e sam e treate dfee ddiet s upo n weanin g a t PND23 . F l offsprin g fee dintak e an dbod y weight s wer e measure dweekl y unti l necropsy . On e mal e fro meac h litte r wa s take n t o necrops y at

PND1 , 22 , 5 0 o r

9 2 t o asses s affect s o n selec t organ weights , histolog y an dhormon e measures . Whole-bod y weight s wer e measured , an dthe n brain , hypothalamus , hippocampus , pituitary , thyroid , liver , testis , epididymis , ventra l prostat e an dseminal vesicle s wer e removed , weighe dan dfixe di n 10 % neutra l buffere dformali n fo r histology . Bloo dwa s collecte dat

PN D 22 , 50 , 9 2 an d99 . Seru msample s wer e collecte dan duse dfo r hormon e analysis . On e testi s an depididymi s fro meac h mal e at PN D 22 , 5 0 an d9 2 obtaine d at necrops y wer e use dfo r

sper mmorphology , count , velocit y an dmotilit y analysis . Seru m level s o f th e thyroi dhormone s T 3 an dT 4 a s wel l a s estadio l an dtestosteron e wer e measure d wit h acommercia l radioimmunoassa y kits . Thyroi dstimulatin g hormon e (TSH ) a s wel l a s luteinizin g hormon e level s wer e measure dusin g specialize dradioimmunoassa y methods . Fo r th e F l males , matin g assay s wer e conducte do n PN D 7 8 o r olde r treate dmales , b y pairin g wit h untreate dvirgi n Wistar Ha n female s PN D 5 6 o r

older . Matin g behavio r

an d matin g succes s wer e measured . Overall , thi s publicatio n describe di n Goetz e t al . 200 7 provide s awid e arra y o f

higher-tie r

test s that allo w determinatio n o f

th e potentia l fo r androgeni c agonis t

o r antagonis t

propertie s o f

propiconazole , whic h i s th e primary purpos e o f th e Hershberge r

assay . Specifically , endpoint s o f androgen-sensitiv e organ weights , spermparameters , hormon e levels , matin g behavior , mal e reproductiv e trac t micropathology , an dreproductiv e succes s ar e al l determine di n thi s study an dwoul db e marker s o f androgeni c o r anti-androgeni c potential . Deviation s t o OPPT S 890.1400 Protoco l OPPT S 890.1400 Requirement s Criteri a Kjaersta de t

al , 2007 ; Taxvi g e t al. , 200 8 Deviation s t o OPPT S 890 . 1400 Justificatio n fo r OSR I Castrat e

peripuberta l mal e rat s Non e

Sam e anima l mode l a s EDS P assay Tes t

substanc e administere d1 0 days , eithe r gavag e o r s c Tes t substanc e administere d7 day s b y gavage . Duratio n o f treatmen t is substantiall y simila r Longe r treatment s evaluate di n Tull y e t al. , 200 6 an d Goet z e t al. , 2007 . Tes t

fo r agonis t propertie s b y direc t administratio n t o castrat e rat s No t teste dfo r agonis t activit y i n thes e reference s Potentia l fo r androgeni c agonis t propertie s full y evaluate di n mal e rat s i n Goetz e t al. , 200 7 an d Tull y e t al. , 2006 . Thes e are higher

tie r studies . Tes t

fo r antagonis m usin g concomitan t

T P administratio n Non e

Sam e evaluatio n desig n a s EDS P protoco l Weig h 5

tissues : VP,SV , LABC , COW , G P Non e

Al l 5 tissue s weighed , a s include di n EDS P protoco l

Report Number : TK002342 3 Pag e 3 4 o f 8 5Conclusions : Th e propiconazol e consortiu mrequests awaive r fo r th e EDS P Tie r

1 Hershberge r

assay becaus e propiconazol e ha s alread y bee n thoroughl y teste di n existin g studie s usin g substantiall y similar methodolog y fo r

th e anti-androgeni c portio n o f th e assa y (Kjaersta de t

al. , 2007 ; Taxvi g e t

al. , 2008) . Whil e thi s Hershberge r Assa y di dno t

includ e a n evaluatio n o f

androgeni c agonis t

respons e i n castrate dmal e rats , higher-tie r studie s wit h propiconazol e suc h a s Goet z e t al. , 200 7 teste dmal e rat s wit h propiconazol e i n a reproductio n study , an dmeasure dth e weight s o f secondary se x organ s (testis , prostate , epididymis , an dsemina l vesicles) , sper mparameters , mal e reproductiv e trac t micropathology , an dhormon e level s includin g testosterone , L H an dFSH . Tull y e t al. , 200 6 provide s man y similar

evaluation s followin g a14-da y exposure t o mal e rats . Al l o f thes e studie s i n combinatio n provid e asensitiv e evaluatio n o f androgeni c agonis t

an dantagonis t potential . I n addition , an OPPT S Guidelin e 2-generatio n reproductio n stud y an d developmenta l toxicity studie s i n rat s an drabbit s hav e no t reveale dan y effect s o n reproductio n o r

developmen t

tha t woul dsugges t

an androgeni c agonis t

o r antagonis t potentia l fo r

propiconazol e (Sectio n 4.0) . 3. 8 Femal e Puberta l - Ra t (OPPT S 890.1450) Purpos e o f

Assay Th e purpos e o f

th e femal e puberta l assa y i s t o hel p identif y chemical s o r

mixture s tha t

hav e th e potentia l t o interac t

with th e endocrin e system , b y evaluatin g effect s o n puberta l developmen t

an dthyroi dfunctio n i n th e intac t juvenile/peripuberta l femal e rat . Thi s assay i s capabl e o f

detectin g anti-thyroid , estrogeni c o r anti-estrogeni c chemical s (includin g agent s whic h ac t vi aalteration s i n recepto r bindin g o r

steroidogenesis) , o r

agent s whic h ca n alte r puberta l developmen t

vi achange s i n luteinizin g hormone , follicl e stimulatin g hormone , prolacti n o r

growt h hormon e level s o r

vi aalteration s i n hypothalami c function . Assa y Methodolog y Summar y Th e OPPT S 890.145 0 scree n use s fiftee n femal e rat s i n eac h o f 3 treatmen t

group s (on e administere dvehicl e an dtw o give n ates t substanc e at

differen t dos e levels) . Th e Sprague

Dawle y rat

strai n i s preferred . Th e highes t dos e leve l shoul db e at

o r jus t belo w th e Maximu mTolerate dDos e (MTD) . Al l dam s mus t

b e pregnan t

fo r th e firs t tim e an dtime dt o delive r o n th e sam e day . T o maximiz e uniformit y i n growt h rates , th e litter s ar e standardize dt o 8 t o 1 0 pup s pe r litte r betwee n post-nata l day s (PND ) 3 an d5 . Th e pup s ar e weane do n PN D 2 1 an dtreatmen t i s initiate dfro mPN D 2 2 throug h PN D 42 . Tes t chemical s ar e administere db y oral gavag e i n 2. 5 t o 5. 0 m l vehicle/k g bod y weigh t at 0700-090 0 daily . Th e followin g observation s ar e made : Vagina l Opening : Beginnin g o n PN D 22 , female s ar e examine ddail y fo r vagina l opening . Estrou s Cyclicity : Beginnin g o n th e day o f vagina l opening , dail y vagina l smear s ar e obtaine dan dclassifie da s diestrus , proestru s o r estrus . Metestru s i s classifie da s (an earl y par t of ) diestrus . A t

th e en do f

th e study , th e overal l patter n o f eac h femal e i s characterize d a s regular cyclin g (recurrin g 4

- t o 5-day cycles) , irregula r cyclin g (some regular

cyclin g bu t Repor t Number : TK002342 3 Pag e 3 5 o f 8 5with aperio do f diestru s longe r

tha n 3 day s o r

aperio do f

cornificatio n longe r

than 2 days ) o r no t cyclin g (havin g prolonge dperiod s o f eithe r vagina l cornificatio n o r

leukocyti c smears) . Th e preferre dmetho do f

sacrific e i s b y injectabl e anesthetic , followe dimmediatel y b y decapitatio n i n orde r

t o obtai n asufficien t

volum e o f

bloo dfo r th e hormon e an dchemistrie s measurements . Trunk

bloo di s collecte dimmediatel y b y inversio n ove r afunnel , i n seru m separatio n tube s (n o anticoagulant) . A t necropsy , th e ovarie s (withou t oviducts) , uterus , thyroi d(wit

h attache dportio n o f

trachea) , liver , kidneys , pituitary an dadrenal s ar e remove d an dth e weight s o f

eac h excep t

th e thyroid/trache aan duteru s recorde d(kidneys , adrenal s an d ovarie s ar e weighe da s pairs) . Th e ovaries , uterus , kidne y an dthyroi dtissu e ar e place di n 10 % buffere dformali n fo r at leas t 2 4 hour s an dthe n store di n 70 % ethano l unti l embedde di n paraffin . Section s ar e cu t an dstaine dwit h hematoxyli n an deosi n (H&E ) fo r subsequen t histologica l evaluations . Hormona l Assays : Immunoassay s o f thyroxin e (T4 ) an dTS H can b e conducte dusin g radioimmunoassa y (RIA) , immunoradiometri c assay (IRMA) , enzyme-linke d immunosorben t

assay (ELISA ) o r

time-resolve dimmunofluorescen t procedures . Bloo dChemistry : An y standar dpane l o f bloo dchemistry test s that include s creatinin e an d bloo dure anitroge n (BUN ) may b e used , a s lon g a s th e measurement s ar e calibrate dfo r rat s an dth e norma l range s fo r control s ar e reported . Histology : Uterus , thyroid , on e ovary an don e kidne y ar e evaluate dfo r pathologi c abnormalitie s an dpotentia l treatment-relate deffects . Thyroi dsection s ar e subjectivel y evaluate dfo r

follicula r cel l heigh t

an dcolloi dare ausin g afiv e poin t gradin g scal e ( 1 = shortest/smallest ; 5 = tallest/largest) , an dan y abnormalities/lesion s noted . Ovarian histolog y shoul dinclud e an evaluatio n o f follicular developmen t

(includin g presence/absenc e o f tertiary/anrral follicles , presence/absenc e o f corporalutea , change s i n corpu s luteu m developmen t

an dchange s i n th e number s o f bot h primary an datreti c follicles ) i n additio n t o an y abnormalities/lesions, suc h a s ovarian atrophy . Uterin e histolog y shoul ddocumen t case s o f uterin e hyper

- o r

hypotroph y a s characterize db y change s i n uterin e ho mdiamete r an d myometrial , stroma l o r

endometria l glan ddevelopment . Othe r Scientificall y Relevan t

Informatio n Propiconazol e ha s bee n previousl y teste di n th e femal e puberta l assa y usin g substantiall y similar methodolog y a s th e EDS P Tie r 1 scree n guidelines . Tie r I scree n Femal e Puberta l OSR I Reference(s ) Rocket t e t al. , 200 6 Comment s

A significantl y mor e encompassin g stud y tha n OPPT S 890 . 145 0 Rocket t e t al. , 200 6 I n thi s study , conducte dat th e laboratorie s o f

th e Offic e o f

Researc h an dDevelopment , U.S . Environmenta l Protectio n Agency , Researc h Triangl e Park , N C 27711 , US A an dth e Departmen t

o f Environmenta l an dMolecular Toxicology , North Carolin aStat e University , Report Number : TK002342 3 Pag e 3 6 o f 8 5

### Raleigh , N C 27695 , USA , propiconazol e a s wel l a s tw o othe r triazol e fungicide s wer e administere dt o timed-pregnan t Wistar-Han rat s (Charle s Rive r Laboratory , Raleigh , NC) . Dam s wer e assigne dt o treatmen t group s b y randomization , t o ensur e equivalen t bod y weigh t distributio n acros s th e groups . Eac h treatmen t grou p consiste do f

approximatel y 3 5 dam s ( a tota l o f 34 8 pregnan t

dam s wa s used) . Dam s an dthei r offsprin g wer e expose dt o propiconazol e administere dat 0 , 100 , 50 0 an d250 0 pp mvi ath e fee dfro mG D 6 t o PN D 99 . Th e hig h dos e o f

250 0 pp mwa s considere dt o b e at o r abov e th e MTD . Dam s wer e allowe d t o delive r naturally , wit h th e da y o f deliver y designate da s postnata l da y (PND ) 0 fo r th e F l offspring . Pup s wer e weighe do n PN D 0 . Th e siz e an dgende r rati o o f eac h litte r

wa s noted . Litter s wer e culle dt o eigh t pup s o n PN D 8 usin g blinde drando mselection , an dth e pup s weane do n PN D 22 . Femal e weanling s wer e house dtw o pe r cag e an dcontinue dunti l terminatio n o n th e same die t a s thei r dam s ha dbee n expose dto . Ear tag s wer e use dt o identif

y weanlings . Bod y weigh t (BW ) an danogenita l distanc e (AGO ) at PN D 0 wer e recorded . Examinatio n fo r vagina l openin g commence do n PN D 29 , an dth e ag e an dB W at vagina l openin g (VO ) wa s recorded . Estrou s cyclicity wa s monitore dfollowin g V O fo r tw o weeks , durin g week s 5- 6 followin g VO , an dfro mPN D 9 2 t o th e firs t proestrou s day afte r

PN D 9 9 (week s 9-10) . Bod y an d orga n weight s (brain , hippocampus , hypothalamus , liver , pituitary , thyroid , ovarie s an d uterus ) wer e measure dat

necropsy . Liver , thyroi d(score dfo r colloi ddepletion , follicula r cel l hypertroph y and/o r

follicula r cel l hyperplasia ) an dovarie s (every twentieth sectio n wa s staine dwith H an dE an dexamine dfo r edema , cysti c follicle s an dretaine doocytes ) wer e rea dfo r histopathology . Estradio l i n seru msample s take n at sacrific e wa s measure dusin g radioimmunoassa

y techniques .

Report Number : TK002342 3 Pag e 3 7 o f 8 5Deviation s t o OPPT S 890.145 0 Protoco l OPPT S 890.145 0 Requirement s Criteri a Rocket t e t al. , 200 6 Deviation s t o OPPT S 890.145 0 Justificatio n fo r OSR I Jus t weane dfemal e rat s Non e Non e neede d Treate dfro mPN D 22-4 2 G D 6 - PN D 9 9 Include s th e require dtreatmen t interval . 2 tes t dose s b y gavage , includin g 1 nea r

MT D 3 dose s vi afeed ; highes t dos e wa s a t o r abov e th e MT D Essentiall y i n lin e with EDS P metho d

Evaluat e VO , estrou s cycl e patterns , seru m T4 , TSH , variou s chemistrie s Evaluate dVO , AGD , estrou s cycl e pattern s an destroge n level s Mos t o f targete dparameter s o f

thi s apica l tes t wer e assessed . Othe r

cite d studie s hav e als o evaluate dseru mT4 , T 3 an dTS H (Kjaersta de t al. , 2007 ; Taxvi g e t al. , 2008 ; Goet z e t

al. , 2007 ) an dseru mchemistry (Sachsse , 1979a ) i n mal e o r

femal e rats .
Histologica l exa mo f

ovaries , uterus , thyroid , kidne y Histologica l exa mo f th e ovaries , thyroi dan dlive r

(uteru s weighed ) Mos t o f targete dparameter s wer e assessed . Histologica l examinatio n o f furthe r tissue s includin g kidneys , uteru s and/o r

vagin awer e performe d als o i n an OPPT S Guidelin e 2

- generatio n reproductio n stud y (Border s & Salamon , 1985 ) an da90

- day rat stud y (Sachsse , 1979a) . Conclusions : Th e propiconazol e consortiu mrequest s awaive r fo r th e EDS P Tier

1 Femal e Puberta l assay becaus e propiconazol e ha s alread y bee n teste di n a n existin g stud y usin g substantiall y similar

methodology . I n combinatio n wit h othe r literatur e studie s an dOPPT S Guidelin e toxicit y studie s i n rat s wit h propiconazole , th e endpoint s t o b e measure di n th e Femal e Pubertal assa

y hav e bee n thoroughl y covered . Ther e appea r to b e n o methodologica

l deviation s tha t

woul dimpac t

th e justificatio n fo r

usin g OSRI i n consideratio n o f propiconazol e activity i n th e femal e rat puberta l screen . 

### 3. 9 Mal e Puberta l - Rat (OPPT S 890.1500 ) Purpos e o f

Assa y Th e purpos e o f

th e mal e puberta l assay i s t o hel p identif y chemical s o r

mixture s tha t

hav e th e potentia l t o interac t

wit h th e endocrin e system , b y identifyin g effect s o n pubertal developmen t

an dthyroi dfunctio n i n th e intac t juvenile/peripuberta l mal e rat . Thi s assa y i s capabl e o f

detectin g anti-thyroid , androgeni c o r anti-androgeni c chemical s (includin g agent s whic h ac t vi aalteration s i n recepto r bindin g o r

steroidogenesis) , o r

agent s whic h alte r puberta l developmen t

vi achange s i n luteinizin g hormone , follicl e stimulatin g hormone , prolacti n o r

growt h hormon e level s o r

vi aalteration s i n hypothalamic function .

Report Number : TK002342 3 Pag e 3 8 o f 8 5

### Assa y Methodolog y Summar y

A summary o f

th e methodolog y use di n th e OPPT S guidelin e study (890.150 0 - Mal e Pubertal (Rat) ) follows . Thi s protoco l use s fiftee n mal e rat s i n eac h treatmen t group ( 1 vehicle-treate dan d2 xenobiotic-treate ddoses) . Th e Sprague-Dawle y o r Wistar strain s ar e preferred . Th e highes t dos e leve l shoul db e at

o r jus t belo w th e Maximu mTolerate dDos e (MTD) , an dth e lowe r dos e at one-hal f th e highe r

dos e unles s justificatio n i s provide dfo r testin g at adifferen t

dos e level . Al l dam s mus t

b e pregnan t

fo r th e firs t tim e an dtime dt o delive r o n th e sam e day . T o maximiz e uniformity i n growt h rates , th e litter s are standardize dto 8 to 1 0 pup s per litte r betwee n post-nata l day s (PND ) 3 an d5 . Th e pup s ar e weane do n PN D 2 1 an dtreatment s initiate dn o late r

tha n PN D 23 . Tes t

substance s ar e administere db y ora l gavag e i n 2. 5 t o 5. 0 m l vehicle/k g bod y weigh t at 0700-090 0 daily , fro mPN D 2 3 throug h PN D 53 . Th e followin g observation s ar e made : Preputia l Separation : Beginnin g o n PN D 30 , male s ar e examine ddail y fo r preputia l separatio n (PPS) . Th e appearanc e o f partial an dcomplete preputial separatio n o r apersistent threa do f tissu e betwee n th e glan s an dprepuc e ar e al l recorde do n th e day s the y ar e observed . Th e day o f

complet e preputia l separatio n i s th e designate dendpoin t fo r th e parameter . Necropsy : Subject s ar e kille do n PN D 53 . Trun k bloo di s collecte dimmediatel y b y inversio n over

afunnel , i n seru mseparatio n tube s (n o anticoagulant) . Th e seru mi s store dat -2 0 ° C o r colde r fo r subsequen t

hormon e an dbloo dchemistry measurements . A t necropsy , th e testes , epididymides , ventra l prostate , dorsolatera l prostate , seminal vesicl e with coagulatin g gland s an dfluid , levato r an i plu s bulbocavernosu s muscles , thyroi d(wit h attache dportio n o f

trachea) , liver , kidneys , pituitar y an dadrenal s ar e remove dan dth e weight s o f

eac h excep t

th e thyroid/trache arecorded . Th e thyroi d(wit h attache dportio n o f th e trachea ) an dasingl e testi s an depididymi s fro meac h anima l ar e prepare dfo r histologica l examination . Testi s an depididymi s ar e place di n Bouin' s fixativ e overnigh t an dstore di n 70 % ethano l unti l embedde di n paraffin . The y ar e the n sectione dan dstaine dwith hematoxyli n an deosi n (H&E ) fo r subsequen t

histologica l evaluations . Th e thyroid , wit h attache dtrachea , i s fixe di n 10 % buffere dformali n fo r at leas t 2 4 hours . The n th e thyroi d (wit h parathyroids ) i s dissecte dfro mth e trachea , blotte dan dweighed , place di n 70 % ethano l unti l embedde di n paraffin , sectioned , staine dwit h H& E an dhistologicall y evaluated . Kidne y i s als o fixe di n 10 % buffere dformali n fo r at leas t 2 4 hours , the n place di n 70 % ethano l unti l embedde di n paraffin , sectioned , staine dwith H& E an dhistologicall y evaluated . Hormona l Assays : Hormona l measurement s can b e conducte dusin g radioimmunoassa y (RIA) , immunoradiometri c assay (IRMA) , enzyme-linke dimmunosorben t assay (ELISA ) o r time-resolve dimmunofmorescen t procedures . Seru mhormon e level s t o b e measure dar e testosterone , thyroxin e (T4 ) an dthyroi dstimulatin g hormon e (TSH) . Report Number : TK002342 3 Pag e 3 9 o f 8 5Bloo dChemistry : An y standar dpane l o f bloo dchemistry test s tha t include s creatinin e an d bloo dure anitroge n (BUN ) ma y b e use da s lon g a s th e measurement s ar e calibrate dfo r rat s an dth e norma l range s fo r control s ar e reported . Histology : Testis , epididymis , thyroi dan don e kidne y ar e evaluate dfo r pathologi c abnormalitie s an dpotentia l treatment-relate deffects . Thyroi dsection s ar e subjectivel y evaluate dfo r

follicula r cel l heigh t

an dcolloi dare ausin g afiv e poin t gradin g scal e ( 1 = shortest/smallest ; 5 = tallest/largest ) an dan y abnormalities/lesion s noted . Othe r Scientificall y Relevan t

Informatio n Propiconazol e ha s bee n previousl y teste di n th e mal e puberta l assay usin g substantiall y simila r methodolog y a s th e EDS P Tie r 1 scree n guidelines . Th e cite dstud y appear s t o hav e evaluate dessentiall y al l o f th e importan t

endocrine-relate dtarget s identifie di n th e OPPT S 890.150 0 protocol . Importantly , th e anima l strain , metho do f administratio n an dduratio n o f dosin g use di n th e Goet z e t al . (2007 ) stud y wer e essentiall y identical t o th e EDS P protocol , bu t i n man y area s exceede dth e requirement s o f th e OPPT S 890.150 0 screen . Ther e appea r t o b e n o methodologica l deviation s tha t woul dimpac t

th e justificatio n fo r

usin g OSR I i n consideratio n o f

propiconazol e activit y i n th e mal e rat puberta l screen . Tie r I scree n Mal e Puberta l Reference(s )

Goetz e t al. , 200 7 Comment s

Significantl y beyon dth e EDS P Tie r 1 scree n Goet z e t al. , 200 7 Thi s stud y wa s conducte dat Offic e o f Researc h an dDevelopment , U.S . Environmenta l Protectio n Agency , Researc h Triangl e Park , NC , US A an dDepartmen t o f Environmenta l an d Molecular

Toxicology , North Carolin aStat e University , Raleigh , NC , USA . Time dpregnan t Wista r Han IG S rat s wer e receive dfro mCharle s Rive r Laboratorie s (Raleigh , NC ) o n gestatio n day (GD ) 1- 3 an dassigne dt o treatmen t group s s o a s t o achiev e equivalen t weigh t mean s acros s treatmen t

groups . Rat s receive don e o f 4 differen t

triazol e treatments , on e o f whic h wa s propiconazole . Dose s o f propiconazol e use dwer e 0,100 , 50 0 an d250 0 ppm . Th e dos e o f

250 0 pp mi s considere dt o b e at o r abov e th e MT D dose . Dam s and/o r thei r F l offsprin g wer e administere dpropiconazol e i n th e die t fro mGD 6 throug h PND99 . Dam s wer e allowe dt o delive r naturally , wit h da y o f

deliver y designate da s postnata l da y (PND ) 0 fo r

th e F l offspring . O n PN D 8 , litter s wer e weighe dan dthe n culle dt o eigh t pup s pe r dam , retainin g male s preferentially , t o maximiz e uniformit y i n growt h rates . F l offsprin g wer e house dwith thei r

respectiv e mother s unti l weanin g at PN D 23 . Male s an dfemale s wer e the n remove dfro mth e dam s an dhouse db y treatmen t i n same-se x pair s unti l PN D 50 . Male s wer e singl e house dafte r PN D 50 , bu t

female s remaine dhouse di n pairs . Th e F l generatio n continue do n th e sam e treate dfee ddiet s upo n weanin g at PN D 23 . F l offsprin g fee dintak e an dbod y weight s wer e measure dweekl y unti l necropsy . On e mal e fro meac h litte r wa s take n t o necrops y at PN D 1,22 , 5 0 an d9 2 t o asses s affect s o n selec t organ weights , histolog y an dhormon e measures . O n PN D 0 , pu p bod y weigh t an danogenita l distanc e (AGO ) wer e measured , an dfootpad s wer e tattooe dfo r identification . F l male s wer e examine dfo r

preputia l separatio n (PPS ) beginnin g o n PN D 38 , continuin g dail y unti l Repor t Number : TK002342 3 Pag e 4 0 o f 8 5

### complet e cleavag e o f th e epitheliu mlinin g th e prepuc e o f th e peni s wa s observe dindicatin g onse t o f puberty wa s achieved . Bod y weight s wer e measure do n th e day o f PPS . A t eac h selecte dtim e poin t

(PND1 , 22 , 5 0 o r

92) , animal s wer e anesthetize db y CO 2 asphyxiatio n followe db y exsanguination . Whole-bod y weight s wer e measured , an dthe n brain , hypothalamus , hippocampus , pituitary , thyroid , liver , testis , epididymis , ventra l prostat e an d semina l vesicle s wer e removed , weighe dan deithe r fixe di n 10 % neutra l buffere dformali n fo r histology , o r

sna p froze n i n liqui dnitroge n fo r molecular

work . Male s withi n eac h treatmen t group wer e randoml y assigne dfo r histologica l o r

molecular

biolog y tissu e collection . Molecular

tissue s include dbrain , pituitary , thyroid , liver , testi s an dventra l prostate . Bloo dwa s collecte dat PN D 22 , 50 , 9 2 an d99 . Seru msample s wer e collecte dan d use dfo r hormon e analysis . On e testi s an depididymi s fro meac h mal e at PN D 22 , 5 0 an d9 2 obtaine da

t necrops y were use dfo r

morpholog y analysis . Brain , pituitary , thyroid , liver , testis , epididymi s an dventra l prostat e wer e collecte dfo r histologica l evaluatio n fro meac h necrops y tim e point . Bloo dsample s wer e collecte di n 6-m l Vacutaine r

tube s containin g SS T Ge l an dClo t Activato r (Becton-Dickinson , Frankli n Lakes , NJ ) an dse t

o n ice . Seru mwa s prepare dfro mth e bloo do n th e sam e day o f necrops y an d store di n siliconize dmicrocentrifug e tube s at -80°C . Estradiol , testosterone , tota l triiodothyronin e (T3 ) an dtota l thyroxin e (T4 ) level s wer e assaye di n duplicat e usin g thei r appropriat e Coat-A-Coun t radioimmunoassa y (RIA ) kit s (Diagnosti c Product s Co. , Lo s Angeles , CA ) accordin g t o manufacturer' s instructions . Luteinizin g hormon e (LH ) level s wer e quantifie dusin g th e ra t L H dissociation-enhance dlanthanid e fluorometri c immunoassay . Ra t

LH , fo r standards , wa s provide db y th e Nationa l Hormon e an dPituitar y Program . Thyroi dstimulatin g hormon e (TSH ) level s wer e quantifie dusin g a n RI A method . Sper mwer e prepare dfro mon e caud aepididymi s pe r animal . Sper

mwer e examine dwit h 34 0 magnificatio n an dphase-contras t illumination . Sper

mwer e assigne dcategorie s base d upo n morphologica l examination : norma l sperm ; abnorma l sperm ; abnorma l hea dan dtail ; o r abnorma l tail . Fiv e hundre dspermpe r anima l wer e categorized . Spermwer e analyze di n eigh t field s fo r eac h biologica l sampl e fo r percen t motile , percen t

progressiv e sper

man d percen t progressiv e tracks . Progressiv e spermwer e use r define da s thos e havin g a n averag e pat h velocit y greate r

tha n 10 0 um/ s an darati o o f straight-lin e velocit y t o smoothe dpath velocit y (SIR ) greate r

tha n 50% . A t

leas t 20 0 spermpe r

anima l wer e tracke dfo r determinatio n o f

mea n velocity an dother

motio n parameters . Matin g assay s were conducte do n PN D 7 8 o r olde r treate dmale s with untreate dvirgi n Wista r Ha n female s PN D 5 6 o r

older . Wista

r Ha n femal e rat s wer e house di n three s unde r th e same condition s describe dabove . Afte r 1 0 day s o f

acclimation , dail y vagina l smear s wer e collecte dan dexamined , an dcycle s were recorded . Female s wit h 4-da y cyclicity wer e use d fo r matin g assays . Upo n secon dproestrus , on e femal e wa s place dpe r treate dmal e jus t befor e light s wer e turne dof f fo r th e duratio n o f

on e dark cycle . Matin g behavio r

wa s observe dat

5 an d2 0 mi n afte r

introduction , an dactiv e mountin g b y th e mal e an d responsivenes s o f

th e femal e wa s noted . Female s were remove dth e followin g morning , vagina l smear s wer e examine dfo r presenc e o f

sperm , an dfemale s were place dbac k i n thei r original housing . Inseminatio n inde x wa s assesse db y th e rati o o f vagina l smear s positiv e fo r sper mt o al l smear s an danalyze dusin g Fisher' s exac t test . I f spermatozo awer e foun di n th e Repor t Number : TK002342 3 " Pag e 4 1 o f 8 5vagina l smea r th e followin g morning , th e femal e wa s designate da s bein g at G D 0 . Female s wer e euthanize dwith CO z asphyxiatio n an dnecropsie dwith remova l o f th e intac t uteru s an d ovarie s o n G D 13-1 6 t o evaluat e fertilit y outcome . Ovarie s wer e remove dan dobserve dfo r gros s abnormalities , an dcorpor alute a(CL ) wer e counted . Uteri wer e excised , number s o f liv e fetuse s an dresorption s wer e recorde dan dfetuse s wer e examine dfo r abnormalities . Absenc e o f

fetuse s o r

resorptio n site s wa s followe du p wit h uterin e stainin g i n 5 m l o f 2 % ammoniu msulfid e solutio n fo r 5 mi n t o distinguis h implantatio n sites , i f any . Fertilit y inde x wa s assesse db y th e rati o o f successfu l pregnancie s t o mate dfemale s an danalyze dusin g Fisher' s exac t

test . Fecundit y o f

th e treate dmale s wa s analyze db y th e numbe r o f liv e fetuse s pe r

litter , th e rati o o f

liv e fetuse s t o th e numbe r o f C L an dth e numbe r

o f implant s relativ e t o th e numbe r

o f liv e fetuses. Deviation s t o OPPT S 890.150 0 Protoco l OPPT S 890.1500 Requirement s Criteri a S D o r Wistar ra t strai n recommende d Dosin g PN D 23-5 3 2 hig h doses , nea r

MT D Weig h th e testes , cpididymidcs , ventra l prostate , dorsolatera l prostate , semina l vesicl e wit h coagulatin g gland s an dfluid , levato r an i plu s bulbocavernosu s muscles , liver , kidneys , pituitar y an dadrenal s Histologi c evaluatio n o f

testis , epididymis , thyroi dan dkidne y fro mal l treate dsubject s Measur e thyroxine , TSH , testosteron e i n

serum .

Measur e seru mclinica l chemistr y includin g

creatinin e an dBU N Goet z e t al. , 200 7 Deviation s t o OPPT S 890.150 0

Non e G D 6 - PN D 9 9 3 doses , includin g 1 at/o r abov e MT D Weighe dbrain , hypothalamus , hippocampus , pituitary , thyroid , liver , testis , epididymis , ventra l prostat e an d

semina l vesicle s Histologi c evaluatio n brain , pituitary , thyroid , liver , testi s an dventra l prostat e fro mal l treate dmale s Measure destradiol , testosterone , T3 , tota l thyroxin e (T4) , TS H and , L H

N o clinica l chemistr y measure s i n thi s

study . Justificatio n fo r OSR I Non e neede d Include s th e require d treatmen t interval . Non e neede d Nearl y al l androge n target s weighe d Specifi c scree n target s wer e evaluate dhistologicall y a t al l level s Non e needed . Mor e extensiv e examinatio n o f hormon e level s tha n EDS P screen . Measure s fo r

EDS P scree n target s includin g hormon e level s an dmicropatholog y wer e full y evaluated . I n addition , seru mchemistr y ha s bee n evaluate da t

multipl e time s points , an dkidne y weigh t an dmicropatholog y wa s evaluate dat

termination , i n a1 3 wee k

stud y i n mal e an d femal e rats . (Sachsse , 1979a) Conclusions : Th e propiconazol e consortiu mrequest s awaive r fo r th e EDS P Tier

1 Mal e Puberta l assay becaus e propiconazol e ha s alread y bee n thoroughl y teste di n existin g studie s usin g substantiall y simila r methodology . I n combinatio n with an OPPT S Guidelin e 13-wee k toxicit y stud y i n rat s wit h propiconazol e (Sachss e e t al. , 1979a) , th e endpoint s t o b e measure di n th e Mal e Puberta l assay hav e bee n thoroughl y covered . There appea r t o b e n o methodologica l deviation s that woul dimpac t

th e justificatio n fo r

usin g OSRI i n consideratio n o f

propiconazol e activit y i n th e mal e rat puberta l screen .

Report Number : TK002342 3 Pag e 4 2 o f 8 53.1 0 Steroidogenesi s - Huma n Cel l Lin e H295 R(OPPT S 890.1550)

Purpos e o f

Assa y Th e H295 RSteroidogenesi s Assay i s intende dt o identif y xenobiotic s tha t

affec t th e steroidogeni c pathwa y beginnin g wit h reaction s occurrin g afte r th e gonadotropi n hormon e receptor s (FSH Ran dLHR ) ar e occupied . Thi s i n vitr o assay i s no t intende dt o identif

y substance s that

ma y affec t Steroidogenesi s du e to actio n o n th e hypothalamu s o r pituitary gland . Assay Methodolog y Summar y Th e OPPT S 890.115 0 assay i s an i n vitr o assa y designe dt o detec t chemical s that

affec t th e synthesi s o f

th e steroi dhormone s b y inducin g o r inhibitin g enzyme s i n th e steroi dhormon e synthesi s pathway . Th e assa y use s H295 Rcell s (ATC C CLR-2128 ) tha t wer e originall y derive dfro mahuma n adrenocortica l carcinoma . Thes e cell s appea r t o synthesiz e an d secrete , spontaneously , al l o f th e principa l androgens , estrogens , glucocorticoids , mineralocorticoid s an dprogestin s normall y produce db y both adrenocortica l an dgonada l tissues . Thi s represent s adistinctio n versu s non-malignan t adrenocortica l tissu e tha t normall y secrete s ver

y littl e estrogen o r

testosterone , an dwhic h require s ACT H to initiat e Steroidogenesis . Thi s assa y i s conducte db y incubatin g H295 Rcell s wit h 7 concentration s o f eac h tes t chemica l i n 24-wel l tissu e cultur e plates . Positiv e contro l incubation s us e forskoli n (a n inducer ) an dprochlora z (an inhibitor ) o f estradio l an dtestosteron e synthesis . Measure s o f IVp-estradio l an dtestosteron e conten t o f th e incubatio n medi aar e performe dusin g appropriate steroi dhormon e assays . Cel l viability , a n important

index o f potential toxicit

y o f tes t chemicals , i s determine dfo r eac h concentration . Othe r Scientificall y Relevan t

Informatio n Propiconazol e ha s bee n previousl y teste di n th e H295 RSteroidogenesi s assay usin g substantiall y simila r

methodolog y a s th e EDS P Tie r 1 scree n guideline s (Goetz e t al. , 2009) . There appea

r t o b e n o methodologica l deviation s i n thi s EP A stud y tha t woul dimpac t

th e justificatio n fo r

usin g OSR I i n consideratio n o f propiconazol e activit y i n th e H295 R Steroidogenesi s screen . Othe r publishe dstudie s hav e als o teste dpropiconazol e i n assay s tha t ar e substantiall y similar

t o th e TI S assay o r

provid e furthe r

supplementa l dat arelate dt o thi s EDS P Tier

1 screen . Tie r I scree n Steroidogenesi s H295 R Steroidogenesi s H295 R Steroidogenesi s H295 R OSR I Reference(s ) Goet z e t al. , 200 9 Kjaersta de t

al. , 200 7 Sanderso n e t

al . 200 2 Comment s

Substantiall y simila r

t o OPPT S 890.155 0 Substantiall y simila r

t o OPPT S 890.155 0 Supplementa l

Report Number : TK002342 3 Pag e 4 3 o f 8 5

### Goet z e t ah , 200 9 Thi s study wa s conducte dat Offic e o f Researc h ari dDevelopment , U.S . Environmenta l Protectio n Agency , Researc h Triangl e Park , NC , US A an dDepartmen t o f Environmenta l an d Molecula r

Toxicology , North Carolin aStat e University , Raleigh , NC , USA . H295 Rhuma n adrenocortica l carcinom acel l line s (ATC C CRL-2128 ) wer e obtaine dfro mth e America n Typ e Cultur e Collection . Cell s wer e grow n i n 75cm2 

### flask s wit h 12. 5 m l o f supplemente d mediu mat

37° C with a5 % CO z atmosphere . H295 Rculture s wer e performe db y Dr . Xiaowe i Zhan g i n th e Departmen t o f Zoolog y at

Michiga n Stat e University , Eas t Lansing , MI . Supplemente dmediu mwa s a1: 1 mixtur e o f Dulbecco' s modifie dEagle' s mediu mwit h Ham' s F-1 2 Nutrien t

mixtur e (Sigma , St . Louis , MO ) wit h 15m M HEPE S buffer . Th e mediu mwa s supplemente dwit h 1. 2 g/ L NaaCOs , ITS+Premi x ( 1 m l Premix/10 0 m l medium ) an d12.5ml/50 0 m l NuSerum . Fina l componen t concentration s i n th e mediu mwer e a s follows : 15m

M HEPES , 6.2 5 ug/m l insulin , 6.2 5 ug/m l transferrin , 6.2 5 ng/m l selenium , 1.25mg/ml bovin e seru malbumin , 5.3 5 ug/m l linolei c aci dan d2.5 % NuSerum . Th e mediu m wa s change dtw o t o thre e time s pe r wee k an dcell s wer e detache dfro mflask s fo r subculturin g b y us e o f

trypsin/ EDT A (Steril e I X Trypsin-EDTA ; Lif e Technologie s Inc. , Gran dIsland , NY) . Cell s wer e expose dt o propiconazol e i n 6-wel l Tissu e Cultur e Plates . T o minimiz e potentia l effect s o f hormone s i n th e seru mdurin g exposures , NuSeru mwa s replace dwit h 2.5 % charcoa l dextran treate dFB S immediatel y befor e cell s wer e expose dt o propiconazol e a

t 0 , 1 , 3 , 10 , 3 0 o r

10 0 u M concentratio n i n dimethy l sulfoxid e (DMSO , Sigma-Aldrich , St . Louis , MO ) fo r 4 8 h . A t th e en do f

thi s culture perio dmedi awa s collecte dan dshippe dfroze n t o EP A fo r hormon e measurements . Estradiol , progesteron e an dtestosteron e level s i n th e H295 Rcultur e medi awer e assaye di n duplicat e usin g appropriat e Coat-A-Coun t radioimmunoassa y (RIA ) kit s (Diagnosti c Product s Co. , Lo s Angeles , CA ) accordin g t o manufacturer' s instruction s afte r extractio n o f

th e medi awith diethy l ethe r

(Goetz , persona l communication) . Establishe dseru mstandards , alow , mediu m an dhig h control , wer e use dt o quantitat e variatio n amon g hormon e assays . Cell s wer e visuall y inspecte dunde r amicroscop e t o evaluat e viabilit y an dcel l numbers . Cel l viabilit y wa s determine dusin g th e live/dea dcel l viabilit y ki t (Molecular

Probes , Eugene , OR) . Kjaersta de t

al. , 200 7 H295 Rcell s (ATCC , CRL-2128 ) establishe dfro mapluripoten t huma n adrenocortical carcinom acel l lin e wer e grow n i n 24-wel l cultur e plate s at 37° C humidifie datmospher e o f 5 % CO2/air . Eac h wel l containe d1 m l DMEM/F1 2 mediu msupplemente dwit h 2.0 % Nu

serum , an dan appropriat e insulin-transferring-seleniu m(ITS+ ) premix . Th e cell s wer e plate dat adensity o f

2 x 10 5 

### cells/wel l an dallowe dt o settl e fo r 2 4 h . Cultur e mediu mwa s remove dan dne w mediu mcontainin g propiconazol e (0 , 0.1 , 0.3 , 1 , 3 , 1 0 an d3 0 uM ) dissolve di n DMS O wa s adde dto th e cell s i n triplicates . A simila r se t o f triplicate well s were incubate dwith prochlora z at concentration s fro m0.0 1 t o 3 |iM . Contro l well s containe dth e sam e amoun t

o f DMS O (0.1% ) a s expose dcells . Afte r incubatio n fo r

4 8 h , th e mediu mwa s remove dan dstore dat

-20° C unti l measure dfo r

testosterone , progesteron e an destradio l levels . Sample s o f

80 0 u l wer e concentrate do n 1S T Isolute ® SPE column s (10 0 mg , C18 , 1 ml) , an dth e steroi dhormone s wer e measure dusin g Delfi akit s (PerkinElme r Lif e Sciences) . Afte r exposure , th e cell s wer e incubate dwith resazuri n solutio n t o tes t fo r cytotoxicity . Report Number : TK002342 3 Pag e 4 4 o f 8 5Sanderso n e t al. , 200 2 Thi s stud y wa s supplementa l t o th e ODS P Tie r 1 screen , becaus e i t

measure dspecifi c parameter s relate dt o aromatas e activit y wit h arang e o f tes t compound s i n H295 Rcells , includin g propiconazole . H295 Rcell s wer e obtaine dfro mth e America n Typ e Cultur e Collectio n (ATC C CRL-2128 ) an dgrow n i n 1: 1 (v/v ) Dulbecco' s modifie dEagl e medium/Ham' s F-1 2 nutrien t mi x (DMEM/F12 ) containin g 36 5 mg/m l L-glutamin e an d1 5 mM Hepes . Th e mediu mwa s supplemente dwit h ITS- G solution s (1 0 mg/L insulin , 6. 7 [ig/ L sodiu mselenite , 5. 5 mg/

L transferrin g 1.2 5 mg/L bovin e seru malbumin , 10 0 U/ L penicillin , 10 0 |xg/ L streptomyci n an d2 % steroid-fre e replacemen t serum . Cell s (abou t

1- 2 x 10 5 cells/well ) i n 24-wel l cultur e plate s containin g 1 m l mediu mpe r wel l wer e expose dt o variou s concentration s o f tes t chemicals , includin g propiconazole , dissolve di n 1 u l o f DMSO . Negativ e contro l cell s receive d1 u l o f DMSO . A s apositiv e contro l fo r aromatas e inhibition, cell s wer e expose dt o 1 u M o f th e know n aromatas e inhibito r 4

- hydroxyandrostenedion e i n DMSO ; a s apositiv e contro l fo r induction , 10 0 u M o f

8-bromo

cycli c adenosin e monophosphat e (8Br-cAMP ) dissolve di n mediu mcontainin g 0.1 % DMS O wa s used . Unexpose dcell s wer e include da s furthe r controls . Al l treatment s wer e teste di n quadruplicate , unles s state dotherwise ; eac h concentration-respons e experimen t wa s performe dthre e times . Propiconazol e wa s teste dat concentration s o f

0 , 0.1 , 1,3 , 10 , 3 0 an d 10 0 uM . Al l exposure s wer e fo r 2 4 h . T o asses s aromatas e activity (increase s o r decreases) , cell s wer e expose dt o 5 4 n M l p [ 3 H]-androstenedion e dissolve di n serum-fre e cultur e mediu man dincubate dfo r 1. 5 h at 37° C i n an atmospher e o f 5 % CO 2 an d95 % air . Aromatas e activity wa s measure dan dexpresse di n picomole s o f androstenedion e converte d pe r hou r pe r milligra mcellular

protein . Th e specificit y o f th e aromatas e assa y base do n th e releas e o f tritiate dwater

wa s verifie db y measurin g th e productio n o f estron e (th e aromatizatio n produc t

o f androstenedione) , usin g a125 I-labele ddouble-antibod y radioimmunoassa y ki t

an db y usin g 4-hydroxyandrostenedione , an irreversibl e inhibito r o f th e catalyti c activit y o f

aromatase , t o bloc k

th e formatio n o f

tritiate dwater . Cel l viabilit y an devidenc e o f cytotoxicit y wa s assesse db y neutra l re duptak e assay , an db y a n assessmen t

o f mitochondrial function . Supplementa l experiment s wit h certai n tes t compound s that

appeare dt o b e aromatas e inducer s measure dmRN A level s o f aromatas e (CYP19) , o r

increase s i n intracellular

cAM P levels , bu t

thes e experiment s were no t performe dfo r

propiconazole .

Report Number : TK002342 3 Pag e 4 5 o f 8 5Deviation s t o OPPT S 890.155 0 Protoco l OPPT S 890.155 0 Requirement s Criteri a H295 Rcell s Evaluat e th e tes t

ite m a t 7 concentration s

Negativ e an dpositiv e control s

Chec k fo r cel l viabilit y

Measure dtestosteron e an destradio l outpu t

i n medi a Goet z e t al. , 200 9 Deviation s t o OPPT S 890.155 0 Non e Substantiall y simila r Evaluate da t

5 concentration s + a negativ e contro l

Negativ e control s evaluated. Positiv e control s no t

described .

Non e None . Measure d testosterone , progesteron e an d estradio l outpu t

i n media . Kjaersta de t al. , 200 7 Deviation s t o OPPT S 890.155 0 Non e Substantiall y simila r Evaluate da t

6 concentration s + a negativ e contro l None . Negativ e control s evaluate dPositiv e control s include d evaluatio n compare d t o prochloraz .

Non e None . Measure d testosterone , progesteron e an d estradio l outpu t

i n medi a Sanderso n e t al.200 2 Deviation s t o OPPT S 890.155 0 Non e Substantiall y simila r Evaluate da t

6 concentration s + a negativ e contro l None . Negativ e control s evaluated . Positiv e control s include d evaluatio n compare d t o prochloraz . Othe r positiv e control s (inducer s an d inhibitors ) were use d i n follow-u p assay s (4-hydroxyandro

stenedione , 8-BrcAMP , forskolin ) Non e Measure donl y aromatas e activity , bu t confirme dbase d o n level s o f

estron e i n medium . Justificatio n fo r OSRI Non e neede d

Non e needed .

Positiv e contro l i n simi l a r structura l clas s a s propiconazol e (prochlora z = inhibitor ) wa s evaluate di n Kjaersta de t

al. , 2007 . Compariso n t o othe r chemical s availabl e i n al l 3 studies .

Non e neede d

Non e neede d

Conclusions : Th e propiconazol e consortiu mrequest s awaive r fo r th e EDS P Tier

1 H295 R Steroidogenesi s scree n becaus e propiconazol e ha s alread y bee n teste di n tw o existin g studie s usin g substantiall y simila r methodology . 3.1 1 Uterotrophi c - Ra t (OPPT S 890.1600 ) Purpos e o f

Assa y Th e Uterotrophi c Assa y i s intende da s an i n viv o screenin g assay providin g dat aabou t a singl e endocrin e mechanism , i.e. , estrogenicity . Th e assay i s base do n th e long-know n principl e tha t

estroge n specificall y stimulate s both hypertroph y an dhyperplasi ao f th e roden t uterus . I t i s als o understoo dtha t

thi s activit y begin s with bindin g o f estroge n t o th e estroge n recepto r (ER) . Thu s th e tes t

i s acomplet e assessmen t o f ates t substance' s abilit y t o bin dt o E Ran dpromot e well-know n an deasil y measure dresponse s t o E Rbinding , i n alivin g animal . N o othe r

hormon e clas s accomplishe s an y for mo f thi s "pur e action" .

Report Number : TK002342 3 Pag e 4 6 o f 8 5Assa y Methodolog y Summar y

Th e goal o f

thi s assay i s t o detec t

estrogehi c chemical s throug h asimpl e i n viv o assay . Validatio n studie s wer e carrie dou t unde r OEC D auspices. Approve dmethod s permi t us e o f eithe r rat s (Sprague-Dawle y o r Wistar strain s preferred ) o r

mice . Subject s ca n b e intac t immatur e (PN D 21-2 4 at

th e beginning ) o r

ovariectomize d(OVX ) adults . Tes t material s can b e administere db y ora l gavag e o r injecte dsubcutaneously . Ther e shoul db e at leas t tw o dos e groups , with th e highe r

dos e at o r nea r th e animals ' maximu mtolerate ddose , plu s agrou p administere dvehicl e only . Ther e shoul db e a t leas t 6 animals/dos e groups . Administratio n o f areferenc e hormone , suc h a s 17a-ethiny l estradio l i s considere dhelpful , bu t i s no t mandatory . I n th e rat , treatmen t

shoul db e administere ddail y fo r 3 consecutiv e days , followe db y sacrific e o n day 4 , approximatel y 2 4 hour s afte r th e las t dose . Th e complet e uterin e organ i s dissecte dat

necropsy , carefull y cleane do f al l fat an dextraneou s connectiv e tissue , cu t ope n t o releas e intralumina l fluid , blotte d(bu t no t dried ) an dthe n weighed . Anima l weigh t shoul d als o b e take n at

sacrific e becaus e expressio n o f orga n weight s pe r

uni t B W may b e importan t i f th e administere dcompoun daffecte dbod y weights . Othe r Scientificall y Relevan t

Informatio n N o specifi c scree n followin g th e OPPT S 890.160 0 guidelin e pe r s e wa s foun dfo r propiconazole . However , th e define dpurpos e o f th e uterotrophi c scree n i s t o detec t estrogeni c chemical s throug h asimpl e i n viv o assay a s state dabov e an do n th e EPA' s EPS P webpag e (Appendi x A l 1-Uterotrophi c http://www.epa.gov/scipolv/oscpendo/pubs/uterotrophi c fs.pdf) . Th e potentia l t o detec t a n estrogeni c effec t

o f propiconazol e ha s bee n evaluate di n othe r EDS P Tier

1 scree n studie s wit h propiconazole , an dth e cite dstudie s i n Tabl e 3 hav e evaluate dth e importan t endocrine

relate dtarget , identifie di n th e OPPT S 890.160 0 protocol , estrogeni c effect s o n th e uteru s a s determine db y weigh t

gain . Thi s i s no t

acomplicate dassessment , an dther e woul dappea r t o b e n o methodologica l deviation s tha t woul dimpac t

th e justificatio n fo r

usin g OSR I i n consideratio n o f

propiconazol e activity i n th e femal e rat puberta l scree n (Rockett

e t al. , 2006) , particularl y considerin g th e extensiv e additiona l endpoint s measure di n thi s publishe d study . Propiconazol e ha s als o bee n assesse di n othe r screen s relate dt o estrogeni c activit y tha t ar e par t o f th e EDS P requirements , a s discusse di n th e OSR I waive r request s fo r

th e Aromatas e Assay , Estroge n Recepto r Binding , Estroge n Recepto r Transcriptiona l Activation , an dSteroidogenesi s i n H295 RCells . I n additio n t o thes e EDS P Tie r 1 screens , both uterin e weigh t an dth e eve n mor e sensitiv e indicato r fo r measurin g estrogeni c potential , uterin e histopathology , hav e bee n examine di n USEP A FIFRA 40CFRPart 15 8 guidelin e studie s wit h propiconazole , a s indicate di n Tabl e 3 .

Report Number : TK002342 3 Pag e 4 7 o f 8 5TABLE 3 OSR I an dFIFR A 40CF RStudie s Conducte dwit h Propiconazol e whic h Examin e Uteru s Weigh t o f Morpholog y

Uterin e endpoint s Weigh t Histolog y Histolog y Histolog y Reference s Rocket t e t al. , 200 6 Border s an dSalamon , 198 5 Sachsse , 1979 a Hunte r etal. , 198 2 Comment s

Part o f asubstantiall y mor e robus t prenatal stud y i n viv o screen . FQ , F | an dp 2 femal e rat s i n a2-generatio n reproductio n study .

1 3-Wee k stud y i n rat s 2-year chroni c toxicity/oncogenicit y i n th e ra t Rockett e t al. , 200 6 Thi s specialize done-generatio n reproductiv e toxicit y stud y i n rats , wit h aparticula r emphasi s o n response s i n F l femal e offspring , evaluate dth e respons e t o thre e differen t tes t substance s includin g propiconazole . Propiconazol e wa s administere dt o timed-pregnan t Wistar-Han rat s (Charle s Rive r

Laboratory , Raleigh , NC ) at 0 , 100 , 50 0 an d250 0 pp mvi ath e fee dfro m gravi dday (GD ) 6 t o postnata l day (PND ) 98 . Eac h treatmen t grou p consiste do f approximatel y 3 5 dams . Dam s wer e allowe dt o delive r naturally , with th e da y o f delivery designate da s PN D 0 fo r

th e F l offspring . Pup s wer e weighe do n PN D 0 . Th e siz e an d gender rati o o f eac h litte r

wa s noted . Litter s wer e culle dt o eigh t pup s o n PN D 8 usin g blinde drando mselection , an dth e pup s weane do n PN D 22 . Femal e weanling s wer e continue do n th e sam e die t a s thei r dam s ha dbee n expose dt o unti l terminatio n o n PN D 99 . Examinatio n fo r

vagina l openin g commence do n PN D 29 , an dth e ag e an dB W at vagina l openin g (VO ) wa s recorded . Estrou s cyclicit y wa s monitore dfollowin g V O fo r tw o weeks , durin g week s 5- 6 followin g VO , an dfro mPN D 9 2 t o th e firs t proestrou s day afte r

PN D 9 9 (week s 9-10) . Bod y an dorgan weight s (brain , hippocampus , hypothalamus , liver , pituitary , thyroid , ovarie s an duterus ) wer e measure dat necropsy . Liver , thyroi d(score dfo r colloi d depletion , follicula r

cel l hypertroph y and/o r

follicula r cel l hyperplasia ) an dovarie s (every twentieth sectio n wa s staine dwith H an dE an dexamine dfo r edema , cysti c follicle s an d retaine doocytes ) wer e rea dfo r histopathology . Estradio l i n seru msample s take n at sacrific e wa s measure dusin g radioimmunoassa y techniques . Border s an dSalamon , 198 5 I n thi s OPPT S Guidelin e 2-generatio n reproductio n stud y (MRI D Numbe r 00151514) , propiconazol e wa s administere dt o Charle s Rive r C D rat s (Sprague-Dawle y origin ) at dietar y concentration s o f

0 , 100 , 50 0 an d250 0 pp macros s tw o generations . Th e uteri o f al l survivin g F O an dF I parenta l female s wer e evaluate db y gros s an dhistopatholog y examination . Afte r

weanin g o n PND21 , 1 0 mal e an d1 0 femal e offsprin g fro meac h treatmen t grou p i n th e Fla , Fib , F2 aan dF2 b litter s wer e randoml y assigne dfo r gros s patholog y examinations , includin g th e uterus . Al l offsprin g tha t died , wer e sacrifice d moribund , o r

ha dabnormalitie s wer e als o evaluate db y gros s patholog y examination . Fro m th e offsprin g wher e tissue s wer e examine dan dpreserved , thos e i n th e Fi b an dF2 b litter s wer e examine db y micropathology , includin g th e uterus , vaginaan dovaries . O f particula r Report Number : TK002342 3 Pag e 4 8 o f 8 5relevanc e t o th e OSRI fo r th e Uterotrophi c Assay screen , gros s an dmicropatholog y examinatio n o f

th e uteru s fro mFi b an dF2 b offsprin g (sacrifice dPND23-27 ) afte r weanin g woul dhav e occurre dbefor e puberty , a t atim e whe n an y difference s fro mcontro l animal s du e t o estrogeni c stimulatio n o f uterin e developmen t

woul dhav e bee n detectabl e i n a n analogou s manne r

t o th e Uterotrophi c Assay . Micropatholog y result s ar e presente di n Appendi x E (F O parent s an dFi b progeny ) an dAppendi x I (F l parent s an dF2 b progeny ) withi n th e stud y report . Sachss e et

al. , 1979 a I n thi s OPPT S Guidelin e 13-wee k rat stud y (MRI D Numbe r

0058606) , propiconazol e wa s administere dt o Tif : RAI f rat s at dietar y concentration s o f 0 , 240 , 120 0 an d600 0 ppm . Th e uteri o f female s i n al l treatmen t

group s wer e evaluate db y histopatholog y at th e en do f

th e study . Hunte r et al. , 198 2 I n thi s OPPT S Guidelin e 2-yea r rat combine dchroni c toxicity/oncogenicity stud y (MRI D Numbe r 00129918) , propiconazol e wa s administere dt o Charle s Rive r C D rat s (Sprague

Dawle y origin ) at

dietary concentration s o f 0 , 100 , 50 0 an d250 0 ppm . Th e uteru s wa s evaluate db y histopatholog y at th e en do f

th e study . Conclusions : Th e propiconazol e consortiu mrequest s awaive r fo r th e EDS P Tier

1 Uterotrophi c scree n becaus e propiconazol e ha s alread y bee n teste di n a n existin g publishe d stud y (Rocket

t e t al. , 2006 ) usin g substantiall y mor e robus t methodolog y t o detec t

estrogeni c effect s tha t

include dth e require dmeasuremen t o f uterin e weight . Othe r

OPPT S Guidelin e studie s with propiconazol e hav e evaluate duterin e micropatholog y a s apotentia l sensitiv e indicato r o f an y estrogeni c effect s afte r differen t duration s o f

treatment , including evaluation s i n pre-puberta l femal e offsprin g i n atwo-generatio n reproductio n study . Additionally , i n vitr o studie s hav e bee n conducte dwit h propiconazol e tha t fulfil l EDS P Tier 1 requirement s fo r

a n Aromatas e Assay , Estroge n Recepto r Binding , Estroge n Recepto r Transcriptional Activation , an dSteroidogenesi s i n ahuma n cel l line , whic h woul db e abl e t o detec t an estrogeni c potential . 

### 4. 0 REPRODUCTIV E AN D DEVELOPMENTA L TOXICIT Y CHARACTERISTIC S O F PROPICONAZOLE Propiconazol e ha s bee n teste dusin g study protocol s whic h ar e identica l t o o r substantiall y equivalen t

t o th e state dpurpos e o f

al l eleve n o f

th e OPPT S Serie s 89 0 EDS P Tier 1 study guidelines . I n additio n ther e i s awealth o f dat afro mhighe r

tie r studie s availabl e fo r propiconazol e whic h evaluate s apica l endpoint s i n intac t animals . Change s i n thes e endpoint s upo n exposur e t o ates t substanc e woul dindicat e th e potentia l fo r th e substanc e t o interfer e wit h endocrin e systems . Th e finding s fro mth e mos t relevan t studie s ar e summarize dbelow . Th e reproductiv e an ddevelopmenta l toxicity databas e fo r propiconazol e ha s bee n reviewe di n a

n FA O Worl dHealt

h Organizatio n repor

t (Dewhurs t

an dDellarco , 2004) , an dals o i n th e USEP A Re-registratio n Eligibilit y Decisio n (USEPA , 2006a , b) . Report Number : TK002342 3 Pag e 4 9 o f 8 5

### Th e FA O toxicolog y revie w an dUSEP A HE D Chapte r (USEPA , 2006b ) conclude dtha t i n a two-generatio n reproductio n stud y i n rat s at dos e level s o f

0,100 , 50 0 an d250 0 ppm , reproductiv e parameter s wer e no t affecte db y treatmen t

wit h propiconazole . Th e NOAE L fo r parenta l toxicit y wa s 10 0 pp m(equivalen t t o 8 mg/kg/day ) o n th e basi s o f reduce dbody

weigh t gai n i n dam s an dhepatotoxicity a t SOOppm . Th e NOAE L fo r offsprin g toxicit y wa s 500pp m(equivalen t

t o 43-5 2 mg/kg/day ) base do n decrease doffsprin g survival an dbod y weight s an dan increase dincidenc e o f hepati c lesion s (cellula

r swelling ) at

250 0 pp m (Border s an dSalamon , 1985 ; USEPA , 2006b) . Thre e studie s o f

developmenta l toxicity wer e conducte di n rat s an don e i n rabbits . I n th e firs t stud y i n rats , at

th e highes t

dos e o f 30 0 mg/kg/da y ther e wa s evidenc e o f materna l toxicity an dretarde ddevelopment , bu t n o malformation s (Fritz , 1979) . I n th e secon dstudy , group s o f 2 4 mate dfemal e rat s wer e give n propiconazol e at ados e o f 0 , 30 , 9 0 o r

360/300mg/kg / day b y gavag e o n day s 6-1 5 o f gestatio n (Gikni s e t

al. , 1987) . Th e NOAE L fo r materna l toxicit y wa s 9 0 mg/kg/day o n th e basi s o f severe sign s o f

toxicity at

30 0 mg/kg/day . Althoug h th e materna l body-weigh t gain s wer e significantl y decrease dat 9 0 mg/kg/da y durin g day s 6- 8 o f

gestatio n only , thi s effec t

wa s considere dt o b e temporar y an dsecondary t o reduce dfoo dconsumption . Th e NOAE L fo r developmenta l toxicit y wa s 3 0 mg/kg/da y o n th e basi s o f

reduce dossificatio n an dth e presenc e o f rudimentar y rib s at

9 0 mg/kg/day . A lo w incidenc e o f

clef t palat e wa s observe dat

9 0 mg/kg/da y (on e fetus ) an dat 30 0 mg/kg/da y (tw o fetuses , 0.7% ) i n th e presenc e o f sever e materna l toxicity . Clef t palat e i s ver y rare , bu t no t unknown , i n C D rats ; incidenc e i n control s range dfro m0 % t o 0.3% . Th e clef t palat e wa s als o seen at

alo w incidenc e i n rat s i n an extensiv e stud y tha t specificall y investigate dth e palat e an dja w at

asingl e dos e leve l o f

30 0 mg/kg/day ; tw o fetuse s (fro mdifferen t litters ) with clef t palat e wer e see n ou t

o f 206 4 fetuse s fro mdam s treate dwit h propiconazol e (0.1%) , versu s zer o ou t

o f 212 2 fetuse s i n th e contro l grou p (Mallows , 1987) . Clef t palat e i s avery rar e bu t occasionall y occur s i n rat contro l groups , an dther e ar e publishe ddat a(Khera , 1985 ) tha t indicat e tha t

testin g compound s at

maternall y toxi c dose s i s associate di n som e wa y wit h th e inductio n o f

anumbe r o f malformations , includin g clef t palate . Th e FA O toxicolog y revie w o f propiconazol e conclude dthat th e lo w incidence s see n i n thes e tw o studie s canno t b e discounte da s bein g relate dt o treatmen t wit h propiconazole , bu t

i t i s considere dtha t

the y ar e unlikel y t o b e see n i n th e absenc e o f materna l toxicit y (Dewhurs t an dDellarco , 2004) . Similarly , i n th e USEP A HE D review , th e HIAR C considere dthat ther e i s aclea

r NOAE L an dwell-characterize ddos e respons e fo r th e developmenta l effect s observe di n rats , an d overal l ther e i s lo w concer n fo r pre - and/o r

postnata l toxicit y resultin g fro mexposure t o propiconazol e (USEPA , 2006b) . Propiconazol e wa s no t

teratogeni c i n rabbits . Ne w Zealan dWhit e rabbit s were administere d dose s o f propiconazol e o n Day s 7-1 9 o f gestatio n a

t 0 , 100 , 200 , o r

40 0 mg/kg/day . Th e NOAE L fo r

materna l toxicit y wa s 10 0 mg/kg/da y o n th e basi s o f reduce dfoo dconsumptio n an dbod y weigh t

los s at 25 0 mg/kg/day . Th e NOAE L fo r developmenta l toxicity wa s 25 0 mg/kg/day o n th e basi s o f increase s i n full y forme dthirteenth rib s an dabortion s at 40 0 mg/kg/day . Th e NOAE L fo r teratogenicity wa s 40 0 mg/kg/day , th e highes t dos e teste d (Raabetal. , 1986) .
Report Number : TK002342 3 Pag e 5 0 o f 8 5TABL E 4 Submitte dReproductiv e an dDevelopmenta l Toxicity Studie s o n Propiconazol e Technica l Stud y Typ e

Rat Reproductio n (Border s an d Salamon , 1985 ) Rat Teratolog y (Fritz , 1979 ) Non-Guidelin e Rat Teratolog y (Gikni s e t

al. , 1987 ) Rat Teratolog y (Mallow s e t al. , 1987 ) Non-Guidelin e Rabbi t Teratolog y (Raabetal. , 1986 ) MRI D Numbe r 0015151 4 0005860 4

4042500 1 4042500 2

4042500 4 Developmenta l /

Offspring . NOAE L (mg/kg/day ) 43-5 2 No t determine d 3 0 No t determine d 25 0 Materna l / Parenta l NOAE L (mg/kg/day ) 8 No t determine d 9 0 No t determine d 10 0 

### 5. 0 CONCLUSIO N Propiconazol e ha s bee n teste dusin g stud y protocol s whic h ar e identica l t o o r substantiall y equivalen t

t o th e state dpurpos e ofte n o f th e OPPT S Serie s 89 0 EDS P Tie r 1 stud y guidelines . Whil e n o stud y tha t specificall y meet s th e desig n criteri ao f th e amphibia n metamorphosi s assay i s availabl e fo r propiconazole , th e propiconazol e consortiu mrequest s a waive r fo r th e Amphibia n Metamorphosi s scree n becaus e propiconazol e ha s bee n evaluate d i n multipl e mammalia n studie s fo r effect s o n th e hypothalamic-pituitary-thyroi dacces s (HPT ) an dsubsequen t

thyroid-relate deffects . A s th e purpos e o f th e Amphibia n Metamorphosi s scree n i s t o evaluat e thyroi dfunctio n i n arepresentativ e vertebrat e mode l (frog) , base do n th e conserve dstructur e an dfunction s o f thyroi dsystems , th e dat afro m multipl e i n vitr o an di n viv o assay s i n rat an dhuma n wit h propiconazol e provid e arobus t assessmen t

i n relevan t

vertebrat e system s tha t

g o beyon dth e Tie r

1 assay . FIFRA 4 0 CF R Part 15 8 guidelin e studie s i n rats , mice , an ddog s als o provid e informatio n o n an y potentia l HP T axis effect s i n multipl e specie s tha t ar e directl y relevan t

t o humans . I n conclusion , propiconazol e ha s bee n thoroughl y teste dfo r al l o f th e endpoint s covere db y th e Tier 1 assay s o f

th e EDSP . Th e propiconazol e consortiu mreques t waiver s fo r

al l o f th e Tie r 1 assay s i n th e EDS P tes t order .

TK0023423Waiver.do c sbh : 5/5/1 0 Verl. l

Repor t Number : TK002342 3 Pag e 5 1 o f 8 5

### 6. 0 REFERENCE S

Balague r P , Fran?oi s F , Comunal e F , Fene t H , Boussiou x AM , Pon s M , Nicola s JC , Casella s C (1999) . Reporte r

cel l line s t o study th e estrogeni c effect s o f xenoestrogens . Sc i Tota l Environ . 199 9 Au g 15 ; 233(l-3):47-56 . Baue r ERS , Bitsc h N , Brun n H , Sauerwei n H , Meye r HH D (2002) . Developmen t o f an immuno-immobilize dandroge n recepto r assa y (IRA ) an dit s applicatio n fo r th e characterizatio n o f

th e recepto r

bindin g affinit y o f

differen t pesticides . Chemosphere 46(7) : 1107-1115 . Bitsc h N , Koerne r

W , Failin g K , Brun n H (2002) . I n vitr o screenin g o f th e estrogeni c activit y o f activ e component s i n pesticides . Umweltwissenschafte n un dSchadstoff-Forschun g 14(2):76-84 . Boehmle r G , Borowsk

i U (2004) . Detectio n o f

estrogeni c activit y b y abiologica l tes t system : Part 2 : Investigatio n o f

food-relevan t

substances . Deutsch e Lebensmittel-Rundschau 100(4) : 133-139 . Boehmle r G , Kohne n R , Borowsk

i U , Rueh e A (2006) . Employmen t o f abiologica l tes t syste m(E-screen ) i n th e officia l foo dmonitorin g fo r detectio n o f

estroge n activ e substances . J Verbraucherschut z undLebensmittelsicherhei t 1(4) : 325-331 . Border s C , Salamo n C (1985) . Two-Generatio n Reproductio n Stud y i n Albin o Rat s wit h CGA-6425 0 Technical ; Toxigenic s Stud y 450-1202 . Projec t No . 790011 . Unpublishe dstud y prepare db y Toxigenics , Inc . 1886p . MRI D No . 00151514 . Bretele r R(1988) . Th e chroni c toxicity o f CGA6425 0 technica l (propiconazole ) t o sheepshea dminno w (Cyprinodo n variegatus) . Report No . 88-04-2685 , Syngent aFil e No . GCA64250/210 . MRI D No . 4088200 1 Dewhurs t I an dDellarc o V (2004) . Propiconazole : Toxicolog y Evaluation . Pesticid e residue s i n Foo d- 2004 . Join t FAO/WH O Meetin g o n Pesticid e Residues . Toxicologica l evaluations : Par t

II . WH O Core Assessmen t Group , Rome , Ital y 20-2 9 Septembe r 2004 . EDSTA C (1998) . EDSTA C Final Report . Chapte r 5 , pag e 52-3 . http://epa.gov/endo/pubs/edstac/chap5vl4.pd f Frit z H (1979) . Report

o n CG A 6425 0 Tech : Teratolog y Stud y (Se g II ) i n Rats . Projec t No . 790011 . Unpublished . Ciba-Geig y Corp. , Greensboro , NC . MRI D No.00058604 . Gerspac h R(1997) . CGA-6425 0 Technical : 18-Mont h Oncogenicity Stud y i n Mice . La b Projec t No . 943126 . Unpublished . Novarti s Cro p Protectio n AG . 45 3 p . MRI D No . 44381401 . Gikni s M , Marcisi n J , Arthu r A an dYau E (1987) . CGA6425 0 Technical : A Teratolog y (Segmen t II ) Stud y i n Rats . Laboratory Projec t I D 86004 . Unpublished . Ciba-Geig y Corporation . 42 5 p . MRI D No . 40425001 . Report Number : TK002342 3 Pag e 5 2 o f 8 5

### Goet z AK , Re n H , Schmi dJE , Blyston e CR , Thillainadaraja h I , Bes t DS , Nichol s HP , Strade r LF , Wol f DC , Narotsky MG , Rbcket t JC , Di x D J (2007) . Disruptio n o f Testosteron e Homeostasi s a s aMod e o f Actio n fo r

th e Reproductiv e Toxicity o f Triazol e Fungicide s i n th e Mal e Rat . Toxico l Sc i 95(1) : 227-239 . Goet z AK , Rockett

JC , Re n H , Thillainadaraja h I , Di x D J (2009) . Inhibitio n o f Rat an d Huma n Steroidogenesi s b y Triazol e Antifungals . System s BiolRepr o Med , 55:214-226 . Hinfra y N , Porche r

JM , Brio n F(2006) . Inhibitio n o f rainbo w trou t

(Oncorhynchu s mykiss ) P45 0 aromatas e activitie s i n brai n an dovarian microsome s b y variou s environmenta l substances . Com

p Bioche mPhysio l C Toxico l Pharmacol . 144(3):252-62 . Hunte r B , Slate r N , Heywoo dR(1982a) . CG A 6 4 250 : Potentia l Tumorigeni c an dToxi c Effect s i n Prolonge dDietary Administratio n t o Rats : CB G 193/828 4 (Tes t No . 789023) . Fina l rept . (Unpublishe dstudy prepare db y Huntingdo n Researc h Centre , Eng. , submitte db y Ciba

- Geig y Corp. , Greensboro , NC) . MRI D No . 00129918 . Hunte r B , Schole y D , Haywoo dR(1982b) . CG A 6 4 250 : Long-termFeedin g Stud y i n Mice . CBG/195/81827 . Fina l Report . (Unpublishe dstud y prepare db y Huntingdo n Researc h Centre , Eng. , submitte db y Ciba-Geig y Corp. , Greensboro , NC) . MRI D No . 00129570 . Hurs t M Ran dSheahan D A (2003) . Th e potentia l fo r oestrogeni c effect s o f

pesticide s i n headwate r stream s i n th e UK . Sc i Total Enviro n 301 : 87-96 . Ile y E (1981) . Flow-throug h Fathea dMinno w earl y life-stag e toxicity tes t with CGA-64250 . Repor t No . PA909 , (1981 ) plu s correctio n t o statisica l data . Syngent aFil e No . CGA-64250 . MRI D No . 00133364 . Johnso n W an dThompso n S (1985) . One-yea r Subchroni c Ora l Toxicit y Stud y i n Beagl e Dog s wit h CGA-6425 0 Technical (Fina l Report) . FDR L Stud y No . 7737 . Unpublished . Foo dan dDru g Researc h Laboratories , Inc . 57 0 p . MRI D No . 00151515 . Judso n R S e t

al . (2009) . I n vitr o Screenin g o f Environmenta l Chemical s fo r Targe t Testin g Prioritization-Th e ToxCas t Projects ; Supplementa l Information . Se e http://www.epa.gov/ncct/toxcast / Supplementa l dat aan dfigure s fo r Judso n e t al . Judso n RS , Houc k

KA , Kavloc k

RJ , Knudse n TB , Marti n MT , Mortense n HM , Rei f DM , Rotrof f DM , Sha h I , Richar dAM , an dDi x D J (2010) . I n vitr o screenin g o f environmenta l chemical s fo r

targete dtestin g prioritizatio n - th e ToxCas t Project . Enviro n Healt

h Perspectives . 118:485-492 . Kher aK (1985) . Materna l toxicity : A possibl e etiologica l facto r i n embryo-feta l death s an d feta l malformation s o f

rodent-rabbi t

species . Toxicology , 31 , 129-153 .

Report Number : TK002342 3 Pag e 5 3 o f 8 5

### Kjaersta dMB , Anderse n HR , Taxvi g C , Has s U , Axelsta dM , Metzdorf f S , Vinggaar dA M (2007) . Effect s o f

azol e fungicide s o n th e functio n o f se x an dthyroi dhormones . Pesticide s Researc h 111 . Danis h Ministr y o f th e Environment , Environmenta l Protectio n Agency , Strandgad e 29 , 140 1 Copenhage n K , Denmark . Kojim aH , Katsur aE , Takeuch i S , NiiyamaK , Kobayash i K (2004) . Screenin g fo r

estroge n an dandroge n recepto r

activitie s i n 20 0 pesticide s b y i n vitr o reporte r gen e assay s usin g Chines e hamste r

ovary cells . Enviro n Healt h Perspect . 112(5):524-31 . Lavill e N P Brio n F , Hinfra y N , Casella s C , Porche r J-M , Ait-Aiss aS (2006) . Modulatio n o f aromatas e activit y an dmRN A b y variou s selecte dpesticide s i n th e huma n choriocarcinom a JEG- 3 cel l line . Toxico l 228 : 98-108 . Mallow s S , Lev y E , Gikni s M , an dYau E (1987) . CGA-64250 : A Modifie dTeratolog y (Segmen t II ) Stud y i n Albin o Rats . Laboratory Projec t I D 86189 . Unpublished . Ciba-Geig y Corporation . 40 8 p . MRI D No . 40425002 . Ohn o K , Araki N , Yanas e T , Nawat aH , lid aM (2004) . A nove l no n radioactiv e metho dfo r measurin g aromatas e activity usin g ahuma n ovaria n granulosa-typ e cel l lin e an da n estron e ELISA . Toxico l Sc i 8 2 443-450 . Raa b D , Youreneff,M , Gikni s M (1986) . Propiconazole : A Teratolog y Stud y i n Ne w Zealan dRabbits : Fina l Report Amendmen t

No . 1 : Laboratory Projec t I D 86043 . Unpublishe d stud y prepare db y Ciba-Geig y Corporation . 12 5 p . MRE D No . 40425004 . Rockett JC , Narotsky MG , Thompso n KE , Thillainadaraja h I , Blyston e CR , Goet z AK , Re n H , Bes t DS , Murrell RN , Nichol s HP , Schmi dJE , Wol f D C an dDi x D J (2006) . Effec t o f conazol e fungicide s o n reproductiv e developmen t i n th e femal e rat . Repr o Toxico l 2 2 647

- 

### 658 . Sachss e K , Suter

P , Luetkemeie r

H (1979a) . CGA-6425 0 Techn . Thre e Month s Toxicity Stud y o n Rats : Projec t

No . 790014 . Fina l rept . (Unpublishe dstud y prepare db y Ciba-Geig y Ltd. , Switzerland , submitte db y Ciba-Geig y Corp. , Greensboro , N.C.) ; MRI D No . 00058606 . Sachss e K , Bath e R , Luetkemeie r H (1979b) . CGA-6425 0 3-Mont h Toxicity Stud y o n Dogs : Report No . 78/5751 . CDL : 244271-R . Fina l rept . (Unpublishe dstudy prepare db y Ciba

Geig y Ltd. , Switzerland , submitte db y Ciba-Geig y Corp. , Greensboro , N.C.) ; MRI D No . 00058607 . Sanderso n JT , Boerm aJ , Lansberge n GWA , an dvan de n Ber g M (2002) . Inductio n an d Inhibitio n o f

Aromatas e (CYP19 ) Activit y b y Variou s Classe s o f Pesticide s i n H295 R Huma n Adrenocortica l Carcinom aCells . Toxico l App l Pharmacol . 182 , 44-54 . Surprenan t

D (1987) . Th e toxicity o f CGA-6425 0 (propiconazole ) t o fathea dminno w (Pimephale s promelas ) embryo s an dlarvae . Report No . 87-8-2476 . Syngent aFil e No . CGA64250/209 . MRI D No . 41720302 . Taxvi g C , Vinggaar dAM , Has s U , Axelsta dM , Metzdorf f S an dNelleman n C (2008) . Endocrine-disruptin g propertie s i n viv o o f widel y use dazol e fungicides . IntlJAndr o 31 , 170-177 . Repor t Number : TK002342 3 Pag e 5 4 o f 8 5

### Troske n ER , Fische r K , Volke l W , Lut z W K

(2006) . Inhibitio n o f

huma n CYP1 9 b y azole s use da s antifunga l agent s an daromafa^ e inhibitors , usin g ane w LC-MS/M S metho dfo r th e analysi s o f

estradio l produc t

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

o f th e Inhibitio n o f

Recombinan t

Huma n CYP1 9 (Aromatase ) b y Azole s Use di n Agricultur e an da s Drug s fo r Humans . Endocrin e Res . 3:387-394 . Tull y DB , Ba o W , Goet z AK , Blyston e CR , Re n H , Schmi dJE, , Strader LF , Woo dCR , Bes t DS , Narotsk

y MG , Wol f

DC , Rocket

t JC , an dDi x D J (2006) . Gen e expressio n profilin g i n live r an dtesti s o f

rat s t o characteriz e th e toxicit y o f triazol e fungicides . Toxico l App I Pharmaco l 215 , 260-273 . USEP A (2006a) . Reregistratio n Eligibility Decisio n (RED ) fo r Propiconazole . Cas e No,3125 , Lis t

C . Unite dState s Environmenta l Protectio n Agency . Prevention , Pesticide s an d Toxi c Substance s (7508P) . EP A 738R-06-027 . Jul y 2006 . USEP A (2006b ) Propiconazole : Phas e 4 , HE D Chapte r o f th e Re-registratio n Eligibility Decisio n Documen t

(RED) . P C Code : 122101 . Reregistratio n Cas e No . 3125 . D P Barcod e D329668.Unite dState s Environmenta l Protectio n Agency , Health Effect s Division . Jun e 28 , 2006 . USEP A (2009a ) EPA' s Approac h fo r Considerin g Othe r

Scientificall y Relevan t

Informatio n (OSRI ) unde r

th e Endocrin e Disrupte r

Screenin g Program . Marc h 26 , 2009 . USEPA (2009b) . Propiconazole ; Pesticide Tolerances . Environmental Protectio n Agenc y (EPA) : Fina l rule . Federa l Registe r / Vol . 74 , No . 5 6 /

Wednesday , Marc h 25 , 200 9 / Rule s an dRegulation s pp . 12606-12613 . Vinggaar dAM , Hni aC , an dLarse n J C (2000) . Screenin g o f selecte dpesticide s fo r inhibitio n o f

CYP1 9 aromatas e activity i n vitro . Toxicol . i n vitr o 14:227-234 . Vinggaar dAM , Niemel aJ , Wedeby e EB , Jense n G E (2008) . Screenin g o f 39 7 Chemical s an dDevelopmen t

o f aQuantitativ e Structure-Activit y Relationshi p Mode l fo r Androge n Recepto r Antagonism . Che mRe s Toxicol . 21:813-823 . Zilliou x E , Johnso n I , e t

al . (2001) . Th e sheepshea dminno w a s an i n viv o mode l fo r endocrin e disruptio n i n marin e teleosts : apartia l life-cycl e tes t with 17alpha-ethynylestradiol . Enviro n Toxico l Che m20(9) : 1968-1978 .

Report Number : TK002342 3 Pag e 5 5 o f 8 5

### APPENDICE S SECTIO N

Report Number : TK002342 3 Pag e 5 6 o f 8 5

### APPENDI X 1 ToxCast TM

ToxCast TM i s aU S EP A progra mtha t include s abatter y o f

i n vitro , high-throughpu t screenin g (HTS ) assay s t o scree n chemical s fo r potentia l huma n toxicity . Ove r 30 0 chemical s hav e undergon e HT S unde r Phas e I o f

th e ToxCast TM program , th e majorit y o f thes e bein g pesticides . A numbe r o f thes e chemicals , includin g propiconazole , hav e curren t o r forthcomin g requirement s fo r Endocrin e Disruptio n Screenin g Progra m(EDSP ) Tie r 1 screening . Potentia l redundancie s exis t betwee n som e o f

th e ToxCast TM assay s an dth e EDS P Tie r 1 screenin g objectives ; thus , ToxCastTM dat ama y b e usefu l OSR

I (Othe r Scientificall y Relevan t Information ) t o submi t

i n lie u o f o r t o supplemen t

Tier 1 require dtests . Informatio n regardin g th e ToxCast TM Programresult s becam e generall y availabl e t o th e publi c throug h Environmenta l Healt h Perspective s (EHP ) an dth e EP A ToxCast TM Program websit e (www.epa.gov/ncct/toxcast / index.html ) i n mid-Decembe r 2009 , with th e ra w dat a fo r man y o f th e assay s poste do n th e ToxCast TM websit e i n mid-February 2010 . Th e dat a presente di n th e EH P articl e (Judso n e t al . 2010 ) an dsupplementary material s (Judson_ehp.0901392.s001.pdf) , an do n th e ToxCast TM Progra mwebsit e i s fairl y opaque , wit h individua l chemica l dat atabulate dan dmuc h o f th e detai l o n assay methodologie s provide donl y throug h websit e links . Thi s revie w wa s develope dt o evaluat e th e dat amos t relevan t t o potentia l endocrin e modulatio n an dth e result s specifi c t o propiconazole . Thi s revie w describe s briefl y th e HT S assa y technologie s incorporate di n ToxCast TM relevan t t o th e EDS P Tie r

1 screenin g requirements , provide s a n overvie w o f th e HT S assay s mos t

relevan t t o potentia l estrogen/androgen/thyroi dhormon e (EAT ) endocrin e modulation , an ddiscusse s th e EDS P Tie r 1 screenin g assay s tha t

coul db e potentiall y fulfilled . 1 . ToxCast TM HT S Tes t Batter y an dRelevanc e t o Potentia l Endocrin e Modulatio n Th e ToxCast TM HT S tes t

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

• Cell-fre e HT S assay s t o measur e bindin g constant s an denzym e inhibitio n values ;

• Cell-base dHT S assay s t o measur e bindin g constant s an denzym e inhibitio n values ;

• Hig h conten t

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

phenotype s i n eithe r th e huma n hepatom acel l lin e HepG 2 o r rat primary hepatocytes ;

• Gen e expressio n i n huma n primary hepatocytes , focuse do n Phas e I an dI I metaboli c enzyme s an dtransporters ;

• Multiple x transcriptio n reporte r assay s t o evaluat e 4 8 transcriptio n facto r bindin g sites ;
Report Number : TK002342 3 Pag e 5 7 o f 8 5• 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 ;

• Phas e I & I I xenobioti c metabolizin g enzym e (XME ) cytotoxicity assay s base do n microarra y technology ;

• High-throughpu t

genotoxicit y screenin g base do n th e GreenScree n hig h conten t GADD45A-GFP HT S genotoxicit y assay ; an d

• Real-tim e cel l electroni c sensin g (RT-CES ) t o measur e cellular change s i n respons e t o chemicals . O f thes e assa

y technologies , thos e that 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 includ e th e cell-fre e HT S assays , th e cell-base dHT S assays , an dth e multiple x transcriptio n reporte r assays . Eac h o f

thes e assay s an dthei r

dat a relevan t t o potentia l endocrin e modulatio n i s describe dbelow . 1. 1 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

Lif e Science s (formall y NovaScreen ; no w Calipe r Lif e Sciences) . O f

th e 23 9 endpoint s examined , bindin g t o th e followin g receptor

protein s relevant

to th e endocrin e systemwa s evaluated : • Bovin e estroge n recepto r fro muterin e membran e (bER) ; • Huma n estroge n recepto r fro mbreas t

cance r cell s (hER) ; • Bovin e progesteron e recepto r fro muterin e membran e (bPR) ; • Huma n progesteron e recepto r fro mT47-D 1 cell s (hPR) ; • Ra t androge n recepto r

(rAR ; recombinant) ; • Huma n androge n recepto r fro mLnCA P cell s (hAR) ; • Rat thyrotropin-releasin g hormon e recepto r fro mforebrai n membrane s (rTRH) ; an d
• Huma n thyroi dhormon e receptor-alph a(hTRa ; recombinant) . Al l o f thes e protein s ar e nuclear receptors , wit h th e exceptio n o f th e rat thyrotropin-releasin g hormon e recepto r

(rTRH) , whic h i s aG-protei n couple dreceptor . Additionally , al l o f these , wit h th e exceptio n o f

th e hTRa , wer e evaluate di n competitiv e bindin g assay s that measure d th 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 ) wa s als o evaluated . Assay parameter s specifi c t o eac h o f thes e endpoint s ar e detaile di n Appendi x Tabl e 1 .

1 EP A indicate dtha t

th e huma n progesteron e recepto r wa s derive dfro mMCF- 7 cells ; however , accordin g t o Calipe r Lif e Sciences , th e recepto r

protei n wa s derive dfro mT-47 D cells . Report Number : TK002342 3 Pag e 5 8 o f 8 5Appendi x Tabl e 1 : Paramete r Specific s Regardin g Endocrine-Relate dEndpoint s o f th e Cell-Fre e HT S Assay s Receptor/enzym e Estroge n Recepto r Progest

eron e recepto r Androge n recepto r
Thyroi d Relate d Aromatas e bE R

hE R bP R hP R rA R hA R rTR H hTRa c hCY P C1 9 Assa y typ e Competitiv e bindin g Competitiv e bindin g Competitiv e bindin g Competitiv e bindin g Competitiv e bindin g Competitiv e bindin g Competitiv e bindin g Recepto r activatio n Enzym e inhibitio n Referenc e

compoun d N/A a 1713-estradio l promegeston e promegeston e methyl

trienolon e methyl

trienolon e thyroi dreleasin g hormon e (TRH ) Bisphenol- A Ketoconazol e Positiv e

contro l compoun d 17B-estradio l 17B-estradio l promegeston e promegeston e methyl

trienolon e methyl

trienolon e TRH N/ A N/ A Ligan d

(cone. , M ) 3 H-estradio l ( 7 x 10-'° ) 3 H-estradio l ( 1 x 10' 10 ) 3 H-promegeston e (7xlO-'° ) 3 H-promegeston e (7xlO- |(> ) b 3 H-methyl

trienolon e ( 1 x 10- 9 ) 3 H-methyl

trienolon e ( 5 x 10' 10 ) 3 H-3-meHis 2 - TRH ( 2 x 10-' ) Triiodo-thyronin e (T 3 ) (l.SxlO' 8 ) Di(benzyloxy

methoxy ) fluorescein d (2. 5 xlO' 6 ) Kd(M )

7x10-' ° 5x10- " 6x10- " 3 x 10- 1 " 4. 6 xlO' 9 3 x 10-' ° 2. 3 x 10' 9 3. 5 xlO' 9 N/ A Bma x

(fmol/m g protein ) 40 6 3,84 4 15 2 15 2 103,92 8 27 0 3 4 N/ A N/ A

aN/A = no t

applicable . b EP A list s th e ligan dconcentratio n a s 3 x 10"' ° M ; however , accordin g t o Calipe r Lif e Sciences , th e ligan dconcentratio n wa s 7xlO' 1 0 M . c Assa y of

antagonis mto ligand-dependen t bindin g of

co-facto r to the receptor . dAromatas e substrat e bindin g affinit y = 4. 4 x 10' 6 M an dvma x = 0.0 3 pmo l product/pmo l CYPC19/min . I n al l cases , th e dat awer e normalize dt o thos e o f th e solven t

control s fro mth e sam e plat e a s th e teste dchemical s an dresult s expresse da s th e percen t o f neutra l control . Fo r

th e parameter s above , chemical s wer e initiall y screene dat asingl e concentratio n o f 2 5 u M (1 0 u M fo r aromatase ) i n duplicat e wells . Thos e chemical s tha t wer e positiv e i n th e initia l scree n wer e the n ru n usin g eigh t concentration s i n th e rang e o f 0.0229-5 0 u M (0.00914-2 0 p. M fo r aromatase ) t o determin e th e ACs o (i.e. , th e hal f maxima l activity concentration)Cell-Base d HT S Assay s 1. 2 Cell-base dHT S Assay s Thes e assay s wer e conducte db y th e NI H Chemica l Genomic s Cente r (NCGC) . Both agonis t an dantagonis t

activit y at

variou s nuclear

receptors , includin g th e huma n androge n recepto r (hAR) , huma n ER a(hERa) , an dhuma n thyroi dhormon e receptor- p (hTRp*) , wer e examine d usin g areporter

gene assa

y system(misidentifie da s Invitrogen TM GeneGLAze r i n EPA materials ; mos t

likely , th e Invitrogen TM GeneBLAzer ® reporte r assay system) . Accordin g t o th e Invitrogen TM websit e (http://www.invitrogen.com/) , th e GeneBLAzer ® technolog y use s a beta-lactamas e reporter

system , bu t

specific s abou t

ho w th e syste mwork s ar e no t provided . Repor t Number : TK002342 3 Pag e 5 9 o f 8 5Th e nuclear recepto r source s an dth e assay parameter s relate dt o eac h o f th e examine d endpoints , a s provide db y th e EPA , ar e detaile di n Appendi x Tabl e 2 . Appendi x Table2 : Paramete r Specific s Regardin g Endocrine-Relate dEndpoint s o f th e Cell-Base dHT S Assay s Androge n Relate d Estroge n relate d Thyroi d relate d Activity

hA Ragonis t hA Rantagonis t hER aagonis t hER aantagonis t hTR p agonis t hTR p antagonis t Targe t sourc e HEK293 H cell s HEK293 H cell s HEK293 H cell s HEK293 H cell s HEK293 T cell s HEK293 T cell s Referenc e

compoun d R188 1 N/ A 17p-estradio l 4-hydroxy-tamoxife n Triiodothyronin e (Ts ) N/ A Positiv e contro l

compoun d N/A 3 N/ A N/ A 4-hydroxy

tamoxife n N/ A N/ A Ligan d

(cone. , M ) N/ A R188 1 ( 1 x 10' 8 ) N/A 17p-estradio l ( 5 x 10' 10) N/ A T 3 ( 4 x ID' 10 ) aN/ A = no t

applicable . Exac t concentration s o f

chemical s teste di n thi s assay syste mar e no t clear . A recen t publicatio n indicate s tha t chemical s wer e teste dat

fiftee n concentration s i n th e rang e o f 0.0012-9 2 u M (Judso n e t al . 2010) . However , th e summar y dat afil e fo r thes e assay s provide do n th e ToxCast TM websit e (http://www.epa.gov/ncct/toxcast/index.html ) indicate s tha t chemical s wer e teste dt o amaximu mconcentratio n o f 20 0 uM . I n th e cas e o f propiconazole , concentration s i n th e rang e o f 0.001 0 - 76. 6 u M wer e tested . A minimu mo f 25 % o f th e contro l efficac y wa s use da s th e cut-of f fo r considerin g achemica l t o b e active . Th e supplemental informatio n t o Judso n e t al . (2010 ) indicate s that , "value s wher e onl y th e highest concentratio n exceede d50 % activity wer e exclude dt o eliminat e wea k o r fals e positives. " Cel l viabilit y wa s als o evaluate di n thi s assay system ; however , th e criteri afo r excludin g concentration s t o b e teste di n th e assay syste mbase do n cytotoxicity ar e no t clear . 1. 3 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 ) librar y 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 activities 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 h chimer 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 ddownstreamo f a5X-UAS-TAT A promoter . I n al l cases , th e reporter 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 h reporte r gene . Th e syste mallowe dfo r reporte r gen e product s t o b e identifie dthroug h sequencin g an dseparatio n vi ahig h resolutio n electrophoresis . Usin g thi s system , th e followin g endpoint s relevan t t o endocrin e modulatio n wer e evaluate d(liste di n Appendi x Tabl e 3 , alon g with thei r

respectiv e referenc e compounds , a s provide di n EP A materials) .

Repor t Number : TK002342 3 Pag e 6 0 o f 8 5Appendi x Tabl e 3 : Endocrine-Relate dEndpoint s an dReferenc e Compound s Teste di n th e Multiple x Transcriptio n Reporte r Assay s Androge n relate d Estroge n relate d Thyroi drelate d Endpoin t Huma n androge n recepto r Huma n ER- a Huma n estroge n respons e element 3 Huma n estrogen-relate dreceptor- a Huma n estrogen-relate dreceptor- y Huma n thyroi dhormon e receptor- a Referenc e Compound s Dioxin , FI C 1 7(3-Estradiol , diethylstilbestro l Estrogen s N/A b 4-Hydroxytamoxife n Triiodothyronin e (T 3 ) 1 Th e huma n estroge n respons e elemen t wa s teste da s aci s element ; al l othe r endpoint s relevan t

t o endocrin e modulatio n wer e teste da s tran s factors . b No t applicable . Cytotoxicit y wa s firs t

determine dusin g th e Dimethy l thiazoly l dipheny l tetrazoliu msal t assay an d24-hou r

chemica l exposur e t o fiv e tes t concentrations , with an uppe r concentratio n o f 5 0 (j. M an d10-fol dserial dilutions . Th e maximu mtolerate dconcentratio n (MTC ) wa s derive da s one-thir dth e cytotoxi c ICso , o r i f n o ICs o valu e wa s determined , the n th e MT C wa s se t at 10 0 uM . Tes t

compound s wer e the n assaye dat seve n differen t

concentration s i n th e rang e o f

0.0014-10 0 uM , startin g at th e MTC , followe db y three-fol dserial dilutions . Afte r a24-hou r

exposure , cell s wer e harvested , reporte r gen e product s isolate dan dreverse transcribe dusin g fluorescent-tagge dprimers , an dresolve db y hig h resolutio n ge l electrophoresis . Fro mth e resultin g data , th e lowes t effectiv e concentratio n tha t activate da cis-actin g respons e elemen t o r transcriptio n facto r

wa s determine d(LEL ; lowes t concentratio n a t

whic h ther e wa s astatisticall y significan t chang e fro mth e concurren t negativ e control) . 2 . Compariso n t o EDS P Tie r 1 I n Vitro Screenin g Requirement s Th e ToxCast TM dat asupplement , an di n some cases , may fulfil l th e i n vitr o dat arequirement s o f th e EDS P Tie r

1 E Rbindin g assay , A Rbindin g assay , E Rtransactivatio n assay an d aromatas e inhibitio n assay . I n addition , thes e dat aprovid e importan t OSRI tha t

shoul db e considere di n aweight-of-the-evidenc e evaluatio n o f functionall y equivalen t

informatio n supportin g th e fulfillmen t o f several EDS P Tie r

1 mammalia n i n viv o screenin g tests . E Rbindin g assay : Th e EDS P Tie r 1 E Rbindin g assay require s tha t bindin g b e evaluate d usin g ra t E Rderive dfro muterin e tissu e take n fro movariectomize dSpragu e Dawle y rat s (EPA 2009d) . I n th e ToxCast TM cell-fre e HT S assays , bindin g t o bovin e an dhuman , bu t no t rat , E Rwa s evaluated . I n th e EDS P Tie r 1 screenin g assay , th e concentratio n o f 3 H-17p

estradio l ligan duse di n th e assay i s 1 n M an dth e tes t chemical s ar e evaluate dat concentration s o f

1 x 10' 1 0 t o 1 x 10" 3 M (EP A 2009d) . I n th e ToxCast TM assay , th e concentratio n o f

radiolabele destradio l varie ddependin g o n th e E Rsourc e (bER : 7 x 10" 1 0 M ; hER : 1 x 10" 1 0 M) , bu t

wa s generall y simila r

t o tha t recommende di n th e EDS P Tier 1 E R bindin g assay ; however , compound s wer e teste dat an initia l concentratio n o f 2. 5 x 10" 5 M , an di f positiv e fo r

bindin g activity , wer e furthe r evaluate dat

concentration s o f

2. 3 x 10" 8 - 5 x 10" 5 M . Thi s concentratio n rang e i s narrowe r tha n th e rang e recommende di n th e EDS P Tie r 1 screenin g assay , bu t

stil l shoul db e adequat e t o detec t activity i n amoderat e concentratio n range . Report Number : TK002342 3 Pag e 6 1 o f 8 5

### Androge n recepto r bindin g assay : Th e EDS P Tie r 1 scree n A Rbindin g assay require s tha t bindin g b e evaluate dusin g ra t A Rderive dfro mprostat e tissu e take n fro mcastrate dSpragu e Dawle y rat s (EP A 2009b) . Th e concentratio n o f 3 

### H-R188 1 ligan duse di n th e assay i s 1 n M an dth e tes t

chemical s ar e evaluate dat concentration s o f

1 x 10" 9 

### t o 1 x 10" 4 

### M . I n th e ToxCast TM cell-fre e HT S assay , bindin g t o both rat an dhuma n androge n receptor s wa s evaluated , s o i t

provide s amore comprehensiv e scree n tha n doe s th e EDS P Tie r 1 battery . Radiolabele dR188 1 wa s use da s th e radioligan di n thi s assa y an dconcentration s varie d dependin g o n th e A Rsourc e (rAR : 4.5 9 x 10" 9 

### M ; hAR : 3 x 10"' ° M) , bu t wer e similar

t o that recommende di n th e EDS P Tie r 1 A Rbindin g assay . Finally , compound s wer e teste di n th e ToxCast TM cell-fre e HT S assay at an initia l concentratio n o f 2. 5 x 10~ 5 

### M , an di f

positiv e fo r bindin g activity , furthe r

evaluate dat

concentration s o f

2. 3 x 10" 8 

### - 5 x 10" 5 

### M . Thi s concentratio n rang e i s slightl y narrowe r tha n tha t recommende di n th e EDS P Tier 1 scree n A Rbindin g assay guidelin e ( 1 x 10" 9 

### - 1 x 10" 4 

### M ; EP A 2009b) , bu t stil l shoul db e adequat e t o detec t activity i n amoderat e concentratio n range . E Rtransactivatio n assay : Th e EDS P Tie r 1 scree n E Rtransactivatio n assay require s tha t th e assa y b e conducte di n hER-HELA-990 2 cells , whic h expres s th e huma n ER- aan dhav e bee n stabl y transfecte dwit h aluciferas e reporte r gen e linke dt o a n upstrea mvitellogeni n estrogen responsiv e element . Tes t

compound s are evaluate dat concentration s o f

1 x 10" 9 

### t o 1 x 10" 3 

### M . Finally , ates t

chemical i s considere dpositiv e fo r E Rtransactivatio n i n th e EDS P Tie r 1 assa y i f th e maximu mrespons e induce di s at leas t 10 % o f tha t induce db y 1 n M l?p-estradio l (EP A 2009a) . In th e cas e o f

th e ToxCast

TM cell-base dHT S assays , detail s regardin g th e gene reporter syste mwer e no t

available ; however , activit y at th e huma n ER- awa s assesse dan dtes t chemical s wer e evaluate dat asimila r rang e o f concentration s (1. 2 x 10~ 9 

### - 0. 9 x 10" 4 

### M ) a s recommende di n th e EDS P Tie r 1 screen . Th e dat aoutput s fro mth e ToxCast TM cell-base d HT S assay s (ACs o values ) ar e differen t tha n thos e require di n Tie r 1 screening , an di n mos t cases , positiv e contro l compound s wer e no t tested . However , 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 cell-base dassay , i t ca n b e assume d tha t i t woul db e negativ e accordin g t o th e EDS P Tie r 1 screenin g criteria . I t

shoul db e note d tha t th e ToxCas t

program' s cell-base dHT S assa y provide s mor e robus t dat atha n tha t require di n th e EDS P Tie r 1 E Rtransactivatio n assay becaus e i t als o examine dth e tes t chemical' s ability t o ac t

a s an antagonis t

at th e ER-a . Th e multiple x transcriptio n reporter assa y als o examine dtransactivatio n o f th e human ER-a . I n thi s case , th e assa y 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 P Tie r

1 screenin g assay . HepG 2 i s aperpetua l cel l line , whic h wa s derive dfro mth e live r tissu e o f a15-yea r ol dCaucasia n American mal e wit h awel l differentiate d hepatocellular

carcinoma . Th e HepG 2 cell s an dit s derivative s ar e ofte n use da s amode l syste mfo r

studie s o f live r metabolis man dxenobiotic s toxicit y becaus e o f thei r metaboli c capabilities . Th e rang e o f tes t chemica l concentration s teste d(1. 4 x 10" 9 

### - 1 x 10" 4 

### M ) i s simila r t o tha t recommende di n th e EDS P Tie r 1 screen . Finally , a s with th e ToxCast TM cell

base dHT S assay , th e dat aoutput s (LE L values ) ar e differen t tha n thos e require di n EDS P Tie r 1 screening , an dpositiv e contro l compound s wer e no t tested . However , i f

achemica l Repor t Number : TK002342 3 Pag e 6 2 o f 8 5elicite dalo w leve l o f activit y at

th e highes t

concentratio n teste di n th e multiple x transcriptio n reporte r

assay , i t can b e assume dtha t

i t woul db e negativ e accordin g t o th e EDS P Tier

1 screenin g criteria . Aromatas e assay : Th e EDS P Tie r 1 scree n aromatas e assa y require s tha t enzym e activity b e evaluate dusin g huma n recombinan t microsome s containin g CYP1 9 plu s reductase . Radio

labele dandrost-4-ene-3,17-dion e at aconcentratio n o f

1 x 10~ 8 M i s use da s th e enzym e substrat e an dtes t

compound s ar e evaluate da t concentration s o f

1 x 10~ 1 0 M - 1 x 10" 3 M (EPA . 2009c) . I n th e ToxCast TM cell-fre e HT S assay , huma n aromatas e activit y wa s evaluate dusin g di(benzyloxymethoxy ) fluorescein , whic h produce s afluorescen t produc t a s th e enzym e substrate . Compound s were initiall y screene dat asingl e concentratio n o f 1 x 10~ 5 M , an dthos e chemical s tha t wer e positiv e i n th e initia l scree n wer e the n ru n usin g eigh t concentration s i n th e rang e o f 9.1 4 x 10~ 9 M - 2 x 10~ 5 M . Thi s concentratio n rang e i s narrowe r tha n th e rang e recommende di n th e EDS P Tier 1 screenin g assay , bu t

stil l adequat e t o detec t activity i n amoderat e concentratio n range . I t shoul db e note dtha t

th e ToxCast TM 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 EDS P Tie r 1 screenin g progra mt o provid e additiona l informatio n o n th e potentia l t o interac t wit h 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 EDS P Tie r 1 steroidogenesi s i n vitr o tes t requirements . 3 Dat aSpecifi c t o Propiconazol e Propiconazol e o f

unknow n purity wa s teste di n al l ToxCast TM assays . I n th e cell-fre e HT S assays , propiconazol e wa s teste dat eigh t concentration s i n th e rang e o f 0.02-5 0 u M at

al l recepto r

protein s o f

relevanc e t o potentia l endocrin e modulation , excep t th e rat thyrotropin-releasin g hormon e recepto r (rTRH) . I n th e ra w dat afile s provide db y EPA , n o rTR H dat afo r

propiconazol e ar e listed , suggestin g tha t eithe r propiconazol e wa s no t teste dfo r bindin g a t

thi s recepto r

protein , o r

mor e likely , tha t

propiconazol e di dno t

provid e a positiv e bindin g signa l at th e initia l tes t

concentratio n o f

2 5 (J. M an dwa s no t

furthe r tested . Thi s include s th e bovin e an dhuma n estroge n receptors ; bovin e an dhuma n progesteron e receptors ; rat

an dhuma n androge n receptors ; an dhuma n thyroi dhormon e receptor-a . I n th e cell-base dHT S assays , propiconazol e wa s teste dat 1 5 concentration s i n th e rang e o f 0.0010

- 

### 76. 6

Report Number : TK002342 3 Pag e 6 3 o f 8 5Appendi x 1 - Reference s

Environmenta l Protectio n Agenc y (EPA) . (2009a) . Endocrin e Disrupto r Screenin g Progra

m Tes t Guidelines . OPPT S 890.1300 : Estrogen Recepto r Transcriptiona l Activatio n (Huma n Cell Lin e (HeLa-9903)) . EPA-740-C-09-006 . Octobe r 2009 . Environmenta l Protectio n Agenc y (EPA) . (2009b) . Endocrin e Disrupto r Screenin g Progra

m Tes t Guidelines . OPPT S 890.1150 : Androge n Recepto r Bindin g (Rat

Prostate Cytosol) . EPA-640-C-09-003 . Octobe r 2009 . Environmenta l Protectio n Agenc y (EPA) . (2009c) . Endocrin e Disrupto r Screenin g Progra

m Tes t Guidelines . OPPTS 890.1200 : Aromatas e (Human Recombinant) . EPA-740-C-09-004 . Octobe r 2009 . Environmenta

l Protectio n Agenc y (EPA) . (2009d) . Endocrin e Disrupto r Screening Progra

m Tes t Guidelines . OPPTS 890.1250 : Estrogen Recepto r Bindin g Assa

y Usin g Ra

t Uterin e Cytoso l (ER-RUC) . EPA-740-C-09-005 . Octobe r 2009 . Environmenta l Protectio n Agenc y (EPA) . (2010) . ToxCast TM Programwebsite . Availabl e at www.epa.gov/ncct/toxcast/index.html . Judso n R.S . e t

al . (2009) . I n vitr o Screenin g o f Environmenta l Chemical s fo r Targe t Testin g Prioritization-Th e ToxCas t Projects ; Supplementa l Information . Se e http://www.epa.gov/ncct/toxcast / Supplementa l dat aan dfigure s fo r Judso n e t al . Judson , R.S. , K.A . Houck , R.J . Kavlock , T.B . Knudsen , M.T . Martin , H.M . Mortensen , D.M . Reif , D.M . Rotroff , I . Shah , A.M . Richard , an dD.J . Dix . (2010) . I n vitr o screenin g o f environmenta l chemical s fo r targete dtestin g prioritizatio n - th e ToxCas t Project . Enviro n Healt h Perspectives . 118:485-492 . Martin , M.T. , D.J . Dix , R.S . Judson , R.J . Kavlock , D.M . Reif , A.M . Richard , D.M . Rotroff , S . Romanov , A . Medvedev , N . Poltoratskaya , M . Gambarian , M . Moeser , S.S . Makarov , an d K.A . Houck . (2010) . Impac t o f environmenta l chemical s o n ke y transcriptio n regulator s an d correlatio n t o toxicity endpoint s withi n EPA' s ToxCast TM Program . Chemical Researc h i n Toxicolog y 23:578-590 .

Report Number : TK002342 3 Pag e 6 4 o f 8 5

### APPENDI X 2 Assay Descriptio n Shee t fro mCalipe r

LifeScience s -Bovin e Estroge n Recepto r

Bindin g Assa y

ESTROGE N (BOVINE ) BINDIN G ASSA Y

O Q . (0 110- , 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 -10

- -13. 0 -10. 5 -8. 0

lo g [drug ] (M ) Referenc e Compoun d I -5. 5 Ki(nM ) » 17,pEstradio l vPromogestron e 0. 2 N/ A

Cat# : Assa y Characteristics : K D (bindin g affinity) : Bma x (recepto r

number) : Material s an dMethods : Recepto r Source : Radioligand : Non-specifi c Determinant : Referenc e Compound : Positiv e Control : Incubatio n Conditions : 100-012 6

0. 7 nM 40 6 fmol/m g protei n

Bovin e uterin e membrane s [ 3 H ] Estradio l Fina l ligan dconcentratio n - [0. 7 nM ] 17P , Estradio l -[10nM ] l?p , Estradio l Reaction s ar e carrie dou t i n 1 0 mM TRIS-HC I (p H 7. 4 containin g 1. 5 m M EDTA , 1. 0 m

M DTT , an d2 5 m

M sodiu mmolybdat e a

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

0-4°C . 

### Report Number : TK002342 3 Pag e 6 5 o f 8 5Th e reactio n mixture s ar e centrifuge dan dth e radioactivit y boun d i n th e supernatan t

i s assesse dan dcompare dt o contro l value s i n orde r t o ascertai n an y interaction s o f tes t compoun dwit h th e estradio l bindin g site . Literatur e Reference : Haji , etal . Age-Relate dChange s i n th e Concentration s o f Cytoso l Receptor s fo r

Se x Steroid s i n th e Hypothalamu s an dPituitar y Glan do f th e Rat . Brai n Research . 204 : 373-38 6 (1980 ) wit h modifications . O'Keefe , J.A . an dHanda , R.J . Transien t Elevatio n o f

Estroge n Receptor s i n th e Neonata l Rat Hippocampus . Develop . Brai n Res . 57 : 119-127(1990) .

Report Number : TK002342 3 Pag e 6 6 o f 8 5

### APPENDI X 3 Assa y Descriptio n Shee t fro mCalipe r

LifeScience s -Huma n Estroge n Recepto r

Bindin g Assa y ESTROGE N (HUMAN ) BINDIN G ASSA Y

0 . V ) 110- 1 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - -10

- -13. 0 -10. 5 -8. 0

lo g [drug ] (M ) Referenc e Compoun d l -5. 5 Ki(nM ) 17,pEstradio l Methyltrienolon e 0.02 5 N/A Cat# : Assay Characteristics : K D (bindin g affinity) : Em TM (recepto r

number) : Material s an dMethods : Recepto r Source : Radioligand : Non-specifi c Determinant : Referenc e Compound : Positiv e Control : Incubatio n Conditions : 100-012 7

0.0 5 nM 3844 fmol/m g protei n

MCF- 7 cell s [ 3 H ] Estradio l Fina l ligan dconcentratio n - [0. 1 nM ] 17p , Estradiol

- [300nM ] 17(3 , Estradio l 17P , Estradio l Reaction s are carrie dou t

i n 1 0 mM TRIS-HC I (p H 7. 4 containin g 1. 5 m M EDTA , 1. 0 m

M DTT , an 2 5 m

M sodiu mmolybdat e a

t 0

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

0-4°C . Th e reactio n mixture s ar e centrifuge dan dth e radioactivit y boun di n th e supernatan t i s assesse dan dcompare dt o contro l value s i n orde r

t o ascertai n an y interaction s o f tes t compoun d wit h th e estradio l bindin g site . 

### Repor t Number : TK002342 3 Pag e 6 7 o f 8 5Literatur e Reference : Haji , etal . Age-Relate dChange s i n th e Concentration s o f Cytoso l Receptor s fo r

Se x Steroid s i n th e Hypothalamu s an dPituitar y Glan do f th e Rat . Brai n Research . 204 : 373-38 6 (1980 ) wit h modifications . O'Keefe , J.A . an dHanda , R.J . Transien t Elevatio n o f

Estroge n Receptor s i n th e Neonata l Rat Hippocampus . Develop . Brai n Res . 57 : 119-127(1990) .

Repor t Number : TK002342 3 Pag e 6 8 o f 8 5

### APPENDI X 4 Titl e Pag e an dMRI D Numbe r o f Cite dStudie s

Repor t Number : TK002342 3 Pag e 6 9 o f 8 5EPAGor e Grad e Minimum ,

BINDE R 1 O F 5 TOXIGENICS 1 STUD

Y 450-123 2 TWO-GENERATIO

NREPRODUCTIO

NSTUD Y ' "I NALBIN

O SAT S .WIT

H CGA-6425 0 TECHNICA L

SUBMITTE D TO i CIBA-GEICJ

yCQRPQRATJO

N AGRICOtTORA i DIVISIO N P.O . Bp x 1830 0 GREENSBORO , N

G 2741 9
SUBMITTE D BY, : TOXIQENICS . INC . 180 0 BAS T PERSHIN

G ROA D DECATUR , I L 6252 6 MARC H 12 , 198 5



### Report Number : TK002342 3 

### ToxiGenic s A SobsWiar

vofWhittake rQorooratio n 

### Pag e 7 0 o f

8 5'MFLI D Hex . 4088:200 1 

# VOLUM E __ _O F _O F SUBMISSIO

N PROMCONAZOL E THE CHRONI C TpXICIT

Y O F C6A-6125 0 TECHNICA

L <PROPICONAZOLE > TO SHEEPSHEA D MINNO W(CYPRINODO

NVARIEGATUS * EP AGUIDELINE S NO , 72- 5 AUTHOR - RONAL D J , BREfELER > PH.B , STUD Y COMPLETE D O NJUL Y i8 > 198 8 CONDUCTE D BY SPRIN0BPR

NLIF E SCIENCE S 79 0 MAI NSTREE T WAREHAM ; M A0257 1 LABORATORY STUD Y NUMBE R88-04-268 5 VOLUM EJ

_O FJ L O FSTUD Y

AGRICULTURA L DIVISIO

N CIBA-6EI6 Y CORPORATIO

N POS T OFFIC E BO

X 1830 0 GREENSBORO , N

C 2711 9

Report Number : TK002342 3 Pag e 7 1 o f 8 5CIBA-GEIG Y BAStE , SWITZERLAN D REPRODUCTIV E TOXICOLOG Y G U 2. 5 /

e k

; MRID . No . .00.05860 4

:EPA Core ; Gradi s Minimu m

REPOR T O N CGA .6. 4 '25 0 tecjih . TERATOLOG

Y STUD Y (Seg . II ) I NRAT S

Report Number : TK002342 3 Pag e 7 2 o f 8 5VOLUH E O F O F SUBMISSIO N MRI D No . 4438i'40 1 ; EP ACor e Grad e .Minimu m

18-HONTHS ONC<X3BNieiTY STUDY I N MICE i 94312 6 CG A6425 0 tech . FINA L REPOR T

Author : Dr. . rer . nat . R . Gerspac h Testin g Facility :

Tes t Guidelines : Nbyarti aCjro p Protectio n A G '(•Successor'

- .ir

i • • busines s -of ; CIBAVGEIG

Y Ltd . an

d Sandb z Ltd. ) Toxicolbgy/ExperimerilEia l Toxicolog

y 433 2 Stei n / Switaerland fIFH A83- 2 OECD 45 1 JAP MAF F Fina l Repor t issued : Marc h 26 , 199 7 Sponsor : Novarti s Cro p Protectio n A

G Huma n Safet

yAssessmen t 406 2 Basl e / Switzerlan d Thi s repor t contains : 44 9 page s

Volume 1 o f 1

Report Number : TK002342 3 Pag e 7 3 o f 8 5Noyarti s Cro p Protectio

n A U Toxicolog

y. , 433 2 STEI N/ SWITZERLAN D MR'iD'Nq . 44381-401 , EP ACor e Grad e Minitnu m FIRS T AMENDMEN

T T O THE FINA

L REPOR

T TES T ARTICLE : TES T NUMBER : STUD Y TITLE S STUD Y INITIATION S DAT E O F ISSU

E O F REPORT : DAT E O F ISSUE O F AMENDMENT : SPONSO R : CG A5425 0 tech . 94312 6 IS^MONTH S ONeQGENICIT Y STUD Y I NMIC E Novembe r 2 , 1994 ^ (protoco l date ) Marc h 26 , 199 7 Novembe r 11 , 199 9 Novartl s Crop Protection Huma n SafetyAssessmen t

STUD Y DIRECTO R Dr . rer . nat . R . Gerspac h date : STUD Y PATHOLOGIS T Die . raed . Ph . Schaett l

RESPONSIBL E FOR PATHOLOG Y Dr . med . vet . ch . Lande s

•FACILIT

Y MANAGEMENT P D Dr . med . vet . G . Winkle r FV H Fundajnenta l Medicin e 

### da i

COPIE S FO R TH E SPONSO R (Dr . T . Hertner ) ARCHIVE S QA U Thi s amendmen t contains 6 0 page s



### Repor t Number : TK002342 3 Pag e 7 4 o f 8 5MRI D No : 4042500 1 EPA : Gor e Grad e Minimu m VOLUME O F O F SUBMISSIO N CGA-6425 0 TECHNICA L TERATOLOG

Y (SEGMEN

T II ) STUD

Y I NRAT S EP AGUIDELINE S NO . 83- 3 AOTHO R - MAR Y GIKNI S STUD Y COMPLETE D O

NJANUAR

Y 28 , 198 7 CONDUCTE D B

Y PHARMACEUTICAL S DIVISIO N CIBA-GEIG

Y CORPORATIO

N 55 6 MORRI S AVENU E SUMMIT , NE

W JERSE Y 0790 1 LABORATOR

Y STUD Y NUMBE R MIN8600 4 VOLUME • „ ;l- : - • O F . 1 . . ,O F STUD Y PKSS i . . 1 .. . O

F 42 5

AGRICULTURA

L DIVISIO

N CIBA-GEIG

Y CORPORATIO

N POST OFFICE BO

X 1830 0 GREENSBORO, , N

C 2741 9

R502SJ0810C

B



### Repor t Number : TK002342 3 Pag e 7 5 o f 8 5CQNFIDENIWl CB G 193/^8 4 > 789023} 

### 1 A GK t

MRI D NO . 0012991 8 Cor e Grade;Minimu

m CG A 'PQ;TE ; NT.[* t 

### ADMINISTRATION ; T O RAT S (Fina l

Addreuee : Profesaa r8 . Hass , 8-1036 . 206 , C H 400 2 Basis , SWITZERLAND , 3 0 Septembe r1982 Author * Brian Hunter , NIgi J ;D . Slater , Ifeilp h Heywood ; Davi d E . Prenfiee , Wlita m A , Gibson , Chirukandot h Gooinoth . Hunt ) ngtte i Researc h Centn , Camfarldgesfiirs , SNGtAND . 

### Report Number : TK002342 3 Pag e 7 6 o f 8 5CONFIDENTIA L



### by : |J ! 

### ;MRHJ NO; , 0012957 0 EP ACor e Grade ; Minimu m

25 0

: (interjmi'f«por : » .<)..-• • 5: 2 I N

Aulhort : Profftuo rR , Hen , R-1036.206 , Obo-Galgy Limired , CH^-4002 , Bojie , SWTZERUNO . 1 8 Septembe r198 ! Dsbora h Scholey , Ralp h Heywood , Ala n E . Street- , Davi d E . Prentice , JdlmOffeT , Wil l tar n Gibson . Huntingdo n Researc h Centre , Huntlnjjdon , Cambrldgeshira , ENGtANO , 

### Repor t Number : TK002342 3 Pag e 7 7 o f 8 5CONFIDENTIA L 

### Rieeetvedby : ADDENPUMT O CBG/19VB182 7

MRI D No , O:0l2957; p EP A Cor e : Grad e Minimu m

ADDENDU M CG A 6 4 25 0 FEEDIN G STUD Y I N MIG E (Amende dpogei }

Addreuee : Professo rR . Hesi , C!bo-G«tgy A.G. , CH-400 2 Sale , SWITZERUND . 2 6 Octobe r1982 . Re-iuue< j - Am . poges 5 Novembe r198 4 1 Aogust 198

5 Authors : Bria n Hunrtr , Deborah Schol»y , Ralp h He/wood , Ala n E . Street , Davi d E . Prentice , Joh n Offer , Willia m Glbion . Huntingdo n Researc h Centre Ltd. , Huntingdon , Combrldgeihire , ENGLAND . 

### Report Number : TK002342 3 Pag e 7 8 o f 8 5MRI D No . 0013336 4 

### ;EP Aconcluded Che stud yfulfilled , the requiremen t fo r a fis h earl ylif > stag e tes t

PA90 9 FLOW-THROUG

H FATHEA D EARL Y : E1F E STAGE TOXICIIY TES T WIT H CGA-6A2S O

Prepared ; f6 r eiBA-GElG

Y Greensboro , North,Carolin a

Submitte d ; b y 

## ;ENVIRONMENTAIRESEARC H& •fEpHNOLOGY , INC .

For t Collin s , Colorad o Prepare d b

y Eugen e S . Iley , Jr . Novembe r 198 1



### Report Number : TK002342 3 Pag e 7 9 o f 8 5FOODJ & DRU G RESEARC H LABORATORIES , INC . ROUT 6 IX X P.O . BO

X 107 . WAVSRLY . N Y 14W2-OIO ? 40 7 5*5*13 1

iMRI D No . 0015151 5 EP AGor e Grad e Minimu m 

## ONE-YEA R SuBCHRONi c Tpxierr vSTUD Y I NEEAGL E DOG S WITH;GGA-6^25 0 TECHNICA L (FINA L REPORT )

i Stud y No. : 773 7 Submitte dto : CIBA-GEIG Y Corporatio n



### :j^Hquliural : Divisio

n Greensboro , N

C 2741 9 Date : Ma

y28 , 198 5 

# n / ^«. ' ,C.."v t >*.•*• 

### Willia mD . Johngon , Ph.D . Manage r o f Patholog

y Directo r

* .^•••A^^rrf J ^^fff..f t f^.^, : ,^ftf-f>v - rf^fi^'.^Jf f r . r Samue l W . Thompson , D.VVMi , M. S Diplomate , America n Colleg e 

### o £iteteririary Patholog y Pete r Jv'BBCci , President

Ikl t n»«r t b ntail W (t rti t «ntosl Ki w « ( IS * ptf»e , fjftttnMf , « wrpsfill w t o rti o I t b KMittsd , M d wltlw rHi t wr t n w ft t nu t « f Ibn e r f It j tUf) , «> | b « ^

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### Repor t Number : TK002342 3 Pag e 8 0 o f 8 5MRI D No . 40:42500 2 EP AStud yAcceptable/Non-guidelin e V0WOM E . ^ Of f _

_OS 1 SCBMISSIO

N CGA-6425 0 TECHKICA L AMODIFIE D TERATOLOG Y (SEGMEN

T II ) STUD

Y INALBIN

O RAT S WIT

H CGA-6425 0 EP AGUIDELINE S NO . 83- 3 ADTHO R - S . MALLOWS , E . LEVY , M . L . A . GIKNI S AN D E . T . XA

O STUD Y GOMPLETE D O

NFEBRUAR

Y 6 , 198 7 CONDUCTE D B

Y PHARMACEUTICAL S DIVISIO N CIBA-GEIG

Y CORPORATIO

N 55 6 MORRI S AVENU E SUMMIT , NEW JERSEY 0790 1 LABORATOR

Y STUD Y NUMBE R 8618 9 VOLUME 1 , O F J L O f STUD Y PAG E 1 O F 40 8

AGRICULTURA

L DIVISIO

N CIBA-GEIG

Y CORPORATIO

N 

### POS T orric s BOX isao o GREENSBORO , NC 2741 9

502SJ0810C B 

### Report Number : TK002342 3 Pag e 8 1 o f 8 5.MRI D No . 404250:0 1 EP A Gore,Grad e Minimu m VOLUMEO FSUBMISSIO N

A TERATOLpG

y STUD Y IN ; NEW^ALAN D WHITE RABBIT S EP A GUIDELINE S NO . 83- 3

AUTHOR S - ^ D;M . RAAB , MiA ; YOU|ENEFF r M.L.A . GIKNIS , AIT . ARTHUR , :A1® ; E^T . YA U STUD Y COMPLETE

D ON AUGUS T 1 , 198 6 FINA L REPOR

T AMENDMENT NO . 1 COMPLETE D JUNE 16 , 198 7 GONDUCTEDiB Y 

### RESEMC H DliPMTMENT , PKARMAGEIJTIGAL S DIVISIO N GIBA-:GEIGY TCQRPORATIO N 55 6 MORRI S AVENUE SUMMIT , NEWJERSEY 0790 1

TOXICOLOGY/PATHOLOGY:REPOR T 8604 3 (MI

N852172 ) (SUPPLEMEN

T T O EP AAccessio n No . 265796 ) VOLUME 1 O F 1 O F AMENDMEN T T O STUD

Y PAGE 1 O F 12 4 AGRICULTyRA

L DIVISIO

N CIBA-GBIG

Y CORPORATIO

N POS T OFFIC E BO

X 1830 0 GREENSBORO , N

C 2741 9



### Repor t Number : TK002342 3 Pag e 8 2 o f 8 5C1B A- GEIG Y LIMITE D BASL E / SWITZERLAN D TOXICOLOGY G U 2

-MR-ID ' NO . 0065860 6

EP AGore : Grade ; Minimu m

64'25 0 TEGHN . THRE E MONTH S TOXIGIT Y vSTUD Y O NRAT S

PINA L REPOR T

AUGUS T 3:0 , 1979/ST U

Report Number : TK002342 3 Pag e 83jp f 8 5GIB A- GEIG Y BASL E / SWITZERLAN D TOXICOLOG Y 6.0 . 2

MRID ; No , a0058S0 7 EP ACor e Grad e Miriimu m

CG A64'25 0 3-MONf H TOXrCJT Y STUD Y O NDOG S

AUGUS T 9 , 197 9



# ** » Report Number : TK002342 3 Pag e 8 4 o f 8 5

## MRI D No, , :4i72q3;p 2 VOLUM E O F O F SUBMISSIO N PROPICONAZOL B TH E TOXICIT

Y O F €GA-r6425 0 fPROPICONAZOLE ) T O FATHEA D MINNO W (PIMBBHALB S PROMELAS ) EMBRYO S AMD LARVA E

EP AGUIDELINE S NO . 72- 4 AUTHOR ! DONALD C . SURPRBNAN T COMPLETE D O

NNOVEMBE R 10 , 198 7

CONDUCTE D B

Y SPRINGBOR

NLIF E SCIENCES , INC . 79 0 MAI NSTREE T WAREHAM , MASSACHUSETT S 0257 1

LABORATOR

Y STUD Y NUMBE R 87-8-247 6

VOLUM E 1 O F 1 O F STUD Y PAGE 1 O F 23 5

AGRICULTURA

L DIVISIO

N CIBA-GEIG

Y CORPORATIO

N POS T OFFIC E BO

X 1830 0 GREENSBORO , N

C 2741 9



### Report Number : TK002342 3 Pag e 8 5 o f 8 5