# N R 481421-0 1 Do w AgroScience s LL C Repor t ID : 101762 Pag e 1 o f 5 5 STUD Y TITL

E Trifluralin : Scientifi c Rational e fo r Citatio n o f

Othe r Scientificall y Relevant

Informatio n (OSRI ) i n Fulfillmen t

of OPPT S 890.1150 , OPPTS 890.1200 , OPPT S 890.1250 , OPPTS 890.1300 , OPPT S 890 . 145 0 an

d OPPT S 890 . 150

0 EDS P Tie

r I Assay s ORDE RNUMBERS : EDSP-036101-221 , EDSP036101-22 3

DATA REQUIREMENT OPPT S 890.1150 , OPPTS 890.1200 , OPPTS 890.1250 , OPPT S 890.1300 , OPPTS 890.145 0 an d OPPT S 890.150

0 EDS P Tie

r I Assay s AUTHO R Sea n C . Gehen , Ph.D . STUD Y COMPLETE

D O N Jun e 22,201 0 PERFORMIN G LABORATORY j...; o 

### Do w AgroScience s LLC 933 0 Zionsvill e Road Indianapolis , IN 4626 8 REPOR T ID 101762

### Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 2 o f 5 5 STATEMEN

T O F N O DATA CONFIDENTIALIT Y CLAIM S Compound : Triflurali n Title : Trifluralin : Scientifi c Rational e fo r Citatio n o f

Other Scientificall y Relevan t

Informatio n (OSRI ) in Fulfillmen t

o f OPPT S 890.1150 , OPPT S 890.1200 , OPPT S 890.1250 , OPPT S 890.1300 , OPPT S 890.145 0 and OPPT S 890.150 0 EDS P Tie

r I Assay s N o clai mo f

confidentiality , o n any basi s whatsoever , i s mad e fo r any informatio n containe d i n thi s document . I acknowledg e that informatio n no t

designate d a s withi n the scop e o f FTFR A Sectio n 1 0 (d)(l)(A)(B ) o r

(C) , an d whic h pertain s to aregistere d o r previousl y registere d pesticide i s no t entitle d to confidential treatment

an d ma y b e released to th e public , subjec t to th e provision s regardin g disclosure t

o multinationa l entitie s under FIFR A Sectio n 10(g) . Company : Do w AgroScience s LL C Compan y Agent :

ritip - Ki 

### Title :

THI S DAT

A MAY B E CONSIDERE D CONFIDENTIA

L OUTSID E TH

E UNITED STATES .Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 3 o f 5 5 STATEMEN

T O F COMPLIANC E WTT H GOO D LABORATORY PRACTIC E STANDARD S Title : Trifluralin : Scientifi c Rational e fo r Citatio n o f

Othe r Scientificall y Relevant

Informatio n (OSRI ) i n Fulfillmen t

of OPPT S 890.1150 , OPPT S 890.1200 , OPPT S 890.1250 , OPPT S 890.1300 , OPPT S 890.145 0 and OPPTS 890.150 0 EDS P Tie

r I Assay s Compound : Triflurali n Thi s repor

t doe s no t mee t th e definitio n o f

aGLP study a s i t

appear s in : Unite d State s Environmenta l Protectio n Agenc y Titl e 4 0 Code o f

Federa l Regulation s Par

t 16 0 Federa l Register , 1 7 Augus t

198 9 Organizatio n fo r

Economi c Cooperatio n an d Developmen t ENV/LMC/CHEM(98)17 , Pari s - January 26,199 8 

# NON-GL P STUD Y

Jun e 22,201 0 Sponsor Do w AgroScience s LLC

I f 

### vj Jun e 22,201 0 Submitter Do w AgroScience s LLC

Sea n C . Gehen , Ph.D . Jun e 22,201 0 Study Director/Autho r Do w AgroScience s LL C

### Do w AgroScience s LL C Report ID : 10176 2 Pag e 4 o f 5 5

QUALIT Y ASSURANC E STATEMEN T Compound : Triflurali n Title : Trifluralin : Scientifi c Rational e fo r Citatio n o f

Othe r Scientificall y Relevan t

Informatio n (OSRI ) in Fulfillmen t

o f OPPT S 890.1150 , OPPT S 890.1200 , OPPT S 890.1250 , OPPT S 890.1300 , OPPTS 890.145 0 an

d OPPTS 890.150

0 EDS P Tie

r I Assay s Repor t Completio n Date : Jun e 22,201 0 NON-GLP STUD YDo w AgroScience s LL C Repor t ID : 10176

2 Pag e 5 o f 5 5


### Tabl e o f Content s 1. 0 Executiv e Summar

y 6 1. 1 Overvie w o f

Triflurali n OSR

I 6 1. 2 Summar

y o f Triflurali n OSRI Rational e 1 1 1.2. 1 OPPT S 890.1150 : Androge n Recepto r Bindin g 1 2 1.2. 2 OPPT S 890.1200 : Aromatas e 1 2 1.2. 3 OPPT S 890.1250 : Estrogen Receptor Bindin g Assa

y 1 3 1.2. 4 OPPT S 890.1300 : Estroge n Recepto r Transcriptiona

l Activatio n 1 3 1.2. 5 OPPT S 890.1450 : Puberta l Developmen

t and Thyroi d Functio n in Intac t Juvenile/Peripuberta

l Femal e Rat s 1 3 1.2. 6 OPPT S 890.1500 : Puberta l Developmen

t and Thyroi d Functio n in Intac t Juvenile/Peripuberta

l Mal e Rat s 1 3 1. 3 Overvie w of

triflurali n OSRI dat a1 4 1. 4 Conclusio n 1 4 2. 0 Introductio n 1 5 3. 0 Indicator s o f

Endocrin e Modulatio n in Regulator y Mammalia

n Toxicologica

l Studie s 1 7 4. 0 Rational e fo r

th e us e o f

Othe r Scientificall y Relevant

Informatio n (OSRI ) i n fulfilmen t o f OPPT S EDS P dat arequirement s 1 9 4. 1 Androge n Receptor

Bindin g (OPPT S 890.1150 ) 1 9 4. 2 Aromatas e (OPPT S 890.1200 ) 2 2 4. 3 Estrogen Receptor

Bindin g (OPPT S 890.1250 ) 2 3 4. 4 Estrogen Recepto

r Transcriptiona

l Activatio n (OPPT S 890.1300 ) 2 8 4. 5 Puberta

l Developmen

t an d Thyroi d Functio n i n Intac t Juvenile/Peripuberta

l Female Rat s (OPPT S 890.1450) 2 9 4. 6 Puberta

l Developmen

t an d Thyroi d Functio n i n Intac t Juvenile/Peripuberta

l Mal e Rat s (OPPT S 890.1500 ) 3 3 5. 0 Discussio n o f

Triflurali n OSR

I dat a3 7 5. 1 Evaluatio n o f

Potentia l Interaction s wit

h th e Estrogen an d Androge n System s i n mammals... . 3 7 5. 2 Evaluatio n o f

Potentia l Triflurali n Interaction s with th e Aromatas e Enzyme System4 0 5. 3 Evaluatio n o f

Potentia l Triflurali n Effect s o n th e Thyroi d 4 0 6. 0 Summar

y an d Conclusion s 4 2 7. 0 References : 4 3 8. 0 Appendi x A-Triflurali n Literatur e Searc h Strateg y 4 5 9. 0 Appendi x B

- Summar y o f Triflurali n Reproductio n Toxicit y Studie s 4 6 10. 0 Appendi x C

- ToxCas t Progra m4 8

### Do w AgroScience s LL C Repor t ID : 10176 2 Pag e 6 o f 5 5 

## 1. 0 Executive Summar

y Thi s document

i s bein g submitte d to EPA i n orde r to addres s th e requirement s under Tie r I o f th e Endocrin e Disruptio n Screenin g Progra m(EDSP ) fo r trifluralin . Studie s conducte d o n triflurali n i n fulfilmen t o f pesticid e registratio n requirement s a s wel l a s studie s publishe d i n th e pee r reviewe d literatur e (see Appendi x A fo r searc h strategy ) wer e carefull y assesse d t o determin e whethe r other scientificall y relevan t

informatio n (OSRI ) comparabl e t o that i n EPA' s Tie

r I batter y i s availabl e fo r th e purpos e o f

assessing th e endocrine-modulatin g potentia l o f trifluralin . Th e triflurali n EDS P consortiu mi s citin g Other Scientificall y Relevan t

Informatio n (OSRI ) t o fulfi l si x o f thes e requirement s becaus e the critica l endpoint s (a s define d i n the OPPT S 890.1150 , OPPT S 890.1200 , OPPT S 890.1250 , OPPTS 890.1300 , OPPT S 890.145 0 an d OPPT S 890.150

0 guidelines ) an

d th e associate d endocrine modes-of-actio n hav e been evaluate d i n on e or mor e o f severa l ways :

• Th e endpoint

ha s bee n directl y assessed i n existin g regulator y mammalian toxicit

y studies ; • Th e endpoint

ha s been directl y assessed i n existin g peer-reviewe d publishe d literature ; • Th e endpoint

ha s bee n directly assesse d a s part o f EPA' s ToxCas t

program ; • Th e endocrine mode-of-actio n i n questio n ha s bee n evaluate d i n existin g higher-tiere d studie s perhap s usin g different , bu t functionall y equivalent

methods ; In addition , asub-set

o f tie r I EDS P assay s wil l b e conducte d to provid e complementar y dat ao n th e endocrin e end-point

o r mode-of-actio n addressed b y th e variou s screening studies . 1. 1 Overview o

f Triflurali n OSR I Tabl e 1.1. 1 an d 1.1. 2 lis t

th e OSRI bein g cite d b y th e triflurali n EDS P consortiumto fulfi l specifi c EDS P Tie r I dat arequirements . Existin g studie s cite d a s OSR I hav e bee n carefull y reviewe d an d categorize d for

their utilit y t o th e EDS P guidelin e studies . Th e categorie s utilize d are :

• Substantiall y similar : som e methodologica

l difference s ma

y exist , bu t

stud y desig n an d endpoint s ar

e identica l o r ver y simila r to th e correspondin g EDS P guideline .

• Functionall y equivalent : Whil e significant methodologica

l difference s ma

y exist , stud y adequatel y addresse s th e mai n purpose(s ) o f th e correspondin g EDS P guideline .

• Functionall y equivalent higher-tiere d Study : Stud

y ma y o r ma y no t directly asses s EDS P guidelin e endpoints , bu t

measure s apica

l o r higher-tiere d functional endpoint s that are sensitiv e to th e endocrine mode-of-actio n bein g assessed b y th e correspondin g EDS P guideline .

• Supplemental : Stud y i s usefu l fo r weight o f evidenc e evaluations , bu t

insufficien t

alone t o fulfi l dat arequiremen t

du e t o significant

methodologica

l difference s o r

lowe r dat areliability .

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 7 o f 5 5

Tabl e 1.1. 1 Existing Pesticid e Registratio n Studie s (OSRI ) Cited i n fulfilmen t

o f Tie r I dat arequirement s 

### OSR I Citatio n
Adam s an d Marham , 199 6

Hoyt , J.A. , 198 6 

### Full OSR I Referenc e Adams , E. ; Markham , J . (1996 ) A One

- Yea

r Two-Generation Reproductio n Study i n C D Rat s Maintaine d o n Diet s Containin g Trifluralin : Supplement

# 1 to MRIDNo . 00162543 : La

b Projec t Numbe r R06384:R13984-S1 : R13984 . Unpublishe d stud y prepare d b y Lill y Researc h Labs . 1 6 p Hoyt , J . (1986 ) A One-yea r Two

generatio n Reproductio n Stud y i n C D Rat s Maintaine d o n Diet s Containin g Triflurali n (EL

- 152 , Compoun d 36352) : Studie s R06384 and R13984 . Unpublishe d stud y prepare d b y Lill y Researc h Laboratories . 77 8 p . 

### Study Typ e
Statistica l reanalysi s o f select endpoint s i n Hoyt , 1986.

Pesticid e regulator

y toxicolog y stud y 

### GLP Study

Ye s

Ye s 

### MRID Numbe r



### an d EPA Classificatio n 4413510 7 EP A Classification : Acceptabl e

0016254 3 EPA Classification : Acceptable/Guidelin e

### Do w AgroScience s LLC Report!) : 10176

2 Pag e 8 o f 5 5 OSR I Citatio n
Becker , H . 198

4

Rubin , Y. , 1986'/198 7

Worth , 1966 

### Ful l OSRI

Referenc e Becker , H . (1984 ) Multipl e Generatio n Stud y i n th e Rat : Triflurali n Substanc e Technica l Grad e (Code : HO E 3847 4 O H AT 2 10) : Projec t No . 008875 . Unpublishe d stud y prepare d b y Researc h & Consultin g Co. , AG . 604 p . Rubin , Y. , Gal , N. , Nyska , A . an d Waner , T . (1987) : Triflure x two-generatio n reproductio n study i n th e rat . Aga

n Chemica l Manufacturers , unpublishe d repor

t No . AGN/125/TR

I ( 9 Augus t 1987) . Worth , HM.eta L 1966 . Effec t

o f Triflurali n Treatment o n Reproductio n i n Rat s an d Dogs . Unpublishe d study prepare d b y Lill y Researc h Laboratories . 

### Stud

y Typ e
Pesticide regulatory toxicolog y stud y

Pesticid e regulator y toxicolog y study

Pesticid e regulator

y toxicolog y study 

### GLP Stud y
Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t

therefor e kno w whethe r

th e stud y wa s conducte d i n accordanc e wit h th e GLP s

Ye s

No , study conducte d prio r to GLP regulation s 

### MRI D Numbe r

an d EP A 

### Classification 0015190 1 EPA Classification : Acceptable/Guidelin e

4040500 7 EPA Classification : Acceptable/Guidelin e

0008364 6 EP A Classification : Supplementar

y



### Range-findin g stud y fo r

198 7 Rubi n study .

### Do w AgroScience s LL C Repor t ID : 101762 Pag e 9 o f 5 5 Tabl e 1.1. 2 Existing 

### OSR I Citatio n Morinag aetal. , 200

4

iudsonetai , 201 0

Kojimaera/.,200 4

Ortone/a/. , 200

9

Jiaoe/a/.,200 8 Literatur e Studie s (OSRI

) Cited i n fulfilmen t

o f Tie r I dat arequirement s 

### Ful l OSR I Referenc e Morinaga , Hidetaka ; Yanase , Toshihiko ; Nomura , Masatoshi ; Okabe , Taijiro ; Goto , Kiminobu ; Harada , Nobuhiro ; Nawata , Hajime . A benzimidazol e fungicide , benomyl , an dit s metabolite , carbendazim , induc e aromatas e activity i n ahuma n ovarian granulosa-like tumo r cel l lin e (KGN) . Endocrinolog y (2004) , 145(4) , 1860-1869 Judso n RS , Houc k

KA , Kavloc k RJ , Knudse n TB , Marti n MT , et

al . 200 9 In Vitro Screenin g o f Environmental Chemical s fo r Targete dTestin g Prioritization : The ToxCas t Project . Enviro n Healt

h Perspectll8(4) : Kojima , Hiroyuki ; Katsura , Eiji ; Takeuchi , Shinji ; Niiyama , Kazuhito ; Kobayashi , Kunihiko . Screenin g fo r

estrogen an d androgen 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 ovar y cells . Environmental Healt

h Perspective s (2004) , 112(5) , 524-531 . Orton , France s [Reprint Author] ; Lutz , Ilka ; Kloas , Werner , Routledge , Edwi n J . Endocrin e Disruptin g Effect s o f Herbicide s an d Pentachlorophenol : I n Vitr o an di n Viv o Evidence . Environmental Scienc e & Technology , (MA

R1 5 2009 ) Vol . 43 , No . 6 , pp . 2 144

- 2150 . Jiao , Baowei ; Yeung , Eri c K . C ; Chan , Ch i Bun ; Cheng , Christophe r H . K . Establishment

o f atransgeni c yeas t screening syste

mfo r estrogenicit y an d identificatio n o f

th e anti

estrogeni c activity o f malachite green . Journal o f Cellular Biochemistr

y (2008) , 105(6) , 1399-1409 . 

### Study Typ e Pee r Reviewe d Literatur e

Pee r Reviewe d Literature / ToxCas t Projec t

Pee r Reviewe d Literatur e

Pee r Reviewe d Literatur e

Pee r Reviewe d Literatur e 

### GLP Stud y Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t therefor e kno w whethe r

th e stud y wa s conducte d i n accordanc e wit h th e GLPs .

Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t therefor e kno w whethe r

th e stud y wa s conducte d i n accordanc e wit h th e GLPs . Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t therefor e kno w whethe r

th e stud y wa s conducted i n accordanc e wit h th e GLPs .

Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t therefor e kno w whethe r

th e stud y wa s conducte d i n accordanc e wit h th e GLP s

Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t therefor e kno w whethe r

th e stud y wa s conducte d i n accordanc e wit h th e GLP s 

### MRI D Numbe r

N/A

N/A

N/A

N/A

N/A

### Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 1 0 o f 5 5 OSR I Citatio n Jun g e l al , 200

4

Jungbaue r an d Beck , 200 2

Nishihanma/.,200 0

Ortonetal. , 2009

Petite / al. , 199 7 

### Ful l OSR

I Referenc e Jung , Joohee ; Ishida , Kunie ; Nishihara , Tsutomu . Anti

estrogeni c activit y o f

fift y chemical s evaluate d b y i n vitr o assays . Lif

e Science s (2004) , 74(25) , 3065-307 4 . Jungbauer , Alois ; Beck , Verena . Yeas t

reporter syste m fo r rapi d determinatio n o f estrogeni c activity . Journa l o f Chromatography , B : Analytica l Technologie s i n th e Biomedica

l an d Lif e Science s (2002) , 777( 1 -2) , 167-178 . Nishihara , Tsutomu ; Nishikawa , Junichi ; Kanayama , Tomohiko ; Dakeyama , Fumi ; Saito , Koichi ; Imagawa , Masayoshi ; Takatori , Satoshi ; Kitagawa , Yoko ; Hori , Shinjiro ; Utsumi , Hideo . Estrogeni c activitie s o f 51 7 chemical s b y yeas t

two hybri d assay . Journa l o f Healt h Scienc e (2000) , 46(4) , 282-298 . Orton , France s [Reprin t Author] ; Lutz , Ilka ; Kloas , Werner ; Routledge , Edwi n J . Endocrin e Disruptin g Effect s o f Herbicide s an d Pentachlorophenol : I n Vitr o an d i n Viv o Evidence . Environmental Scienc e & Technology , (MA R1 5 2009 ) Vol . 43 , No . 6 , pp . 2 144

- 2150 . Petit , F. ; L e Goff , P. ; Cravedi , J.-P. ; Valotaire , Y. ; Pakdel , F . Tw o complementar

y bioassay s fo r

screenin g th e estrogeni c potenc y o f xenobiotics : recombinant yeas t fo r trou t estroge n recepto r an d trou t hepatocyte cultures . Journa

l o f Molecula r Endocrinolog y (1997) , 19(3) , 321-335 . 

### Study Typ e Pee r Reviewe d Literatur e

Pee r Reviewe d Literatur e

Pee r Reviewe d Literatur e

Pee r Reviewe d Literature

Pee r Reviewe d Literature 

### GLP Stud y Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t therefor e kno w whethe r

th e stud y wa s conducte d i n accordanc e wit h th e GLP s Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t therefor e kno w whethe r

th e stud y wa s conducte d i n accordanc e wit h th e GLP s Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t therefor e kno w whethe r

th e stud y wa s conducte d i n accordanc e wit h th e GLP s

Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t therefor e kno w whethe r

th e stud y wa s conducte d i n accordanc e wit h th e GLP s

Triflurali n consortiu m di d no t sponso r o r conduc t the stud y and d o no t therefor e kno w whethe r

th e stud y wa s conducted i n accordanc e wit h th e GLP s 

### MRI D Numbe r

N/A

N/ A

N/A

N/A

N/A

### Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 1 1 o f 5 5 OSR I Citatio n Saghir etal. , 200 8

Sotoe/a/. , 199 5 

### Ful l OSR I Referenc e Saghir , Shaki l A . (correspondence) ; Charles , Grantle y D. ; Bartels , Michae l J. ; Kan , Lyn n H.L. ; Dryzga , Mar k D. ; Break , Kath y A. ; Clark , Am y J . Mechanis mo f trifluralin-induce d thyroi d tumor s i n rats . Toxicolog y Letters , (3 0 Ju l 2008 ) Vol . 1 80 , No . 1 , pp . 38-45 . Soto , A.M . et

al . The E

SCREE N Assa y a s aToo l t o Identif y Estrogens : A n Updat e o n Estrogeni c Environmental Pollutant s 

### Study Typ e Pee r Reviewe d Literatur e

Pee r Reviewe d Literature 

### GLP Stud y Ye s

Triflurali n consortiu m di d no t sponso r o r conduc t th e stud y an d d o no t therefor e kno w whethe r

th e stud y wa s conducte d i n accordanc e wit h th e GLP s 

### MRID Numbe r

N/ A

N/ A



### 1. 2 Summar

y o f Triflurali n OSR I Rational e Th e state d purpos e o f

th e Endocrine Disrupter

Screening Progra

mi s ". . .t o determin e whethe r

certai n substance s ma

y hav e a n effec t i n human s that

i s simila r to a n effec t produce d b y anaturall y occurrin g estrogen , o r

suc h other

endocrin e effec t

a s th e Administrato r

ma y designate. " Th e state d purpos e o f Tie r I testin g ". . .i s to identif

y substance s tha t

hav e th e potentia

l to interac t

with th e estrogen , androgen , o r

thyroi d hormon e system s usin g abatter y o f assays. " Th e triflurali n EDS P consortiu mbelieve s that conductin g asub-set

o f th e Tie r I batter y o f screening studie s wil l provid e complementar y dat aan d sufficien t

scientifi c redundanc y t o assur e that an y potential endocrine-relate d effect s o f triflurali n woul d b e clearly identified . Tabl e 1.2. 1 identifie s thos e assay s fo r whic h th e triflurali n consortiumi s submittin g OSR I an d thos e that

w e wil l conduct . Th e consortiumbelieve s that

th e Androgen Recepto

r bindin g (OPPTS 890.1150) , Aromatas e (OPPTS 890.1200) , Estrogen recepto r bindin g (OPPT S 890.1250) , Estrogen Recepto r Transcriptiona

l Activatio n (OPPT S 890.1300) an d Mal e an d Femal e Puberta l assay s (OPPT S 890.145 0 & OPPT S 890.1500 ) ar e no t require d to mak

e thes e determination s sinc e existin g dat afro mmammalia n regulator y toxicit y studie s an d publishe d literature studie s cite d a s OSR I i n combinatio n with th e dat a fro mothe r Tie r I studie s make thes e assay s redundan t an d unjustifie d fro mth e standpoint o f anima l use . Consequently , consisten t wit h EPA' s mandate to avoi d duplicativ e testing , thes e studie s shoul d no t b e conducte d an d shoul d b e considere d fulfille dfo r th e purpose s o f

th e EDS P Tie

r I dat a requirements . W e respectfull y as k

that EPA carefull y examin e th e OSR I cite d i n thi s rational e an d reques t tha t base d o n it s evaluatio n o f

th e OSR I an d th e othe

r EDS P assay s to b e conducted , th e Agenc y conside r th e requirement

fo r triflurali n fo r

conductin g th e abov e mentione d EDS P Tie r I screening studie s to hav e been met .Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 1 2 o

f 5 5

Table 1.2.1 : EDS P Tie

r I batter y and requeste d EP

A Action s 

### Endocrin e Disrupte r

Screenin g Progra

mTie r 1 Test Guidelin e OPPTS 890. 1 100 : Amphibia n Metamorphosi s OPPT S 890. 1 150 : Androge n Receptor Bindin g OPPT S 890.1200 : Aromatas e OPPT S 890.1250 : Estrogen Recepto r Bindin g Assa

y OPPT S 890.1300 : Estroge n Receptor Transcriptional Activatio n OPPT S 890.1350 : Fis h Short-Ter mReproductio n Assa y OPPT S 890.1400 : Hershberge r Bioassa y OPPT S 890.1450 : Puberta

l Development

an d Thyroi d Functio n i n Intac t

Juvenile/Peripubertal Femal e Rat s OPPTS 890.1500 : Puberta

l Developmen

t and Thyroi d Functio n i n Intac t

Juvenile/Peripubertal Mal e Rat s OPPT S 890.1550 : Steroidogenesi s OPPT S 890.1600 : Uterotrophi c Assa y 

### Triflurali n EDS P ConsortiumProposa l Th e triflurali n consortiu mwil l conduc t thi s stud y Th e triflurali n consortiu mi s submittin g OSR I t o fulfi l thi s dat arequiremen t Th e triflurali n consortiu mi s submittin g OSR I t o fulfi l thi s dat arequiremen t Th e triflurali n consortiu mi s submittin g OSR I t o fulfi l thi s dat arequiremen t Th e triflurali n consortiu mi s submittin g OSR I t o fulfi l thi s dat arequiremen t Th e triflurali n consortiu mwil l conduc t thi s study Th e triflurali n consortiu mwil l conduc t thi s study Th e triflurali n consortiu mi s submittin g OSR I t o fulfi l thi s dat arequiremen t Th e triflurali n consortiu mi s submittin g OSR I t o fulfi l thi s dat arequiremen t Th e triflurali n consortiu mwil l conduc t thi s study Th e triflurali n consortiu mwil l conduc t thi s study 

### 1.2. 1 OPPT S 890.1150 : Androge n Receptor Bindin g Th e triflurali n consortiu mi s citin g th e followin g OSR I an d requestin g tha

t th e requiremen t

t o conduc t

an androgen receptor

bindin g assa

y (OPPT S 890.1150

) b e considere d fulfille dbase d o n th e sufficienc y o f thes e existin g studies : 1 . Judso n et

aL , 2010 : multipl e studie s conducte d that ar e substantiall y similar , functionall y equivalent . OLSupplemental t

o OPPT S 890.1150 . 2 . Kojim aet

a/. , 2004 : Study provide s functionall y equivalen t informatio n to OPPTS 890.1150 . 3 . Orto n e

t aL , 2009 : Stud

y provide s supplementa

l informatio n t

o OPPTS 890.1150 . 

### 1.2. 2 OPPT S 890.1200 : Aromatas e Th e triflurali n consortiu mi s citin g th e followin g OSR I an d requestin g tha

t th e requiremen t

t o conduc t

an aromatas e assa

y (OPPTS 890.1200) b e considere d fulfille dbased o n th e sufficienc y o f thes e existin g studies : 1 . Moring aet

al. , 2004 : Study provide s informatio n that i s substantiall y simila

r t o OPPTS 890.1200 . 2 . Judso n et

aL , 2010 : Stud

y provide s informatio n that i s substantiall y simila

r t o OPPT S 890.1200 .Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 1 3 of

5 5 

### 1.2. 3 OPPT S 890.1250 : Estroge n Recepto r Bindin g Assa y Th e triflurali n consortiu mi s citin g th e followin g OSR I an d requestin g tha

t th e requiremen t

t o conduc t

an estroge n recepto r

bindin g assa

y (OPPT S 890.1250

) b e considere d fulfille dbase d o n th e sufficienc y o f thes e existin g studies : 1 . Judso n e t

aL , 2010 : multipl e studie s conducte d tha t ar e substantiall y similar , functionall y equivalent . o r supplementa

l t o OPPT S 890.1250 . 2 . Kojim aet

al. , 2004 : Stud

y provide s functionall y equivalen t informatio n t

o OPPT S 890.1250 . 3 . Jiao e t aL , 2008 , Jun g e

t al , 2004 , Jungbaue r

an d beck. , 2002 , Nishihar ae t al , 2000 , Peti t

e t al , 1997 , Soto , e

t al. , 1995 , Orto n e

t aL , 2009 : Eac h study provide s informatio n tha t i s supplemental t

o OPPT S 890.1250 . 

### 1.2. 4 OPPT S 890.1300 : Estroge n Receptor Transcriptiona

l Activatio n Th e triflurali n consortiu mi s citin g th e followin g OSR I an d requestin g that

th e requirement

t o conduc t

a n estroge n receptor

transcriptional activatio n assa y (OPPT S 890.1300

) b e considere d fulfille dbased o n th e sufficienc y o f

thes e existin g studies : 1 . Judso n et

aL , 2010 : multipl e studie s conducte d that ar e either substantiall y simila

r t o or supplemental t o OPPT S 890.1250 . 2 . Kojim aet

al. , 2004 : Stud y provide s substantiall y simila r t o informatio n t

o OPPT S 890.1250 . 3 . Jiao et

aL , 2008 , Jun g et

aL , 2004 , Jungbaue r

an d beck. , 2002 , Nishihar aet al , 2000 , Peti t

et al , 1997, Soto , e

t al. , 1995, Orto n e

t al , 2009 : Eac h stud

y provide s informatio n tha

t i s supplementa

l t o OPPT S 890.1250 . 

### 1.2. 5 OPPT S 890.1450 : Pubertal Development an d Thyroi d Functio n i n Intac t Juvenile/Peripuberta

l Femal e Rat s Th e triflurali n consortiu mi s citin g th e followin g OSR I an d requestin g that

th e requiremen t

t o conduc t

Femal e Pubertal assa

y (OPPT S 890.1450

) b e considere d fulfille dbase d o n th e sufficienc y o f thes e existin g studies : 1 . Hoyt , J.A . 1986

, Becker , H . 1984

, Rubin , Y . e

t a l 1987 , an

d Wort h e t a l 1966 : Thes e ar

e higher ; tiere d reproductio n studie s tha t provid e functionall y equivalen t an d supplementa

l informatio n t o OPPT S 890.1450 . 2 . Saghir , e

t a l 2008 : Thi s i s asupplementa l mode-of-actio n stud

y tha t assesse d the effect s o f

triflurali n o n th e thyroi d 

### 1.2. 6 OPPT S 890.1500 : Pubertal Development an d Thyroi d Functio n i n Intac t Juvenile/Peripuberta

l Mal e Rat s Th e triflurali n consortiu mi s citin g th e followin g OSR I an d requestin g tha t

th e requiremen t

t o conduc t

Mal e Pubertal assa

y (OPPT S 890.1500

) b e considere d fulfille dbase d o n th e sufficienc y o f thes e existin g studies :Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 1 4 o f 5 5 3 . Hoyt , J.A . 1986

, Becker , H . 1984

, Rubin , Y . e

t a l 1987 , an

d Wort h e t a l 1966 : Thes e ar

e higher ; tiere d reproductio n studie s tha t provide functionall y equivalen t an d supplementa

l informatio n to OPPT S 890.1500 . 4 . Saghir , et

a L 2008 : Thi s i s asupplemental mode-of-actio n study that assessed th e effect s o f

triflurali n o n th e thyroi d

1. 3 Overview o

f triflurali n OSR I data Examinatio n o f

th e existin g triflurali n data-set doe s no t revea l concer n fo r specifi c modulatio n o f

th e estrogen , androgen , or

aromatas e assa

y systems . Base d o n weigh t o f evidenc e evaluatio n o f

bot h higher-tiere d anima

l studie s a s wel l a s mode-of-actio n literatur e studies , the followin g conclusion s ca n b e drawn :

• Triflurali n doe s no t

demonstrat

e th e capacit

y to bin d to th e androge n receptor . • Triflurali n doe s no t

demonstrate th e capacit

y to bin d to th e estrogen recepto r no r doe s i t appea r t o demonstrat

e estrogeni c or

anti-estrogeni c activit

y • Triflurali n doe s no t

demonstrat

e th e capacit

y to alte r aromatas e activity . • Th e effect s o f

triflurali n o n th e thyroi d hav e bee n wel l characterize d i n existin g studies ; th e principl e effec t

o n th e thyroi d observe d i n ra t studie s i s no t

considered to b e relevan t to humans .

1.4 Conclusio n Th e conclusio n fro mthi s process , afte r carefu l consideratio n o f

th e weight

o f th e evidenc e provide d b y existin g toxicit

y studie s an d endpoint s availabl e fo r triflurali n (i.e. , OSRI) , combine d wit h th e EDS P studie s that

wil l b e conducted , i s that th e AR-binding . aromatase . ER-binding . ER

transcriptiona

l activation , an d femal e an d mal e pubertal assay s are redundant whe n compare d with existin g relevant

information , an d are , therefore no t justifie d fro ma

n anima l us e o r scientifi c need s perspective . In grantin g a n exemptio n fro mperformin g thes e studies . EPA wil l preven t th e unnecessar

y us e o f a t leas t 9 0 animals . Furthermore , th e OSRI cite d suppor t tha t ther e i s n o specifi c concer n fo r estrogenic/anti-estrogeni c o r androgenic/anti-androgeni c effect s o f trifluralin . Th e effect s o f triflurali n o n th e thyroi d hav e been wel l characterize d an d additiona l testin g i n rat s i s no t

needed .

### Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 1 5 o f 5 5 

### 2. 0 Introductio n Th e EPA document

entitled : "EPA' s Approac h fo r Considering Other

Scientifically Relevant Informatio n (OSRI) unde r

th e Endocrin e Disrupter

Screenin g Program " (EPA , Marc h 2009 ) provide s guidanc e to registrant s o n ho w existin g dat ao r ". . .othe r scientifically relevant

informatio n " (OSRI ) are to b e considere d i n ful l o r partial fulfilmen t

o f Tie r IEDS P dat arequirements . OSRI i s define d as". . .informatio n tha t inform s th e determinatio n a s to whether th e substanc e ma

y hav e a n effec t that i s simila r to a n effec t produce d b y asubstanc e that interact s wit

h th e estrogen , androgen , and/o r thyroi d hormonal system s (USEPA , 2009). " Accordin g to th e EPA' s OSR I guidanc e document , th e followin g type s o f informatio n ma

y b e usefu l a s OSRI : studie s that

correspon d to th e assay s i n Tie r 1 or Tie r 2 , metabolis mstudies , an d othe r vertebrate o r

invertebrat

e toxicit y studies , including , bu t

no t limite d to , developmenta

l toxicit y tests , carcinogenicit

y tests , toxicogenomi c data , an d reproductiv e toxicit y tests . In additio n to th e type s o f

dat anote d previousl y a s relevant OSR I information , th e OSR

I guidanc e document give s specifi c mentio n to toxicit y studie s conducte d i n fulfilmen t o f th e dat arequirement s fo r pesticid e registratio n (4 0 CF

R Par t 158) . Evaluatio n o f

thes e dat ain addressing Tie r I dat a requirement s i s asignifican t priorit y sinc e thes e dat adelineat

e abroad rang e o f

robus t toxicit y studie s that mus t b e complete d an d critically assessed b y th e EPA prior to registratio n o f

apesticid e activ e ingredient i n th e Unite d States . Amon g th e require d data-se t ar e multipl e repeat-dos e toxicit y studie s o f variou s duration , fro m4-1 3 week s to exposure s encompassin g th e majority o f thes e organism' s lifespan . In addition , studie s ar

e conducted under

4 0 CF RPar t 15 8 to determin e any potentia l for treatment relate d effect s o n reproductio n a s wel l a s pre - an d post-nata l development . The dose s use d i n all o f thes e studie s appropriatel y challeng e th e tes t organism s an d th e dose s employe d ar e muc h higher

than anticipate d human exposur e levels . Examinatio n o f

thi s complet

e data-set , whic h i s alread

y availabl e for

al l pesticid

e activ e ingredients , allow s fo r determinatio n o f

target orga n toxicit y an d an y othe

r advers e effect s that

ma y b e elicite d b y exposure . Thes e existin g studie s ar e capabl e o f

detectin g advers e biological effect s o f treatment that coul d b e elicite d b y on e o r mor e modes-of-action , includin g th e endocrine modes-of-actio n bein g addresse d b y th e EDSP . Potentia l advers e effect s resultin g fro mmodulatio n o f th e endocrine system hav e already bee n intrinsicall y evaluate d i n th e exitin g registratio n toxicit y studies . Base d o n th e no effec t level s determine d b y thes e studies , applicatio n o f th e principle s o f

ris k assessmen

t allow s fo r appropriate margin s o f

exposure i n th e us e o f

thes e activ e ingredients . Bot h i n term s o f

goo d scientifi c principle s an d particularl y i n light o f th e scientifi c community' s responsibilit

y fo r judiciou s us e o f

anima l resources , i t

i s appropriate to conside

r existin g dat ai n th e contex t o f the EDS P requirements , an d EP A should grant

exemption s fro mscreening wher e existin g dat aalread

y provid e adequate information . I n fact , accordin g to Federa l Food , Dru g an d Cosmeti c Ac t (FFDC A section 408(p ) (5 ) (B)) , EP A i s oblige d t

o ". . ..minimiz e duplicativ e testin g o f the sam eDo w AgroScience s LL C Repor t ID : 10176

2 Pag e 1 6 o f 5 5 substanc e fo r

th e sam e endocrin e effect. " Thi s polic y i s echoed b y th e Offic e o f Management

an d Budget' s respons e to EPA' s EDS P Informatio n Collectio n Respons e (ICR ) whic h direct s th e Agenc y to ". . .promot

e an d encourag e tes t

order recipient s to submi t

Othe r Scientifically Relevan t Informatio n (OSRI

) in lie u o f performin g all o

r som e o f the Tie r I assays , and EP

A shoul d accep

t OSR I a s sufficien t

to satisf y th e tes t order s to th e greates t

extent possible. " A s with othe

r pesticide activ e ingredients , th e 4 0 CF R Par t 15 8 require d studie s hav e been conducte d fo r trifluralin . Whil e th e entk e dat ase t i s som e what larger , th e studie s considere d mos t relevan t fo r assessing any potentia

l for interactio n wit h th e endocrine syste mar e foun d i n Table s 1.1. 1 an d 1.1. 2 an d includ e severa

l regulator

y multi-generatio n reproductio n studie s a s wel l a s anumbe r o f studie s availabl e i n the publishe d literature . The regulator y studie s wer

e conducte d i n accordanc e wit h internationall y accepted tes t guideline s i n plac e a

t th e time , an d th e mor

e recen t studie s adhere d to GLP standards . Th e studie s utilize d rat s and , therefore , ar e considere d th e mos t

relevant to th e mammalian Tie

r I dat arequirements . Wher e appropriate , dat ai n othe r specie s ma

y als o b e cite d whe n considere d relevant . Th e multi-generatio n reproductio n studie s provid e th e foundational data-set fo r triflurali n relativ e to th e i n viv o Tier I EDS P assays . Taken together , thi s suit e o f studie s involvin g numerou s endpoint s o f

interest , an d wit

h th e additiona

l strengt h afforde d throug h direc t

analysi s o f effect s o n potentia

l endocrine target

organ s an d inclusio n o f

clinica l chemistr y an d histopathology , provide s th e basi s fo r

arobus t determinatio n o f

any potentia

l effec t o n genera

l toxicity , developmen

t an d maturation , a s wel l a s other endpoint s that

coul d signif

y a n interactio n o r

effec t o n th e endocrin e system . I n summary , fo r

pesticide activ e ingredient s ther e i s awealt

h o f existin g informatio n availabl e tha t ca n reduc e o r eliminat e th e nee d fo r

Tie r I screening . I n particular , th e multi-generatio n reproductio n study alread

y availabl e fo r

pesticid e activ e ingredient s i s ahigher-tiere d assessmen t o f potentia l endocrine-mediate d toxicity , an d EPA i s i n fac t plannin g to us e i t

a s th e mammalian Tie

r n testin g requirement . Logi c therefore dictate s that havin g th e higher-tiere d study complete d lessen s o r eliminate s th e nee d fo r

supplyin g lower-tiere d screening assay s o f ales s robus t

nature . Wit

h higher

tier dat aavailable , th e practica l utilit y o f performin g screenin g studie s i s highl y questionable . I n addition , EPA ha s alread

y evaluate d thes e existin g studie s fo r pesticid e activ e ingredients , includin g any potentia

l advers e effect s resultin g fro mmodulatio n o f th e endocrine system , an d th e Agenc y ha s already mad e adeterminatio n that thes e activ e ingredient s ca n b e use d withou t

undu e ris k

t o human health .Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 1 7 o f 5 5 

### 3. 0 Indicator

s o f Endocrin e Modulatio n i n Regulator y Mammalia n Toxicologica l Studie s Table s 3.1,3.2 , an d 3. 3 belo w provid e example s o f end-point s commonly assessed i n 4 0 CF R 15 8 guidelin e studie s tha

t ca n serv e a s surrogate s fo r

other endpoint s that

asses s estrogenicity , anti

estrogenicity , an d anti-androgenicit y (androgenicit

y no t include d a s xenobioti c androgen agonist s ar e rare) . Thes e table s presen t aclea r an d predictabl e patter

n o f advers e biologica

l outcome s followin g exposur e to chemical s actin g throug h estrogenic , anti-estrogeni c o r anti-androgeni c modes-of-action . I f apatter n consisten

t with th e effect s presente d i n thes e table s i s no t observed , the n ates t

chemica l ca n b e considere d negativ e fo r advers e effect s elicite d throug h variou s endocrine mode(s)-of-action . Therefore , individual endpoint s tha t ar e lackin g o r

even isolate d statisticall y significan t finding s ar

e o f limite d importanc e i n determinin g achemical' s potentia l to interac t

wit h th e endocrine syste

msinc e i t i s th e array o r

patter n o f effect s tha t

i s mos t usefu l i n makin g thi s determination . Tabl e 3.1 : Advers e effect s elicited b y estrogeni c substance s

Endpoin t Estrou s cyclicit

y Time to matin g Uterin e weigh t Vaginal patenc y 

### PP S Ovaria n follicl e count Fertilit y Matin g index Corporalute a Implantation s Gestationa

l lengt h Litter siz e Anogenita

l distanc e Epidydimi s sper

mcount Testi s Histopatholog y Epidydimi s histopatholog y Prostat e histopatholog y Ovary weigh t Ovary histopatholog y Mammar y histopatholog y Expecte d Chang e Decreas e frequency ; anestrou s Increas e Increas e Accelerate d Delaye d Decreas e Decreas e Decreas e Decreas e Increas e increas e Decreas e Decreas e Decreas e Atroph y Lesions , decreased sper

m content Involutio n o f

ventral prostat

e epithelium ; stroma

l proliferatio n Decreas e Stroma l hyperplasia ; follicula

r cyst s Increase d Reference s Terry , 2004 ; Abbott , 2006 ; Deli aSeta , 2008 ; Sawaki , 200 3

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 1 8 o f 5 5 Tabl e 3.2 : Advers e effect s elicite d b y anti-estrogeni c substance s 

### Endpoin t Estrou s cyclicit

y Time to matin g Testi s weigh t Vaginal patenc y Corpor alute a Litter siz e Testi s Histopatholog y Epidydimi s histopatholog y Prostat e histopatholog y Semina l Vesicl e histopatholog y Ovar y histopatholog y Expecte d Chang e Decreas e frequency ; anestrou s Increas e Decreas e Delaye d Decreas e or

absent Decreas e Leydi g cell atrophy Degenerativ e change s Degenerativ e change s Degenerativ e change s Decreas e primordia

l follicles ; hemorrhagi c follicles ; interstitial/stroma

l hyperplasi a Reference s

Vanage , 1997 ; Abbott , 2006 ;

Tabl e 3.3 : Advers e effect s elicite d by anti-androgeni c substance s 

### Endpoin t Preputia l separatio n Sper mcount , motilit

y Testi s Weigh t Vaginal patenc y Epididymi s weigh t Prostat e weight Semina l Vesicle s Weight Testi s histopatholog y Epididymi s histopatholog y Prostat e histopatholog y Semina l Vesicl e histopatholog y Anogenita l distanc e Uterin e histopatholog y Mammar y glan d Estrou s cyclicit

y Tim e t o matin g Fertilit y Ovar y histopatholog y Uterin e histopatholog y Expected Chang e Delaye d Decreas e Generall y Decreas e Delaye d Decreas e Decreas e Decreas e Carcinoma ; leydi g cell hypertrophy/hyperplasia ; germina l degeneratio n Agenesis ; spermati c depletio n Fibrosis , cellula

r infiltration ; epithelia l hyperplasia ; agenesi s Fibrosis , cellula

r infiltration ; epithelia l hyperplasi a Decreas e Cysti c endometria

l hyperplasi a Decreas e adenomas , adenocarcinoma s Persistent estru s Increas e decreas e Hypertroph y Cysti c endometria

l hyperplasi a Reference s

Gray , 2001 ; Shin , 2002 ; Lund , 2004 ; Va n De r Schoot, 1992 ; York , 2006 ; Bowman , 2005 ; Horsley , 2005 ; Rider , 2008 ; O'Connor , 2002 ;

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 1 9 o f 5 5



### 4. 0 Rational e fo rth e us e of Othe rScientificall y Relevan t Informatio n (OSRI ) i n fulfilmen t o

f OPPTS EDS P data requirement s I n preparin g thi s scientifi c rationale , th e followin g proces s wa s followed : 1 . Evaluate appropriate existin g triflurali n dat a(OSRI) , includin g primaril y guidelin e toxicit y studie s an d othe

r publishe d studies . 2 . Examin e existin g dat afo r endpoint s relevan t

to EDS P Tie

r I . 3 . Determin e whic h Tie

r I endpoint s an d assay s ar

e adequately addresse d b y OSRI . For registere d activ e ingredient s lik e trifluralin , there i s a

n abundanc e o f

availabl e informatio n that i s relevant to determinin g i f

any endocrine-mediate d effect s occu r wit h treatment . Studie s submitte d a s OSR I i n fulfilmen t

o f Tie r I requirement s shoul d b e carefull y considere d an d no t dismisse d i f

onl y on e o r afe w specifi c endpoint s requeste d b y th e Tie r I screening studie s wer

e no t evaluated . Fo r

example , TS H and T

4 measurement s ar

e require d endpoint s under OPPTS 890.145 0 and OPPT S 890.150 0 assays , but

thes e measurement s provide only on e insigh t (an d on e based o n asnapshot i n time ) o n potential thyroi d toxicity . It i s well-establishe d that

assessmen

t o f orga n weight an d histopathologica

l revie w o f th e thyroi d glan d are mor e deterministi c an d informativ e relativ e to thi s orga n bein g atarget an d potentia

l implicator

o f endocrine-relate d toxicity . In thi s specifi c case , thes e type s o f dat aar e superio r to discret e measurement s o n thyroi d hormones . Further to thi s point , i t

shoul d b e note d that i n the cas e o f

the puberta

l assays , man y o f

the endpoint s ar

e no t specifi c t o the endocrine syste

man d ca n b e altere d a s aresul t

o f genera l systemi c toxicit

y (Stoker , 2000) . For

example , endpoint s evaluate d i n th e pubertal assay s ca n b e altere d secondar

y to effect s o n body weight (Carney , 2004) . Thi s i s significant

a s th e guideline s fo r

pubertal assay s defin e reduce d bod y weigh t a s evidenc e fo r havin g achieve d a

n appropriate hig h dose-level fo r testing . Give n th e lack

o f specificit y fo r man y o f the apica l puberta l endpoints , it

i s appropriat

e t o conside r surrogat e endpoint s in plac e o f

thes e less

specifi c endpoint s whic h ma y no t hav e been examine d i n th e existin g databas e fo r agiven chemica

l (se e Table s 3.1-3.3) . Thi s approac h wil l allo w fo r arobus t weight o f evidenc e determinatio n base d upo n th e totalit

y o f th e availabl e data . Whil e al l o f thes e endpoint s ma

y no t b e o f equa l sensitivity , consisten t result s foun d acros s multipl e studie s an d end-point s wil l ofte n provid e ahig h degre e o f confidenc e i n th e conclusion withou t th e need for

conductin g additiona

l testing . 4. 1 Androge n Recepto rBindin g (OPPT S 890.1150

) Assa y Overvie w Th e androge n recepto r

(AR ) i s involve d i n th e developmen t o f mal e sexua l characteristics . Th e A RBindin g assay identifie s chemical s tha t affec t th e endocrin e syste mb y bindin g t o hormon e receptor s t o either mimi c th e actio n o f

th e natura l hormon e o r bloc k acces s o f th e hormon e t o th e site an d thu s bloc k hormon e controlle d activity .

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 20 o f

5 5 Rational e Th e triflurali n consortiu mrequest s that th e requirement

to conduc t

a n androge n recepto r

(AR ) bindin g assa

y b e considere d complet

e du e to th e availability o f

existin g dat a(OSRI ) tha t specificall y addres s thi s dat arequirement . Cell-fre e assay s conducte d a s part o f th e EPA ToxCas t progra m(Judson e

t al. , 2010 ) ar e considered t

o b e substantially simila

r t o the OPPTS 890.115 0 requirement . Cell-base d ToxCas t assay s a s wel l a s dat aprovide d b y Kojim aet al . ar e functionall y equivalent

o r supplementa

l to Tie r I AR-bindin g an d i n som

e case s provid e mor e comprehensiv e informatio n a s the y evaluate potentia l AR-mediate d gene regulation . Orto n et al . utilize d ayeast-base d gene reporte r metho d to evaluat

e ARactivity . Thes e dat aar e considere d supplementa

l onl y a s th e relevanc e an d utility o f th e yeast-base d metho d ar e no t clear . Together , thes e availabl e studie s provid e sufficien t informatio n a s requeste d b y OPPT S 890.115 0 (Tabl e 4.1) . Tabl e 4.1 : AR-bindin g OSR I OSR I Referenc e Judso n et al. , 201 0

Kojim aet al. , 200 4

Orto n e t al. , 200 9 OSR

I Typ e 1 . Substantiall y similar : Huma n an d ra

t A R competitiv e bindin g assay s (cell-fre e HTS

- Calipe

r Lif e Sciences/NovaScreen) . 2 . Functionall y equivalent : huma n reporter

gen e agonis t an d antagonis t assays , (NCGQ . 3 . Supplemental : Multiple x transcriptio n reporte

r assa y (Attagen e assays )

Functionall y eouivalent : huma n A R reporte

r gen e (luciferase ) agonis t

an d antagonis t assa y Supplemental : Study utilize d recombinan t

yeas t reporte r scree n fo r evaluatio n o f androgeni c an d anti

androgeni c activity . Deviation s fromOPPTS Guideline s Bindin g t o bot

h ra t an d huma n A R receptor s evaluate d an d sourc e o r

A R differen t

Di d no t evaluat e bindin g t o th e AR , bu t

receptor agonis m an d antagonis m Di d no t evaluat e bindin g t o th e AR , bu t

reporte r gen e regulation ; Us e o f

chimeri c protei n multiple x reporter syste mdiffer s significantl y fro mOPPTS 890 . 115

0

Di d no t evaluate bindin g t o th e AR , bu t

recepto r agonis m an d antagonis m Di d no t evaluate bindin g potential , bu t

AR-mediate d reporter gene regulation . Impac t o f deviations/comment s Evaluatio n o f

bindin g t o bot

h ra t an d huma n AR a s wel l a s evaluatio n o f AR-dependent

gene regulatio n shoul d provide acomprehensiv e scree n fo r AR-bindin g potential . Differen t A R sourc e shoul d hav e n o impac t Stud y provide s importan t

informatio n a s i t evaluate s gene regulatio n resultan t fro mpotentia

l bindin g Relevanc e o f

reporter systemi s no t clea r relativ e t o OPPTS 890 . 1 15 0 an d shoul d b e furthe r

assessed .

Stud y provide s importan t

informatio n a s i t evaluate s gen e regulatio n resultan t fro mpotentia

l binding ; study desig n i s simila r t o OPPT S 890 . 1300 . Informatio n considere d supportiv e only . Yeas t

reporte r system s wer

e no t selected for

EDS P an d ar

e o f questionabl e relevance .Do w AgroScience s LL C Repor t ID : 101762 Pag e 2 1 o f

5 5 

### .Tudso n e t al . (2010 ) Thi s stud y provide s informatio n that i s substantiall y simila

r (NovaScree n HT S assays) , functionall y equivalent

(NCG C reporte

r gen e assays) , an d supplementa l (Attagen e multiple x assays ) t o OPPT S 890 . 1150 . Thi s publicatio n provide s a n overvie w o f

the ToxCas t

program , th e purpos e o f

whic h i s to conduc t

high-throughpu t

screening an d thu s allo w fo r prioritizatio n o f testin g fo r

environmenta

l chemical s (see Appendi x C fo r mor e informatio n o n th e ToxCas t progra man d assays) . A chemica l librar y o f 32 0 substance s (includin g trifluralin ) wa s evaluate d i n 46 7 cell-fre e or cell-base d assays . O f

these , severa

l ar e relevan t to EDS P Tie

r I an d i n particula

r AR-binding . Thes e includ e cell-fre e an d cell-base d HT S assays . Th e cell

fre e assay s evaluate d bindin g to th e huma n an d ra t androgen receptor s whil e th e cell-based assay s wer

e designed t

o asses s reporte

r gen e agonis man d antagonis ma t the human AR . In addition , amutiple x transcriptio n reporter assa y systemutilizin g alibrar

y o f transcriptio n facto r bindin g site s wa s performe d t o evaluat e th e activitie s o f

endogenou s (cis-acting ) or exogenou s (trans-acting ) chimeri c transcriptio n factors . Th e specifi c assay s utilize d to asses s AR-bindin g an d A R activatio n are : Novascreen' s (Calipe r Lif e Sciences ) ra

t androge n recepto r an d human androge n receptor binding , NI H Chemica

l Genomic s Center' s (NCGC ) human AR_Agonis t

an d human AR_Antagonis t assays , an d Attagene ' s human androgen receptor multi-ple x reporte

r gen e assay . (2004 ) Thi s study i s functionall y equivalen t t o OPPT S 890. 1 150 . The stud y teste d 20 0 chemicals , includin g trifluralin , fo r

agonis man d antagonis mto hERa , hERp , an d hAR . Th e metho d utilize d Chines e hamster

ovar y cell s transientl y transfecte d with luciferas e reporter gen e construct s containin g eithe r a n estrogen-responsive-elemen t (ERE ) or

a n androgen

responsive-elemen

t (ARA) . Afte r

transfection , cell s wer

e treate d wit

h 1 0 " 8 - 1 0 " 5 M o f

each tes t compoun d o r

0.1 % DMS O (vehicl e control) . Th e stud y als o evaluate d antagonis t activit y b y treatment

wit h tes t compound s i n th e presenc e o f E 2 (10" 1 * M o r

10"'° M fo r

ER aan d ER

p respectively ) or

10" 10 M DH T for

AR antagonism . Whil e th e study doe s no t

directl y asses s AR-binding , i t i n fac t provide s mor

e robus t an d comprehensiv e informatio n becaus e i t evaluate s AR-mediate d gene agonis man d antagonism

activity that

woul d b e expecte d to b e specifi c fo r AR-binders . Th e luciferas e reporte r gene systemuse d her

e i s simila r tha t use d for th e ER transactivatio n assa y (OPPT S 890.1300) . Th e concentratio n range s utilize d wer e narrower

tha n indicate d b y th e 890. 1 15 0 guideline , but stil l provid e sufficien t

dose-respons e information . Thi s publicatio n shoul d b e considere d a s functionall y equivalent

t o the OPPT S 890 . 1 15 0 guideline . 

### Ortone t a£ . 200 9 Thi s study i s considered supplementa l i n nature . Orto n e t

al . utilize d arecombinan t yeas t reporter systemt o evaluat e E

R an d AR agonis t

an d antagonis t

activitie s o f

1 2 pesticides , triflurali n inclusive . Th e human ER an d A R gen e sequence s wer e stabl y incorporate d into th e yeas t genome . Th e principl e o f th e tes t systeminvolve s estrogen - or androgen-mediate d stimulatio n o f

th e lac- Z gen

e encodin g th e B - galactosidas e enzyme whic h i n tur n act s a s a reporter gen e resultin g i n acolo

r chang e i n th e yeas t

culture media . Th e degre e o f activatio n correlate s with absorbanc e a t 54 0 nm .

### Do w AgroScience s LL C Report!) : 10176

2 Pag e 2 2 o f

5 5 Th e concentratio n range s utilize d wer e narrower

tha n indicate d b y th e 890.115 0 guideline , bu t stil l provid e dose-respons e information . Utilizatio n o f th e yeast-base d metho d i s o f questionabl e relevance . EPA di d no t select yeas t model s fo r

us e i n EDS P likel y base d i n par t o n th e recommendatio n o f

NTP/NICEATM an d als o becaus e mammalia n cell-base d assay s ar e considered mor

e relevant i n particula

r du e t o difference s i n tes t

chemica l uptake and/o r eliminatio n (Gaid o et

al , 1997 , NI H Publicatio n No : 03-4505,2002) . 4. 2 Aromatas e (OPPT

S 890.1200

) Assa y Overvie w Aromatas e i s an enzym e comple x responsibl e fo r estroge n biosynthesi s that convert s androgen s int o estrogens , estradiol , an d estrone . Th e Aromatas e i n vitr o assay focuse s o n thi s portio n o f

th e steroidogeni c pathwa y t o detec t substance s tha t

inhibi t aromatas e activity . Rational e Th e triflurali n EDS P consortiu mrequest s that th e requirement

to conduc t

a n aromatas e assa

y b e considere d complet

e du e to th e availabilit

y o f existin g dat a(OSRI ) that

specificall y addres s thi s dat arequirement . The tw o studie s cite d a s OSR I (Tabl e 4.2 ) ar

e substantiall y simila

r t o OPPT S 890.1200 . Moring aet al . publishe d a

n aromatas e assa

y i n whic h triflurali n wa s teste d i n KGN cell s (granulosa-like tumo r cell line) . Thi s study i s considere d to b e substantiall y simila r t o th e OPPT S 890.120

0 method . In addition , Judso n et al . (see supplementa

l data ) performe d acell-fre e fluorometri c hCYP1 9 (aromatase ) inhibitio n assa y a s part o f th e ToxCas t battery o f high-throughpu t

assays . Thi s study i s considere d to b e substantiall y simila r to th e OPPT S 890.120

0 method . Together , thes e availabl e studie s provid e sufficien t informatio n a s requeste d by OPPT S 890.1200 . Tabl e 4.2 : Aromatas e OSR I OSR I Referenc e Moring aetai. , 200

4

Judso n e t al. , 201 0 OSR

I Typ e Substantiall y similar : Cell

basedassa y utilizin g KGN granulosa-like tumo r

cell line . Substantiall y similar : cell

fre e HT S scree

n fo r hCY P 1 9 inhibition . Deviation s fro mOPPTS Guideline s OPPTS 890 . 120

0 utilize s huma n recombinan t aromatas e (cell-free ) no t

cell

base dKG N system . OPPTS 890 . 120

0 utilize s of 3 H

- androstenedion e a s a substrate ; Judso n e t

al . use da fluorometri c approac h (di(benzyloxymethoxy ) fluorescein ) Impac t o f deviations/comment s KGN cell s appea

r t o b e avali d method ; other

parameter s are simila

r includin g us e o f

3 H

- androstenedion e a s asubstrate . Bot h method s expectedt

o b e equall y vali d



### Moring aeta L Thi s study i s considered t

o b e substantially simila

r t o the OPPTS 890.120

0 metho d a s it directl y measure d th e potentia l o f triflurali n to inhibi t

th e aromatas e enzyme . Whil e th e metho d differ s fro mth e OPPTS method , assessmen t o f aromatas e activit

y utilizin g th e KGN

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 2 3 o f

5 5 cell-lin e metho d i s considere d vali d a s th e cel l lin e generall y provide s expecte d result s with aromatas e inhibitor s (see Ohno , 2004) . The assa y utilize d 10" 5 

### M o f

trifluralin ; assay s wer e conducte d i n triplicate . Dose-responsivenes s with thi s mode l wa s confirme d wit h expected aromatase-inhibitor s (Ohno , 2004) . 

### .Tudso n e t al . Judso n et al . als o provid e informatio n that i s substantiall y simila

r t o th e OPPT S 890.120

0 aromatas e assay . Simila

r to th e OPPT S 890.120

0 guideline , th e author s utiliz e acell-fre e aromatase/CYP1 9 inhibitio n assay . Th e Judso n publicatio n utilize d afluorescent metho d whil e radio-labelle d androstenedione i s th e substrat e requeste d i n th e OPPT S guideline . Th e differenc e i s substrate shoul d no t hav e any impac t

o n th e study especially a s expecte d result s were observe d wit

h know n inhibitor s o f

aromatas e (see supplementa l dat ao n ToxCas t

web

site) . Compound s were initiall y screene d a t asingl e concentratio n o f 1 x 10" 5 

### M , an d thos e chemical s that

wer e positiv e i n th e initia l screen were the n ru n usin g eigh t concentration s i n th e rang e o f 9.1 4 x 1 0 9 

### M - 2 x 10" 5 

### M . Thi s concentratio n rang e i s narrowe r tha n th e rang e recommende d i n th e Tier 1 screenin g assay , bu t

stil l adequat e t o detec t activit y i n amoderate concentratio n range . Th e assa y relevan t fo r aromatas e activit y i s acell-fre e NovaScreen/Calipe r Lif e Science s assa

y fo r CYP1 9 (CYP19A1 ) enzym e activit y inhibitio n (Appendi x C) . 4. 3 Estroge n Recepto rBindin g (OPPT S 890.1250

) Assa y Overvie w Th e estroge n recepto r

(ER ) i s involve d i n femal e maturatio n an d reproductiv e function . Th e E R Bindin g assay measure s th e abilit y o f achemical t o bin d t o th e estroge n receptor . Rational e Th e triflurali n consortiu mrequest s that th e requirement

to conduc t

a n estrogen receptor bindin g assa

y b e considered complet

e du e to th e availabilit

y o f existin g dat a(OSRI ) that specificall y addres s thi s dat arequirement . Th e existin g studie s that ar e bein g cite d ar

e substantiall y simila

r to ( 1 study) , functionall y equivalen t to ( 2 studies) , an d supplementa l ( 8 studies ) to th e OPPT S 890.125 0 guideline . Sinc e multipl e studie s ar e availabl e tha t

ar e essentiall y the sam

e a s or functionall y equivalent

t o OPPT S 890.1250 , conductin g the OPPT S 890.125 0 study shoul d no t b e necessar

y to obtai n th e necessar

y informatio n usin g aweigh t o f evidenc e approac h (Tabl e 4.3) .

### Do w AgroScience s LL C Repor t ID : 101762 Pag e 2 4 of 5 5 Table 4.3 : Estroge

n Receptor

Binding OSR

I OSR I Referenc e Judso n etai , 201 0

K.ojimae/a/.,200 4

Jia o era/. , 200 8

Jun g et al. , 200 4 Jungbaue r

an d beck. , 2002

Nishihar aetai , 200

0

Peti t etai , 199 7

Orto n etai , 200

9

Soto.etal. , 199 5 OSR I Typ e 1 . Substantiall y similar . Huma n an dbovin e E R competitiv e bindin g assay s (cell-fre e HTS

Calipe r Lif e Sciences/NovaScreen) ; 2 . Functionall y equivalent : huma n reporter

gene agonis t an dantagonis t assays , (NCGC ) 3 . Supplemental multiple x transcriptio n reporter assa y (Attagen e assays )

Functionall y equivalent : huma n ER aan dER( 3 reporter gen e (luciferase ) agonis t an dantagonis t

assa y Supplemental : assa

y utilize d recombinant

yeas t reporte r scree n fo r evaluatio n o f estrogeni c activity Supplemental : assa

v utilize d yeas t 2-hybri dmetho dt

o asses s anti-estrogenicit

y Supplemental : assa

v utilize d recombinant

yeas t reporte r screen fo r evaluatio n o f estrogeni c activit y Supplemental : assa

v utilize d yeas t 2-hybri dmetho dt

o asses s agonis t

activity a t th e E R Supplemental : assa

v utilize d recombinant

yeas t reporter scree n for evaluatio n o f estrogeni c activit

y Supplemental : assa

v utilize d recombinan t

yeas t reporter scree n fo r evaluatio n o f estrogeni c an danti

estrogeni c activit

y Supplemental : Assa

v assesse d the effect s o f treatment o n a n estrogen sensitiv e cell-lin e (MCF-7 ) Deviation s fro mOPPTS Guideline s E Ri s fro madifferen t

sourc e

Di dno t evaluat e bindin g to th e ER , bu t receptor agonis m an dantagonis m Di dno t evaluate bindin g t o th e ER , bu t recepto r agonis m an dantagonism ; Us e o f chimeri c protei n multiple x reporter systemdiffer s significantl y fro mOPPTS 890.115 0 Di dno t evaluate bindin g t o th e ER , bu t receptor agonis m an dantagonis m Di dno t evaluate bindin g potential , bu t

ER-mediate d reporte r gen e regulatio n Di dno t evaluate bindin g per th e guideline , bu t

decrease d reporte r gen e activit y du e to ant-estrogeni c effect s Di dno t evaluate bindin g potential , bu t

ER-mediate d reporte r gene regulatio n Di dno t evaluate bindin g potential , bu t

ER-mediate d reporte r gene regulatio n Di dno t evaluate bindin g potential , bu t

ER-mediate d reporter gene regulatio n Di dno t evaluate bindin g potential , bu t

ER-mediate d reporter gen e regulatio n Di dno t evaluate bindin g potential , bu t

di dasses s downstrea meffect s (cel l proliferation ) Impac t o f deviations/comment s Bot h method s expected to b e equall y vali d

Study provide s important

informatio n a s i t evaluate s gen

e regulatio n resultan t fro mpotential bindin g Relevanc e o f

reporte r systemi s no t clea r relativ e t o OPPTS 890 . 125 0 and furthe r evaluatio n i s needed .

Provide s additiona l informatio n than tie r I bindin g assa

y a s i t evaluate s gene regulatio n resultan t

fro mpotential bindin g Informatio n considere dsupplementa l only . Yeas t reporter system s wer

e no t selectedfor EDS P an dar

e o f questionabl e relevance . Informatio n considere d supplemental only . Yeas t reporte r system s wer

e no t selectedfor EDS P andar

e o f questionabl e relevance . Informatio n considere d supplemental only . Yeas t reporte r system s wer

e no t selected fo r EDS P an dar

e o f questionabl e relevance . Informatio n considere d supplemental only . Yeas t reporte r system s wer

e no t selectedfor EDS P an dar

e o f questionabl e relevance . Informatio n considere dsupplemental only . Yeas t reporte r system s wer

e no t selectedfor EDS P an dar

e o f questionabl e relevance . Informatio n considere dsupplemental only . Yeas t reporte r system s wer

e no t selectedfor EDS P an dar

e o f questionabl e relevance . Measuremen t

o f estrogen-dependen

t cel l proliferatio n i s considere dto supplemental informatio n t o E R bindin g a s th e specificit

y o f th e system i s no t clea r

### Do w AgroScience s LL C Repor t ED : 10176

2 Pag e 2 5 of

5 5 

### .Tudson e t a L Thi s study provide s informatio n that i s substantiall y simila

r (NovaScreen HT S assays) , functionall y equivalen t

(NCG C reporte

r gene assays) , an d supplementa l (Attagen e multiple x assays ) t o OPPT S 890.1250 . Thi s publicatio n provide s a n overvie w o f

th e ToxCas t

program , th e purpos e o f

whic h i s to conduc t

high-throughpu t

screening , an d thu s allo w fo r prioritizatio n o f testin g fo r

environmenta

l chemical s (see Appendi x C fo r mor e informatio n o n th e ToxCas t progra man d assays) . A chemica

l librar y o f 32 0 substance s (includin g trifluralin ) wa s evaluate d i n 46 7 cell-fre e or cell-base d assays . O f

these , severa

l ar e relevant t o EDS P Tie

r I an d i n particula

r ER-binding . Thes e include cell-fre e an d cell-based HT S assay s (NovaScreen/Caliper) . Th e cell-fre e assay s evaluate d bindin g t o th e human an d bovin e ER whil e th e cell-base d assay s (NCGC ) wer e designe d t

o asses s reporte

r gen e agonis man d antagonis ma

t th e huma n ER . I n addition , amutiple x transcriptio n reporte r assa y system (Attagene ) utilize d alibrar

y o f transcriptio n facto r

bindin g site s wa s utilize d to evaluate th e activitie s o f

endogenou s (cis-acting ) or exogenou s (trans-acting ) chimeri c transcriptio n factors . Th e specifi c assay s relevant to ER-bindin g an d ER-activit y are : Novascreen' s (Calipe r Lif e Sciences ) bovin e estrogen recepto r binding , human estrogen recepto r binding ; NIH Chemica

l Genomic s Center' s (NCGC ) human estrogen receptor aagonis t an d antagonis t

assays , an d Attagene ' s human ER-a , human estrogen-respons e element , human estrogen-relate d receptorex , human estrogen-relate d recepto r y multi-ple x reporte

r assays . Koiimagfa/.(2004 ) Thi s study i s functionall y equivalen t t o OPPT S 890.1250 . Th e study teste d 20 0 chemicals , includin g trifluralin , fo r

agonis man d antagonis mto hERa , hERp , an d hAR . Th e metho d utilize d Chines e hamste

r ovar y cell s transientl y transfecte d wit h luciferas e reporte

r gen e construct s containin g eithe r a n estrogen-responsive-elemen t (ERE ) or a n androgen

responsive-elemen

t (ARA) . Afte r

transfection , cell s wer

e treate d wit

h 1 0 " 8 

### - 1 0 " 5 

### M o f

each tes t compoun d or

0.1 % DMS O (vehicl e control) . Th e study als o evaluate d antagonis t activit y b y treatment

with tes t compound s i n th e presenc e o f E 2 (10" U

### M o r

10"'° M fo r

ER aan d ERP respectively ) or

10"'° M DH T for

ARantagonism . Whil e th e study doe s no t

directly asses s ER-binding , i t i n fac t provide s mor

e robus t an d comprehensiv e informatio n becaus e i t evaluate d ER-mediate d gene agonis man d antagonis m a t bot h th e hER aan d hERp . Th e luciferas e reporte r gen e systemuse d her

e i s simila r that use d for th e ERtransactivatio n assa y (OPPTS 890.1300

) Th e concentratio n range s utilize d wer e narrowe r tha n indicate d b y th e 890. 1 15 0 guideline , but stil l provide sufficien t

dose-respons e information . Thi s publicatio n shoul d b e considere d a s functionall y equivalent to th e OPPT S 890 . 125 0 guideline . Thi s study i s considere d supplementa l i n nature . Jia o e t

al . utilize d atransgeni c yeas t mode l to asses s estrogeni c potentia

l o f tes t chemicals . Th e assa

y utilize d agoldfis h ER ayeas t expressio n vecto

r an d areporte

r plasmi d (p-galactosidase) . Tes t chemical s wer

e evaluate d fo r estrogeni c activity a

t 10" 9

### , 10" 7 

### , and 10"5 

### M . Thi s concentratio n rang e i s somewha t narrower than tha t requeste d b y OPPT S 890.1250 , bu t i s stil l sufficien t

to provid e dose-respons eDo w AgroScience s LLC Repor t ID : 10176

2 Pag e 2 6 o f

5 5 information . Th e yeas t

model system s provid e supplementa l informatio n that

ca n b e use d i n a weigh t o f evidenc e assessment , but ar e no t primar y dat asources . EPA di d no t select yeas t model s fo r

us e i n EDS P likel y base d i n part o n th e recommendatio n o f NTP/NICEATM an d als o becaus e mammalian cell-base d assay s are considere d mor e relevan t i n particula

r du e to difference s i n tes t

chemica l uptake and/o r

eliminatio n (Gaido et

al , 1997 , NI H Publicatio n No : 03-4505 , 2002) . 

### Jun g etal . 200

4 Thi s stud y i s considere d supplementa l i n nature . Fift y chemicals , includin g trifluralin , wer e evaluate d fo r

anti-estrogenicit

y i n ayeas t

two-hybri d systemexpressin g th e ra t ERa . Th e tes t systemutilize d th e reporte r gene (3-galactosidas e linke d to a n estrogen-responsiv e promote r region . Yeas t

cell s wer e treate d wit

h 10" 9 -10" 3 M tes t

chemical s togethe

r with 5 xlO" 9 M E 2 fo r 4 hours . Reporte

r gen e activit y wa s determine d an d furthe r screening assay s wer

e performe d i n th e event

o f positiv e result s only . Th e yeas t mode l system s provid e supplemental informatio n tha

t ca n b e use d i n aweigh t

o f evidenc e assessment , bu t ar e no t primar y dat a sources . EPA di d no t

selec t yeas t model s fo r

us e i n EDS P likely base d i n part o n th e recommendatio n o f

NTP/NICEATM an d als o becaus e mammalian cell-base d assay s ar e considere d mor

e relevan t i n particula

r du e to difference s i n tes t

chemica l uptake and/o r eliminatio n (Gaido e

t al. , 1997 , NIH Publicatio n No : 03-4505 , 2002) . .Tungbauer

an d Beck . 2002 Thi s study i s considere d supplementa l i n nature . Jungbaue r

an d Beck use d ayeas t

two-hybri d syste mexpressin g human ER a(under contro l o f

th e metallothionin e promoter) an d areporter plasmi d expressin g p-galactosidas e (unde r contro l o f

th e vitellogeni n respons e element ) wa s utilize d t o asses s estrogeni c potentia l o f variou s chemicals . Limite d informatio n i s availabl e o n assa y parameter s includin g concentration s o f tes t chemical s utilize d (authors ' stat e that seria l dilution s were utilized) . Th e yeas t mode l system s provid e supplementa l informatio n that ca n b e use d i n aweight

o f evidenc e assessment , bu t ar e no t primar y dat asources . EPA di d no t selec t yeas t model s fo r

us e i n EDS P likel y base d i n part o n th e recommendatio n o f NTP/NICEATM an d als o becaus e mammalian cell-base d assay s ar e considere d mor

e relevan t i n particula

r du e to difference s i n tes t

chemica l uptake and/o r

eliminatio n (Gaid o et

al. , 1997 , NI H Publicatio n No : 03-4505 , 2002) . Nishihar at'M/. . 200

0 Thi s study i s considere d supplementa l i n nature . 51 7 chemical s includin g triflurali n wer e teste d fo r agonis t activit y a t th e estrogen recepto

r i n ayeas t

two-hybri d system(|3

- galactosidas e reporte

r under contro l o f

estrogen-responsiv e promote r region) . Littl e informatio n i s availabl e o n tes t concentration s use d or

othe r parameters . Th e yeas t

mode l system s provid e supplementa l informatio n that

ca n b e use d i n aweight

o f evidenc e assessment , bu t

ar e no t primar y dat asources . EPA di d no t select yeas t model s fo r

us e i n EDS P likel y base d i n part o n th e recommendatio n o f NTP/NICEAT

M an d als o becaus e mammalian cell-base d assay s ar e considered mor

e relevant i n particula

r du e to difference s i n tes t chemica l uptake and/o r

eliminatio n (Gaid o et

al. , 1997 , NIH Publicatio n No : 03-4505 , 2002) .

### Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 27 o f

5 5 Orio n et o£,200 9 Thi s stud y i s considere d supplementa l i n nature . Orto n et

al . utilize d arecombinant

yeas t reporte r systemt o evaluat e E Ran d ARagonis t

an d antagonis t

activitie s o f

1 2 pesticides , triflurali n inclusive . Th e human ERan d ARgene sequence s wer e stabl y incorporate d into th e yeas t genome . The principl e o f

the tes t systeminvolve s estrogen - or androgen-mediate d stimulatio n o f

th e lac- Z gene encodin g th e B - galactosidas e enzym e whic h i n tur n act s a s a reporte r gen e resultin g i n acolo

r chang e i n the yeas t

culture media . The degree o f activatio n correlate s wit

h absorbanc e a

t 54 0 nm . The concentratio n range s utilize d wer e narrower

than indicate d b y the 890.115 0 guideline , but stil l provid e sufficien t

dose-respons e information . Utilizatio n o f th e yeast-base d metho d i s o f questionabl e relevance , henc e th e us e o f mammalian-base d cel l system s for EDS P assays . EP A di d no t select yeas t model s for

us e i n EDS P based o n the recommendatio n o f NTP/NICEAT

M an d sinc e mammalian cell-base d assay s ar e considere d mor

e relevant i n particula r du e t o difference s i n tes t

chemica l uptake and/o r

eliminatio n (Gaid o et

al. , 1997 , NI H Publicatio n No : 03-4505,2002) . Petit etaL , 199 7 Thi s study i s considered supplementa l i n nature . Petit

et al . utilize d ayeast-base d reporte r gen e (P-galactosidase ) assa

y fo r activit y a t th e rainbo w trou t

estrogen receptor . Th e study als o assesse d th e ability o f

tes t compound s t o induc e vitellogeni n mRN A expressio n wa s evaluate d usin g primary hepatocyt e culture s derive d fro mmal e rainbo w trout . Th e tes t concentration s wer e slightl y narrowe r tha n wha t

i s requeste d i n th e OPPTS . 125 0 tes t guideline , bu t

provid e informatio n o n dose-response . Th e yeas t model system s provid e supplementa

l informatio n tha

t ca n b e use d i n aweigh t

o f evidenc e assessment , bu t

ar e no t primar y dat asources . EPA di d no t select yeas t model s for

us e i n EDS P likel y base d i n part o n the recommendatio n o f

NTP/NICEATM an d als o becaus e mammalia n cell-base d assay s ar

e considere d mor

e relevant i n particula

r du e to difference s i n tes t

chemica l uptake and/o r eliminatio n (Gaido e

t al. , 1997 , NIH Publicatio n No : 03-4505,2002) . Soto . e t al. . 199 5 Thi s study i s considere d supplementa l i n nature . Variou s chemical s includin g triflurali n wer e evaluate d fo r

estrogeni c potentia

l i n th e E-Scree

n utilizin g MCF- 7 cells . Whil e exac t concentration s wer

e no t reported , arang e o f

tes t chemica l concentration s wer

e utilized . Afte r 6 day s o f incubation , cell s wer

e lyse d an d nuclei wer

e counte d (cell number) . Proliferativ e capacit y wa s compare d to that

induce d b y E 2 (relativ e proliferativ e potency) . At leas t fiv e separat e experiment s utilizin g duplicate sample s wer e included .

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 2 8 of

5 5 4. 4 Estroge n Recepto rTranscriptiona l Activatio n (OPPT S 890.1300

) Assa y overvie w Th e estroge n recepto r

(ER ) i s involve d i n femal e maturatio n an d reproductiv e function . Th e E R Transcriptiona l Activatio n i s acell-base d assay tha t measure s th e abilit y o f

a chemica l t o bin d t o th e E R an d activat e transcriptio n resultin g i n th e synthesi s o f th e enzym e luciferase . 

### Rational e Th e triflurali n consortiu mrequest s that th e requirement

to conduc t

a n estrogen recepto

r bindin g assa

y b e considered complet

e du e to th e availabilit

y o f existin g dat a(OSRI ) that specificall y addres s thi s dat arequirement . Th e existin g studie s that ar e bein g cite d ar

e substantiall y simila

r t o ( 2 study) , an d supplementa l ( 8 studies ) t

o the OPPT S 890.130

0 guideline . Sinc e multipl e studie s ar e available , conductin g th e OPPT S 890.130 0 stud y shoul d no t b e necessar

y to obtai n th e necessar

y informatio n usin g aweigh t o f evidenc e approac h (Tabl e 4.4) . Tabl e 4.4 : Estroge

n Receptor

Transcriptiona

l Activatio n OSR

I OSR I Referenc e Judsonefa/,,201 0

Kqjim aetal. , 200

4

Jia o era/. , 200 8

Jun g e l at. , 200

4 OSR

I Typ e 1 . Substantiall y Similar : huma n reporte

r gen e agonis t an dantagonis t assay s (NCGC Assays ) 2 . Supplemental : Huma n an d bovin e ERcompetitiv e bindin g assay s (cell-fre e HTS-CaliperLif

e Sciences/NovaScreen) ; multiple x transcriptio n reporte r assa y (Attagen e assays ) Substantiall y similar ' human ER aan dERf

l reporter gen e (luciferase ) agonis t

an d antagonis t assa y Supplemental : assa

v utilize d recombinant

yeas t reporte r scree n for evaluatio n o f estrogeni c activit

y Supplemental : assa

v utilize d yeas t 2-hybri dmetho dto asses s anti-estrogenicit

y Deviation s fro mOPPTS Guideline s Cel l lin e an dreporte

r system ar e differen t fro mOPPTS 890.130 0
Cel l fre e assay s do no t

asses s transcriptiona

l activation , bu t binding ; For

Attagen e assays , us e o f chimeri c protei n multiple x reporter

syste m differ s significantl y fro m OPPTS 890 . 1 1 5 0

Cell lin e systemwa s differen t fro mOPPTS 890 . 1300 ; utilize dtransien t

transfectio n Utilize dayeast-base dsyste

m an dno t mammalian cell line . Utilize dayeast-basedsystem an dno t mammalian cell line . Impac t o f deviations/comment s Bot h method s expectedto b e equall y vali d

Bindin g coul doccu

r withou t gen e activatio n (antagonism) ; Relevanc e o f reporter systemi s no t

clea r relativ e to OPPTS 890.125 0 an dshoul db e furthe r evaluated .

Bot h method s expecte dto b e equall y vali d Informatio n considere dsupplemental only . Yeas t reporter system s were no t selectedfor EDS P an dar

e o f questionabl e relevance . Informatio n considere dsupplemental only . Yeas t reporte r system s were no t selectedfo r EDS P an dar

e o f questionabl e relevance .

### Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 2 9 o f

5 5 OSR I Referenc e Jungbaue r an d beck. , 2002
Nishiharaeia/,,200 0

Petite / al , 199 7

Orto n era/. , 2009

Soto , e t al. , 199 5 OSR

I Typ e Supplemental : assa

v utilize d recombinant

yeas t reporte r scree n fo r evaluatio n o f estrogeni c activit

y Supplemental : assa

v utilize d yeas t 2-hybri dmetho dto asses s agonis t

activity a t th e ER Supplemental : assa

v utilize d recombinant

yeas t reporte r scree n for evaluatio n o f estrogeni c activit

y Supplemental : assa

v utilize d recombinan t yeas t reporte r scree n for evaluatio n o f estrogeni c an danti

estrogeni c activit

y Supplemental : Assa

v assesse d the effect s o f treatmen t o n a n estrogen sensitiv e cell-lin e (MCF-7 ) Deviation s fro mOPPT S Guideline s Utilize dayeast-basedsyste

m an dno t mammalian cell line . Utilize dayeast-base dsystem an dno t mammalian cel l line . Utilize dayeast-base dsystem an dno t mammalian cel l line . Utilize dayeast-base dsystem an dno t mammalia n cel l line . Di dno t evaluate gen

e regulatio n directly , bu t

di d asses s downstrea

meffect s (cell proliferation ) Impac t o f deviations/comment s Informatio n considere dsupplemental only . Yeas t reporter system s were no t selectedfor EDS P an dar

e o f questionabl e relevance . Informatio n considered supplementa l only . Yeas t reporter system s were no t selectedfor EDS P an dar

e o f questionabl e relevance . Informatio n considere dsupplemental only . Yeas t reporte r system s wer

e no t selected for EDS P an dar

e o f questionabl e relevance . Informatio n consideredsupplementa l only . Yeas t reporte r systems wer

e not selectedfor EDS P an dar

e o f questionabl e relevance . Measurement

o f estrogen-dependen

t cel l proliferatio n i s considere dto b e supplementa

l informatio n to ER binding/transactivatio n a s th e specificit y o f th e systemi s no t

clea r 

### For stud y summaries , se e sectio n 4. 3 (ER binding ) 4. 5 Puberta l Developmen t an d Thyroi d Functio n i n Intac t Juvenile/Peripuberta l Femal e Rats (OPPT

S 890.1450

) Assa y Overvie w Th e Puberta

l Femal e assa

y involve s th e us e o f

rat s to screen fo r

estrogeni c an d thyroi d activity i n female s durin g sexua l maturation . Thi s assa y examine s abnormalitie s associate d with se x organ s an d pubert y markers , a s wel l a s thyroi d tissue . Rational e Th e triflurali n consortiu mrequest s tha t th e requiremen t

fo r th e femal e puberta l assay b e considere d fulfille d du e t o th e availabilit y o f Othe r Scientificall y Relevan t

Information

primaril y higher-tiere d functionall y equivalen t studie s (Tabl e 4.5.1) . Whil e aspecifi c stud y correspondin g t

o the OPP T 890.145 0 guideline ha s no t bee n conducted , higher-tiere d multi

generatio n reproductio n studie s ar e availabl e whic h mee

t bot h o f EPA' s definition s o f

what constitute s OSRI : "OSR

I may eithe r

b e functionall y equivalen t t o informatio n obtaine d fro mth e Tier

1 assays—that

is , dat afro massay s tha t performth e sam

e functio n a s EDS P Tier 1 assays—o r

may includ e dat atha t

provid e informatio n o n apotentia l consequenc e o r effec t tha t coul d b e du e t o effect s o n th e estrogen , androge n o r thyroi d system s (USEPA , 2009). " Th e critical component s o f OPPT S 890.145 0 hav e bee n assesse d a s th e higher-tiere d multi-generatio n studie s ar e designe d t o detec t an y potentia l advers e effect s o n fertility , reproductiv e performanc e o r developmen t

tha t coul d occur

throug h an endocrine-mediate d mode-of-action . Th e exposur e time-frame s i n th e reproductio nDo w AgroScience s LL C Repor t ID : 10176

2 Pag e 3 0 of

5 5 studie s overla

p wit h that

o f th e Femal e Puberta l Assa y an d i n fac t ar e more comprehensiv e a s the y als o asses s perinata l exposure . Whil e non e o f th e individua l studie s fulfill s th e requirement , together the y provid e robus t

functionall y equivalen t informatio n t o th e OPPT S 890.145 0 guidelin e study. Redundanc y i n th e databas e als o increase s confidenc e i n th e abilit y t o utiliz e thes e studies . Table 4.5.1 : Female Puberta l OSR I OSR I Referenc e Hoyt , J.A . 198

6 (Adam s an

d Markham , 1996

)

Becker , H . 198

4

Rubin , V.e t

oi 198 7

Wort h eta L 196 6

Saghir,<tfo£200 8 OSR I Typ e Functionall y equivalen t Higher-Tier

Studv : Thi s wa s amulti-generatio n reproductio n study tha t evaluate dpost-nata

l growt h an ddevelopment . Functionall y equivalen t Hieher-Tie r Studv : Thi s wa s amulti-generatio n reproductio n stud y that evaluate dpost-nata

l growt h an ddevelopment . Functionall y equivalen t Hieher-Tie r Studv : Thi s wa s amulti-generatio n reproductio n study that evaluate dpost-nata

l growt h an ddevelopment . Supplemental Higher-Tier : Thi s wa s amulti-generatio n reproductio n stud y that evaluate dpost-nata

l growt h an ddevelopment . Supplemental : Thi s stud y investigate dth e mode-of

actio n o f thyroi dtumour s i n rat s Deviation s fro mOPPT S Guideline s Vagina l openin g no t

assessed ; estrou s cyclicity no t

assessed ; thyroi dhormon e measurement s an d histopatholog y no t

evaluated ; bloo dchemistr

y no t evaluated ; orga

n weight s no t take n unknow n

Vagina l openin g no t

assessed ; thyroi dhormon e measurement s no t

taken ; onl y ovarian weight s taken ; kidne y an d thyroi dhistopatholog y no t evaluated ; bloo d chemistr y no t evaluate d Vagina l openin g no t

assessed ; estrou s cyclicit

y no t assessed ; thyroi dhormon e measurement s no t

taken ; Pituitary weight s no t

taken ; bloo d chemistr

y no t evaluate d Doe s not directly addres s OPPTS 890 . 1450 except

fo r thyroi dendpoint s Impac t o f deviations/comment s Assessmen t o f treatment o n fertility , reproduction , growt

h an d development valid . Lackin g endpoint s hav e bee n assesse d in othe r way s (se e sectio n 3.0)

Th e triflurali n EDS P consortiu mdi d no t conduc t thi s study .

Assessmen t o f treatment o n fertility , reproduction , growt

h an ddevelopmen t valid . Lackin g endpoint s hav e been assesse d in other way s (se e sectio n 3.0 ) Assessmen t o f treatmen t o n fertility , reproduction , growt

h an ddevelopmen

t valid . Lackin g endpoint s hav e been assesse d in othe r way s (se e sectio n 3.0) N/A

Hovt..T.A . 1986 Thi s wa s atw o generatio n reproductio n study i n Crl : C D (SD ) rats . Triflurali n wa s administere d t

o 2 5 rats/sex/group a

t dietar y level s o f

0,0.02,0.06 3 or

0.20 % for

eithe r 1 0 or 2 0 week s prior

to mating , throughou t

mating , gestatio n an d lactatio n periods . Tw o breedin g trial s wer e conducte d (fl aan d fi b progeny) . F l parent s (derive d fro mfi a progeny ) wer e als o maintaine d o n tes t

diet s throughou t

tw o breedin g trial s (f2 aan d f2 b ) . Parameter s evaluate d i n thi s study included : mortality/clinica l observations , foo d consumptio n an d foo d utilization , bod y weight s an d growth , matin g performanc e an d fertility , reproductio n parameter s (precoita l periods , gestatio n length , numbe r o f liv e an d dea

d progeny) , progenc y survival , progenc y se x distribution , progen y bod y weigh t an d growth , histopathologica l evaluatio n o f

variou s tissue s

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 3 1 of

5 5 (vagina , uterus , ovaries , mammary gland , testes , epididymus , semina l vesicles , prostat

e an d kidneys) . Thi s i s higher-tier

stud y whic h evaluate d th e effect s o f triflurali n o n mating , fertility , reproduction , an d pre - an d post-nata l developmen

t al l o f whic h coul d b e impacte d throug h endocrin e mode s o f

action . Th e study provide s aportio n o f th e end-point s tha

t make up th e OPPT S 890.145 0 guidelin e includin g postnatal growt h an d histopathologica

l evaluatio n o f variou s organs . Adam s an d Markham . 1996 fMRI D #44135107 ) Thi s repor

t wa s astatistica

l reanalysi s o f

afe w endpoint s i n th e Hoyt , 199 6 report . Th e reanalysi s doe s no t

chang e th e conclusion s o f th e study . Becker . H . 198

4 Thi s i s amulti-generatio n stud y submitte d to EPA . Dose-level s wer e 0,200,65 0 an d 200

0 ppm . Body weights , kidne y weight s an d kidney histopatholog y wer e evaluated . N o additiona l informatio n o n thi s study i s availabl e to th e consortium . 

### Rubin , \.eta l 198

7 In thi s two-generatio n reproductio n study , 2 5 C D rats/sex/group receive d dietar y level s o f Triflure x (trifluralin ) o f

0,50,450 , o r

400 0 pp m(HD L determine d to exceed a n MTD) . Exposure wa s for

1 0 week s prio r

to mating , throughou t

mating , gestation , an d lactation . Twenty-fiv e Fl pups/sex/group wer e randoml y selected fo r

matin g to provid e a

n F 2 generation . Parameter s evaluate d included : clinica l sign s an d mortality , bod

y weight an d foo d consumption , oestru s cycles , pre-coita l intervals , duratio n o f

gestation , haematolog y parameters , ovar

y an d teste s weight s a s wel l a s histopathologica l evaluatio n o f

variou s tissues . Thi s i s higher-tier

stud y whic h evaluate d th e effect s o f triflurali n o n mating , fertility , reproduction , an d pre - an d post-nata l development

al l o f whic h coul d b e impacte d throug h endocrine mode s o f

action . Th e stud y provide s aportio n o f th e end-point s tha t

mak e up th e OPPT S 890.145 0 guidelin e includin g postnata l growth , estrou s cyclicity , orga n weight s an d histopathology . Wort h eta L 196

6 Thi s wa s a3-generatio n reproductio n stud y where 6 mal e an d 1 2 femal e Harlan rats/group receive d dietar

y level s o f 0,0.02,0.2 % trifluralin . Tw o male s were house d pe r each female . Weanling s fro mth e second litter wer e use d to generat

e th e F 2 generatio n an d asimila r protoco l wa s use d to generat e th e F 3 generation . Parameter s evaluate d included : bod y weig h an d foo d consumption , reproductiv e indices , haematology , variou s orga n weight ratio s an d histopatholog y o n variou s tissues . Thi s i s higher-tier

stud y whic h evaluate d th e effect s o f triflurali n o n mating , fertility , reproduction , an d pre - an d post-nata l development

al l o f whic h coul d b e impacte d throug h endocrine mode s o f

action . Th e study provide s aportio n o f th e end-point s that

make up th e OPPT S 890.145 0 guidelin e includin g postnata l growth , orga

n weight s an d histopathology .

### Do w AgroScience s LLC Repor t ID : 101762 Pag e 3 2 o f

5 5 Saehi r e t al. . 200 8 Thi s study i s cite d becaus e it assesse d thyroi d end-point s include d i n OPPT S 890.1450 . The stud y wa s conducte d to addres s th e mode-of-actio n o f trifluralin-induce d thyroi d tumor s i n rats . Group s o f

1 5 mal e Fische

r 34 4 rat s receive d 0 o r

650 0 pp mtriflurali n i n th e diet fo r 2 weeks . Bod

y weight an d fee d consumptio n wer e monitore d durin g th e treatment period . At the en d o f the dosin g perio d an d prior t o termination , T3 , T

4 an d TS H wer

e measure d i n treate d an d contro l animals . Live r an d thyroi d weight s wer

e evaluated . UGT enzyme activit y a s wel l a s biliar

y eliminatio n rate s wer

e evaluated . Tabl e 4.5. 2 list s th e endpoint s requeste d b y th e OPPT S 890.145 0 guidelin e an d describe s ho w each individua l endpoin

t ha s bee n assessed b y existin g OSRI bein g cited . Wher e aspecifi c endpoint

ha s no t bee n directl y evaluate d i n existing studies , othe r surrogat e or alternativ e endpoint s that

hav e bee n evaluate d ar

e listed . Thes e provide simila r informatio n o n th e sam

e potentia l endocrine-mediate d effect s a s th e guidelin e endpoints . Evaluatin g severa l o f thes e surrogate endpoint s i n multipl e studie s shoul d provid e sufficien t

confidenc e i n th e data , (refe r to table s 3.1-3.3) . Table 4.5.2 : OPPTS 890.145 0 Endpoint s Evaluate d i n Regulator

y Toxicolog

y Studie s 

### OPPT S 890.145

0 Endpoin t

Growt h Ag e a t Vagina l Openin g

Uterin e Weight Ovaria n Weight Thyroi dWeigh t Liver Weight Kidney Weigh

t Pituitar y Weigh t

Adrenal Weigh t Uterin e Histopatholog y 

### Functionall y

Equivalen t OSR I (Citatio n code s fro mstudie s i n Tabl e 2 ) Hoyt,J.A.1986 , Rubin , Y . r

t o i 1987, Wort h eta L 196

6 No t evaluate d i n existin g studie s

Wort h efo i 196

6 Rubin , V.eta L 1987 , Wort h eta L 196

6 Wort h eta L 196

6 Wort h eta L 196

6 Wort h etaL 196

6 No t evaluate d i n existin g studie s

Wort h eta L 196 6 Hoyt,J.A.1986 , Rubin , Y.^/o i 1987 , Wort h eta L 196 6 

### Other

endpoint s i n Regulator y Toxicolog

y Studie s that

ca n b e used a s surrogate s

Se e appendix B : Time t o matin g Uterin e Weight Fertilit y Ovar y weight Ovar y histopatholog y Testi s histopatholog y Epididymi s Histopatholog y 

### Additiona l data/comment s

Compound s havin g estrogeni c activit y woul db e expecte dto promote precociou s vagina l openin g whil e anti-estrogeni c o r androgeni c compound s woul ddela y vagina l opening . Othe

r studie s whic h addres s thes e modes-of-actio n wil l provide th e necessar

y datao n estrogenicit y o r anti-estrogenicity .

Base do n existin g understandin g o f triflurali n effect s o n HP T axis , pituitar y weigh t i s considere do f

lesse r importanc e a s astand-alon e endpoint

sinc e th e detaile dMo A ha s alread

y bee n establishe d(Saghir , 2008) .Do w AgroScience s LLC Repor t ED : 10176

2 Pag e 3 3 o f

5 5 OPPT S 890.145

0 Endpoin t

Ovaria n Histopatholog y Thyroi d Histopatholog y Kidne y Histopatholog y Live r histopatholog y TSHandT 4 Estrou s Cyclicity Clinica l Chemistry Functionall y

Equivalen t

OSR I (Citatio n code s fro mstudie s i n Tabl e 2 ) Hoyt,J.A.1986 , Rubin , \.e t

a t 1987, Wort h e/a i 196

6 Hoyt,J.A.1986 , Rubin , \.e t

a L 1987 , Worth* * a t 196 6 Hoyt,JA.1986 , Wort h eta L 196

6 Hoyt,J.A.1986 , Rubin , Y . era/ . 1987 , Wort h e t a L 196 6 Saghir,«/aI2008(T 4 andTSH ) Rubin , Y.eta L 198

7 Othe r

endpoint s i n Regulator

y Toxicolog

y Studie s tha t

ca n b e used a s surrogate s Additiona l data/comment s

Clinica l chemistr y endpoint s hav e been assesse d i n multipl e triflurali n repeat-dos e studie s

4. 6 Puberta l Development an d Thyroi d Functio n i n Intac t Juvenile/Peripuberta l Mal e Rats (OPPT

S 890.1500

) 

### Assa y Overvie w Th e Puberta

l Mal e assa

y involve s th e us e o f

rat s to scree

n fo r androgenic , anti-androgenic , an d thyroi d activit

y i n male s durin g sexua

l maturation . Thi s assa

y examine s abnormalitie s associated with se x organ s an d pubert y markers , a s wel l a s thyroi d tissue . 

### Rational e Th e triflurali n consortiu mrequest s tha t th e requiremen t

fo r th e Mal e Puberta l assa

y b e considere d fulfille d du e t o th e availabilit y o f Othe r Scientificall y Relevan t

Information

primaril y higher-tiere d functionall y equivalen t studie s (Tabl e 4.6.1) . Whil e aspecifi c stud y correspondin g t

o th e OPP T 890.150

0 guidelin e ha s no t

bee n conducted , higher-tiere d multi

generatio n reproductio n studie s ar e availabl e whic h mee

t bot h o f EPA' s definition s o f

wha t constitute s OSRI : "OSR

I ma y eithe r b e functionall y equivalen t to informatio n obtaine d fro mth e Tier

1 assays—that

is , dat afro massay s tha t perfor mth e same functio n a s EDS P Tier 1 assays—o r

may includ e dat atha t

provid e informatio n o n apotentia l consequenc e o r effec t tha t coul d b e du e t o effect s o n th e estrogen , androge n o r thyroi d system s (USEPA , 2009). " Th e critica l component s o f OPPT S 890.150 0 hav e bee n assesse d a s th e higher-tiere d multi-generatio n studie s ar e designe d t o detec t an y potentia l advers e effect s o n fertility , reproductiv e performanc e o r developmen t

tha t coul d occur

throug h an endocrine-mediate d mode-of-action . Th e exposur e time-frame s i n th e reproductio nDo w AgroScience s LLC Repor t ID : 10176

2 Pag e 3 4 of

5 5 studie s overlap wit h tha t o f th e Mal e Puberta l Assay an d i n fac t ar e more comprehensiv e a s the y als o asses s perinata l exposure . Whil e non e o f th e individua l studie s fulfill s th e requirement , togethe r

the y provid e th e functionall y equivalen t informatio n t o th e OPPT S 890.150 0 guidelin e study . Redundanc y i n th e databas e als o increase s th e confidenc e i n thes e studies . Se e OSRI data-set

for OPPTS 890.145 0 Femal e Puberta l Assay , (sectio n 4.5 ) Table 4.6.1 : Male Puberta l OSR I OSR I Referenc e Hoyt,JA.198 6

Becker, H . 198

4

Rubin , \.eta L 198

7

Wort h eta l 196 6

Saghir,e*a£200 8 OSR I Typ e Functionall y equivalen t Higher-Tier Study : Thi s wa s amulti-generatio n reproductio n study tha t evaluate dpost-nata

l growt h an ddevelopment . Functionall y equivalent Hieher-Tie r Studv : Thi s wa s amulti-generatio n reproductio n stud y tha t evaluate dpost-nata

l growt h an ddevelopment . Functionall v equivalen t Hieher-Tie r Studv : Thi s wa s amulti-generatio n reproductio n study tha

t evaluate dpost-nata

l growt h an ddevelopment . Supplementa

l Hieher-Tie

r : Thi s wa s amulti-generatio n reproductio n stud

y that evaluate dpost-nata

l growt h an ddevelopment . Supplemental : Thi s stud v investigate dth e mode-of

actio n o f thyroi dtumour s i n rat s Deviation s fro mOPPTS Guideline s PP S not assessed ; hormon e measurement s no t

evaluated ; bloo dchemistr

y no t assessed ; orga n weight s no t

taken ; n o thyroi dhistopatholog y unknow n

PP S no t evaluated ; hormon e measurement s no t

taken ; onl y teste s weigh t

taken ; kidne y an dthyroi dhistopatholog y no t evaluated ; bloo d chemistr y no t evaluate d PP S no t evaluated ; hormon e measurement s no t

taken ; semina l vesicle , levator

ani plu s bulbocavernosus , epididymous , pituitary weight s no t taken ; bloo d chemistr y no t evaluated ; epididymu s no t

evaluate d histologicall y Doe s no t directl y addres s OPPTS 890.150

0 except fo r thyroi dendpoint s Impac t o f deviations/comment s Assessmen t o f treatmen t o n fertility , reproduction , growt

h an ddevelopment valid . Lackin g endpoint s hav e been assesse d in other way s (se e sectio n 3.0) Th e triflurali n EDS P consortiu mdi d no t conduct thi s study .

Assessmen t o f treatmen t o n fertility , reproduction , growt

h an ddevelopmen t valid . Lackin g endpoint s hav e been assesse d in othe r way s (sectio n 3.0

) Assessmen t o f treatment o n fertility , reproduction , growt

h an ddevelopmen

t valid . Lacking endpoint s hav e bee n assesse d in other way s (sectio n 3.0

)

N/A

Tabl e 4.6. 2 list s th e endpoint s requeste d b y th e OPPT S 890.150 0 guidelin e an d describe s ho w each individual endpoint

ha s bee n assessed b y existin g OSR I bein g cited . Wher

e aspecifi c endpoint

ha s no t bee n directl y evaluate d i n existin g studies , othe r surrogate o r

alternativ e endpoint s that hav e bee n evaluate d ar

e listed . Thes e provide simila r informatio n o n the sam

e potentia l endocrine-mediate d effect s a s th e guidelin e endpoints . Evaluatin g severa l o f thes e surrogate endpoint s i n multipl e studie s shoul d provid e sufficien t

confidenc e i n th e data , (see table s 3.1-3.3) . In addition , conduc t o f th e Hershberge

r assa y wil l provid e additiona

l supportiv e information .Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 3 5 of

5 5 Table 4.6.2 : OPPTS 890.150 0 Endpoint s Evaluate d i

n Regulator

y Toxicolog

y Studie s OPPTS 890.145

0 Endpoin t

Growt h Ag e an dweigh t

a t preputial separatio n

Semina l Vesicl e Weigh t Ventra l prostat

e weight Dorsolatera l Prostat e Weight levato r ani/bulbocavernosu s muscl e comple x weigh t Epididymide s weigh t Teste s weight Thyroi dWeight Live r Weight Kidne y Weight Pituitary Weight

Adrenal Weight Epididymi s Histopatholog y Teste s Histopatholog y Thyroi dHistopatholog y Kidne y Histopatholog y Functionally Equivalen t OSR I (Citatio n code s fro m studie s i n Tabl e 2 ) Hoyt,J.A.1986 , Rubin , \.eta L 1987, Wort h eta L 196 6 No t evaluate di n existin g studie s

No t availabl e Prostat e weight

Wort h eta L 196 6 Prostat e weight

Wort h eta L 196 6 No t availabl e No t availabl e Rubin , Y.r t

a t 1987 , Wort h eta L 196 6 Wort h eta L 196 6 Wort h eta L 196 6 Wort h eta L 196 6 No t evaluate di n existin g studie s

Wort h eta L 196 6 Hoyt,J.A.1986 , Rubin , \.eta L 1987, HoytJ.A,1986 , Rubin , \.eta L 1987, Wort h eta L 196 6 Worthe d 196

6 Hoyt,J.A.1986 , Rubin , \.eta L 1987 , Worthed . 196

6 Othe

r endpoint s i n Regulator y Toxicolog

y Studie s tha t

ca n b e used a s surrogate s

Se e appendix B : Tim e to matin g Uterin e Weigh t Fertilit y Ovar y weigh t Ovar y histopatholog y Testi s histopatholog y Epididymi s Histopatholog y Prostat e Histopatholog y Vaginal histopatholog y Testi s weigh t Prostat e weigh t Semina l Vesicl e histopatholog y Uterin e histopatholog y Additiona l data/comment s

Compound s havin g estrogeni c o r anti

androgeni c activity woul db e expecte dto dela y PPS . Other

existin g dataan d EDS P assay s whic h measur

e thes e modes-of-action wil l provide th e necessar y datao n estrogenicit

y or anti

androgenicity .

T o b e evaluate di n th e Hershberge r Als o evaluate di n th e Hershberge r Als o evaluate di n th e Hershberge r T o b e evaluate di n th e Hershberge r T o b e evaluate di n th e Hershberge r

Base do n existin g understandin g o f triflurali n effect s o n HP T axis , pituitary weight i s considere do f

lesse r importanc e a s astand-alon e endpoin t

sinc e th e detaile dMo A ha s alread

y bee n establishe d(Saghir , 2008) .

### Do w AgroScience s LLC Repor t ID : 10176

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5 5 OPPT S 890.145

0 Endpoin t

TSHandT 4 Seru mTestosterone Clinica l Chemistr

y Functionally

Equivalen

t OSR I (Citatio n code s fro m studie s i n Tabl e 2 ) Saghir , era/ . 200 8 (T4andTSH ) No t evaluate d i n existing studie s Other endpoint s i n Regulator

y Toxicolog

y Studie s tha t

ca n b e used a s surrogate s Additiona l data/comment s

to b e evaluate d i n steroidogenesi s an d Hershberge

r Clinica l chemistr

y endpoint s hav e bee

n assesse d i n multipl e triflurali n repeat dos e studie s

### Do w AgroScience s LL C Repor t ED : 101762 Pag e 3 7 of

5 5 

### 5. 0 Discussio n of Triflurali n OSR I dat a Examinatio n o f

th e existin g triflurali n data-set doe s no t revea l concer n fo r specifi c modulatio n o f

th e estrogen , androgen , or

aromatas e systems . Base d o n weigh t o f evidenc e evaluatio n o f

bot h higher

tiere d anima

l studie s a s wel l a s mode-of-actio n literature studies , th e followin g conclusion s ca n b e drawn :

• Triflurali n doe s no t

demonstrat

e th e capacit

y to bin d to th e androgen recepto r • Triflurali n doe s no t

demonstrat

e th e capacit

y to bin d to th e estrogen receptor no r doe s i t appea r to demonstrat

e estrogeni c o r

anti-estrogeni c activit

y • Triflurali n doe s no t

demonstrat

e th e capacit

y to alte r aromatas e activit

y Fo r trifluralin , thre e guideline/acceptabl e multi-generatio n reproductio n studie s hav e bee n conducted . Thes e serv e a

n importan t

functio n i n that

the y allo w fo r

th e integratio n an d cooperativ e functio n o f th e variou s endocrine pathway s require d fo r prope r reproductio n an d development . Th e findin g that triflurali n i s no t

selectively toxi c to reproductiv e processe s i n multipl e higher-tiere d studie s i s stron g dat ai n suppor

t o f th e conclusion s above . Th e higher-tiere d reproductio n studie s cite d a s OSRI shoul d therefore b e give

n appropriately hig h weigh t i n EPA' s overall assessmen t o f triflurali n i n relatio n t

o th e EDS P dat arequirements . Consisten t with othe

r dinitroanilin e herbicides , triflurali n target s th e liver , kidney s an d thyroi d glan d i n rats . Th e effect s o f

triflurali n o n th e thyroi d hav e bee n wel l characterize d i n existin g studie s where thyroi d weight

an d histopatholog y hav e bee n examined . I n addition , astudy investigatin g th e mode

of-actio n (M O A ) o f

thyroi d tumor s ha s been conducte d to furthe r elucidate th e high-dos e thyroi d effect s (Saghi

r etal. , 2008) . The result s ar e consisten

t wit h aMo A involvin g inductio n o f UDP-GT , increase d clearanc e o f

thyroi d hormone s (T 3 an d T4) , increased productio n o f TSH , chroni c stimulatio n o f

follicula r cell s an d increase d neoplasi a(Fischer , 1991,1993 ; Saghir , 2005) . The key event s responsibl e fo r

thyroi d toxicit

y i n rat s d o no t

occu r i n human s du e to th e presenc e o f high

affinity thyroxin e bindin g protei n (TBG ) i n human s an d alac k o f i t i n rat s (Hil l e t

al. , 1989,1998) . Du e to th e lac k

o f TB G i n rats , fre e T4 i s muc h large r i n rat s than i n human s an d i s muc h mor e susceptibl e to remova

l fro mth e blood , whic h trigger s th e cycle o f TS H stimulatio n an d follicula r cell stimulation .
5. 1 Evaluatio n o

f Potentia l Interaction s wit h th e Estroge n an d Androge n System s i n mammals

• Wo E evaluatio n o f

th e OSR I cite d her e doe s no t point to aconcer

n fo r estrogenic , anti

estrogenic , androgeni c or

anti-androgeni c effect sDo w AgroScience s LL C Repor t ID : 10176

2 Pag e 3 8 of

5 5

• I n th e multi-generatio n reproductio n studie s cite d a s OSRI , triflurali n di d no t adversel y affec t endocrine-sensitiv e parameter s a t dose-level s that

di d no t excee d a n MTD . Significan t

weigh t shoul d b e give n t

o thes e result s a s thes e ar

e definitiv e apica

l studie s designe d t

o detect advers e effect s elicite d thoug h endocrine-mediate d toxicity . o Becker , 1984 : There wer e n o effect s o f treatmen t

o n precoital interval , gestatio n length , behavio r

o f dam s durin g parturitio n an d lactation , pu p mortality , o r th e proportio n o f

parent s tha t

mated , becam e pregnant , delivered , o r reare d litter s t o weaning . Th e LOAE

L fo r reproductiv e toxicit

y wa s no t observed . Th e NOAEL fo r reproductiv e toxicit y i s 200 0 pp m(10 0 mg/kg/day) . o Hoyt , 1986 : N o effect s o f treatment o n matin g performance , fertility , pre-coita l periods , reproductio n parameters , histopatholog y o f reproductiv e organ s (vagina , uterus , ovaries , testes , epididymus , semina l vesicles , prostate) . o Rubi n e t

al. , 1987 : Estrou s cyclicity , pre-coita l intervals , matin g performance , fertility , an d duratio n o f

gestatio n wer

e unaffecte d b y treatment . Effect s a t th e high-dos e leve l (400 0 ppm) include d uterin e atrophy , decrease d ovaria n weigh t and decrease

d number o f implantations . Thes e effect s wer e considere d t

o b e secondar

y t o genera l materna l toxicit y an d no t adirec t endocrine-mediate d effec t a s 400 0 pp mwa s clearl y a

n excessively hig h dos e (11.6 % decreas e BW , 35.5 % decreas e BWG , anemia) . The othe r multi-generatio n reproductio n studie s utilize d ahigh-dos e leve l o f 200 0 pp mand simila r effect s were no t

observed . o Worth , 1966 : Fertility , gestation , viability , an d lactatio n wer e unaffecte d b y treatment . Ther e wer e n o effect s o f

treatment o n reproductiv e orga

n weight s (adrenals , ovaries , uterus , prostate , testes ) or histopatholog y o f

th e endocrine/reproductiv e organ s (thyroid , adrenal , uterus , ovaries , prostate , semina l vesicle s an d testes )

• Base d o n weigh t

o f evidenc e evaluatio n o f

th e pee r reviewe d literature , triflurali n doe s no t demonstrat

e the potentia

l t o bin d t o the androgen recepto

r (se e Table 5.1.1) . • Th e Hershberge

r assa y wil l b e conducte d t o more full y investigat e androgenic/anti-androgeni c potentia l i n vivo . Tabl e 5.1.1

- A

R Binding data : Mai n Conclusion s OSR I Referenc e Judsone/a/. , 201 0 OSR I Typ e Substantiall y similar : Huma n an drat A R competitiv e bindin g assay s (cell-fre e HTS

Caliper Lif e Sciences/NovaScreen) ; Functionall y equivalent : human reporte r gen e agonis t an dantagonis t

assays , (NCGC ) Supplemental : Multiple x transcriptio n reporte r assa y (Attagen e assays ) Mai n Conclusion s Triflurali n di dno t

demonstrate th e potential t o bin dhuma n o r

rat androge n recepto r Triflurali n di dno t

demonstrat e agonis t or antagonis t activit y a t th e huma n androgen receptor Triflurali n di dno t

induc e reporte

r gene activity a t th e huma n androge n receptor usin g th e chimeri c reporter

system

### Do w AgroScience s LLC Repor t ID : 101762 Pag e 3 9 of

5 5 OSR I Referenc e Kojimae/a/.,200 4 Ottonetal. , 2()0 9 OSR I Typ e Functionall y equivalent : huma n A Rreporte r gene (luciferase ) agonis t

an dantagonis t

assa y Supplemental : assa

y utilize drecombinan t yeas t reporter scree n fo r evaluatio n o f androgeni c an danti-androgeni c activit y 

### Overal l Conclusions : Mai n Conclusion s Triflurali n di dno t

demonstrat e activity at th e huma n androge n receptor

usin g th e luciferas e reporter

gen e assa y Triflurali n di dno t

demonstrat e androgeni c activity . A sligh t

anti-androgeni c resul t wa s reported ; effec t

appear s to b e relativel y mino r

an doveral l relevanc e o f th e yeas t reporte r syste mi s no t

clea r Base do n th e mos t

relevan t dat a (substantiall y simila

r an dfunctionall y equivalen t studies) , triflurali n doe s no t demonstrate th e capacit

y t o bin dt o th e androge n receptor .



### • Base d o n weigh t

o f evidenc e evaluatio n o f

th e pee r reviewe d literature , triflurali n doe s no t demonstrate estrogeni c o

r anti-estrogeni c potentia

l (see Table 5.1.2) . • Th e uterotrophi c assa

y wil l als o b e conducte d to mor e full y investigat

e estrogeni c potentia

l i n vivo .

Table 5.1.2

- E R Binding and transcriptiona l activatio n data : Mai n Conclusion s OSR I Referenc e Judsonetal. , 201 0

Kojim aetal. , 200

4

JiaoefaA,200 8

Jun g etal . ,200 4 OSR I Typ e Substantiall y simila

r (E Rbinding ) o r Supplemental (ERtranscriptional activation') : Huma n an dbovin e E R competitiv e bindin g assay s (cell-fre e HTS

- Calipe

r Lif e Sciences/NovaScreen) ; Functionall y equivalen t

(E Rbinding ) o r Substantiall y simila

r (ERtranscriptiona

l activation) : huma n reporter

gen e agonis t an dantagonis t

assays , (NCGC ) Supplemental multiple x transcriptio n reporter assa y ( Attagen e assays )

Functionall y equivalen t

(E Rbinding ) o r substantiall y simila

r (ERtranscriptional activation) : huma n ER aan dERf } reporte r gene (luciferase ) agonis t

an dantagonis t assa y Supplemental : assa

y utilize drecombinant yeas t reporte r scree n for evaluatio n o f estrogeni c activity Supplemental : assa

y utilize dyeas t

2

- hybri dmetho dto asses s anti-estrogenicit y Mai n Conclusion s Triflurali n di dno t

demonstrat e th e potential to bin dhuma n o r

bovin e estroge

n receptor

Triflurali n di dno t

demonstrate agonis t

o r antagonis t activit y at th e huma n androge n receptor N o apparent activity fo r th e human estroge

n respons e element , huma n estrogen-relate d recepto r y , huma n estrogen-relate dreceptor a . Fo r ER-a , a n approximat

e 3-fol dincreas e i n activity reported ; relevanc e o f multiple x syste mi s no t

clear . Triflurali n di dno t

demonstrate activity at th e huma n ER- ao r

ER- 0 receptor s usin g th e luciferas e reporter

gen e assa y Triflurali n di dno t

demonstrate estrogeni c o r anti-estrogeni c reporter

gen e activity ; relevanc e o f th e yeas t reporter systemi s no t clea r Triflurali n di dno t

demonstrate estrogeni c reporter gen e activity ; relevanc e o f

th e yeas t reporte r syste mi s no t clea r

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2 Pag e 4 0 of

5 5 OSR I Referenc e Jungbaue r an d beck. , 2002 Ntshiharae/a/.,200 0 Petite / al , 199 7 Ononetal.,200 9 Soto , etal. , 199

5 OSR I Typ e Supplemental : assa

y utilize drecombinant yeas t reporte r scree n fo r evaluatio n o f estrogeni c activit y Supplemental : assa

y utilize dyeas t

2

- hybri dmetho dto asses s agonis t activity a t theE R Supplemental : assa

y utilize drecombinan t yeas t reporter scree n for evaluatio n o f estrogeni c activit y Supplemental : assa

y utilize drecombinan t yeas t reporter scree n fo r evaluatio n o f estrogeni c an danti-estrogeni c activity Supplemental : Assa

y assesse d the effect s o f treatmen t o n a n estrogen-sensitiv e cell

lin e (MCF-7 ) 

### Overal l Conclusion : Mai n Conclusion s Triflurali n di dno t

demonstrate estrogeni c reporte r gen e activity ; relevanc e o f

th e yeas t reporter systemi s no t

clea r Triflurali n di dno t

demonstrat e estrogeni c reporter gen e activity ; relevanc e o f

th e yeas t reporte r systemi s no t

clea r Triflurali n di dno t

demonstrat e estrogeni c reporte r gene activity ; relevanc e o f

th e yeas t reporte r systemi s no t

clea r Triflurali n di d no t

demonstrat

e estrogeni c reporte r gen e activity ; relevanc e o f

th e yeas t reporte r syste mi s no t clea r Triflurali n di d no t

demonstrat e estrogenic activity ; relevanc e o f

th e E-Scree

n system i s no t clea r Base do n th e mos t

relevan t dat a (substantiall y simila

r an dfunctionall y equivalen t studies) , trifiurali n doe s no t demonstrat e th e capacit

y t o bin dt o th e estrogen receptor

nor doe s it uosses s estropeni c o r

anti-estrogeni c activity . 

### 5. 2 Evaluatio n o

f Potentia l Triflurali n Interaction s wit h th e Aromatas e Enzym e Syste m

• Base d o n existin g dat afro mpee r reviewe d literature studies , trifiurali n doe s no t inhibi t th e aromatas e enzyme (Tabl e 5.2) . • Th e steroidogenesi s assa y wil l als o b e conducte d to furthe r investigat

e any potentia

l effect s o n th e steroi d biosynthesi s pathways . Tabl e 5.2

- Aromatas e Activit

y data : Mea

n Conclusion s OSR I Referenc e Moring aet at. , 200 4 Judsone/a/. , 201 0 OSR I Typ e Substantiall y similar

Cell-basedassa

v utilizin g KG N granulosa-lik

e tumo r cel l line . Substantiall y simila

r cell-fre e HT S scree

n for hCY P 1 9 inhibition . 

### Overall Conclusion s Mai n Conclusion s Triflurali n di dno t

affec t aromatas e activit y Triflurali n di dno t

affec t aromatas e activity Base do n tw o substantiall y simila r studies , trifiurali n doe s no t

demonstrate th e capacit

y t o alter aromatas e activity . 

### 5. 3 Evaluatio n o

f Potentia l Triflurali n Effect s o n th e Thyroi d

• A n increas e i n thyroi d tumor s wa s observe d i n Fische r 34 4 rat s followin g chroni c exposure to hig h level s o f

trifiuralin . Th e mod e o f

actio n ha s bee n wel l investigated , an d i s no t considere d relevan t to human s (Saghir , et

al . 2008) .Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 4 1 o f

5 5

• Effect s o f

treatment o n thyroi d hormon e level s i n rat s ha s bee n wel l investigate d (Saghir , e t al . 2008) .

• Thyroi d weight s an d histopatholog y hav e bee n investigate d i n variou s ra t repeat-dos e studie s (Hoyt , J.A . 1986 , Rubin , Y . e t al . 1987 , Wort

h e t al . 1966) .

• Th e potentia

l o f triflurali n to interac t

with th e thyroi d hormon e receptor o r relate d protein s ha s bee n investigate d b y th e ToxCas t progra m(Appendi x C) . Relevan t assay s include : NovaScreen' s Ra

t thyrotropin-releasin g hormon e recepto r fro mforebrai n membrane s (rTRH ) an d Human thyroi d hormon e receptor-alph a(hTRa ; recombinant) ; NCG C hTR p agonist/antagonis t

assays ; Attagen e Huma

n thyroi d hormon e receptor- amulti-ple x transcriptio n assay .

• Potentia

l effect s o f

treatment o n th e thyroi d wil l als o b e furthe r evaluate d i n th e Amphibian Metamorphosi s Assa

y

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 4 2 o f

5 5 

### 6. 0 Summar

y an d Conclusion s Fo r trifluralin , al l o f

th e critica l endpoint s define d i n th e guideline s fo r AR-receptor

binding , aromatase , ER-receptor

binding , ER-transcriptiona l activation , femal e pubertal an d mal e pubertal assay s a s wel l a s th e associate d endocrin e mode(s)-of-actio n hav e been evaluate d i n on e or mor e o f severa l ways :

• Th e endpoin

t ha s bee n directl y assessed i n existin g regulator y mammalian toxicit

y studies ; • Th e endpoin

t ha s been directl y assesse d i n existin g peer-reviewe d publishe d literature ; • Th e endpoint

ha s bee n directl y assessed a s part

o f EPA' s ToxCas t

program ; • Th e endocrin e mode-of-actio n i n questio n ha s been evaluate d i n existin g higher-tiere d studie s perhap s usin g different , bu t functionall y equivalent

methods ; I n addition , asub-set

o f Tie r IEDS P assay s wil l b e conducte d to provid e complementar y dat ao n th e endocrin e end-point

o r mode-of-actio n addressed b y th e screening study . Th e confidenc e i n th e existin g studie s cite d i n thi s rational e an d ability to us e the mi n lieu o f performin g ne w studie s i s increase d b y examinin g th e totalit y o f availabl e informatio n an d performin g aweigh t o f evidenc e evaluation . Th e existin g databas e fo r trifluralin , doe s no t

demonstrat

e any patter

n o f effect s consisten

t with advers e endocrine-mediate d effect s o n reproductiv e organ s or processes . Effect s o f triflurali n o n th e thyroi d hav e bee n wel l characterize d an d wil l b e furthe r assesse d i n th e Amphibian Metamorphosi s Assay . The state d purpos e o f

the Endocrin e Disrupter

Screening Progra

mi s "t o determin e whethe

r certai n substance s ma

y hav e a n effec t i n human s that

i s simila r t o a n effec t produce d b y anaturall y occurrin g estrogen , o r

suc h other

endocrine effec t

a s th e Administrator

ma y designate. " Th e purpos e o f Tie r I screening "i s to identif

y substance s that

hav e th e potentia

l to interac t

wit h th e estrogen , androgen , o r thyroi d hormon e system s usin g abatter y o f assays. " Base d o n th e informatio n presente d above , i t i s clea r that th e AR-receptor

binding , aromatase , ER-receptor binding , ER-transcriptiona l activation , femal e pubertal an d mal e pubertal assay s are no t require d to mak

e thi s determination . Adequate informatio n i s availabl e base d o n existin g dat aan d throug h other Tie r I studie s that

wil l b e conducte d fo r trifluralin . Thes e screening assay s are , therefore , redundant , no t justifie d fro ma

n anima l us e position , an d shoul d no t

b e conducte d per

EPA' s mandate to avoi d duplicativ e testing . W e encourag e EP A t o carefull y examin e th e OSRI cite d i n thi s rational e an d reques t that th e requirement

fo r conductin g thes e assay s b e considere d complet e fo r trifluralin .

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5 5



### 7. 0 References : Abbott , DH , V Padmanabhan , an d D A Dumesic . (2006) . Contribution s o f androge n an d estrogen to feta l programmin g o f

ovarian dysfunction . Repro d Bio l Endocrino l 4:17 . Bowman , CJ . (2005) . Validatio n o f th e Puberta

l Mal e Assa

y i n Rat s (W

A 4-15) . WIL-431005 . Fina l report . 1 3 Decembe

r 2005 . Carney , E.W. , C . L . Zablotny , M . S . Marty , J . W . Crissman , P . Anderson , M . Woolhiser , an d M . Holsappl e Th e Effect s o f

Feed Restrictio n durin g i n Uter o an d Postnata l Developmen

t i n Rat s Toxicol . Sci. , No v 2004 ; 82 : 23 7 - 249 . Deli aSeta , D , F Farabollini , F Dessi-Fulgheri , an d L Fusani . (2008) . Environmental-lik e exposure to lo w level s o f

estrogen affect s sexua

l behavio r an d physiolog y o f

femal e rats . Endocrinolog y 149(11):5592-5598 . Gaido , K.W. , Leonard , L.S. , Lovell , S. , Gould , J.C. , Babai , D. , Portier , C.J . an d McDonnell , D.P . 1997 . Evaluatio n o f

chemical s wit h endocrin e modulatin g activit y i n ayeast-base d steroi d hormon e recepto r

gen e transcriptio n assay . Toxicol . Appl . Pharmacol . 143:205-212 . Gray , Jr , LE , J Ostby , J Furr , C J Wolf , C Lambright , L Parks , D N Veeramachaneni , V Wilson , M Price , A Hotchkiss , E Orlando , an d L Guillette . (2001) . Effect s o f environmenta

l antiandrogen s o n reproductiv e developmen

t i n experimenta

l animals . Hu mRepro d Update 7(3):248-264 . Horsley , EMT . (2005) . Assessmen t o f Puberta l Developmen

t an d Thyroi d Functio n i n Juvenil e Mal e Rats . Study No . 04-4276 . Fina l report . 2 2 Decembe

r 2005 . Lund , TD , D J Munson , M E Haldy , KD RSetchell , ED Lephart , an d R J Handa . (2004) . Equo l i s a nove l anti-androge n that

inhibit s prostat

e growt h an d hormon e feedback . Biol Repro d 70:1188-1195 . O'Connor , JC , S RFrame , an d G S Ladies . (2002) . Evaluatio n o f a15-da y screenin g assa

y usin g intac t mal e rat s for

identifyin g antiandrogens . Toxicol Sc i 69:92-108 . Ohno , K. , Araki , Yanase , N.T. , Nawata , H an d lida , M . A Nove l Nonradioactiv e Metho d fo r Measurin g Aromatas e Activit y Usin g aHuma

n Ovaria n Granulosa-Lik

e Tumor Cel l Lin e an d a

n Estron e ELISA . Toxicol . Sci. , Decembe r 1,2004 ; 82(2) : 44 3 - 450 . NI H Publicatio n No : 03-4505,200 2Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 4 4 o f 5 5 Rider , CV , J Furr , V S Wilson , an d LE Gray , Jr . (2008) . A mixtur e o f seven antiandrogen s induce s reproductiv e malformation s i n rats . In t J Andro l 31:249-262 . Sawaki , M , S Noda , T Muroi , H Mitoma , S Takakura , S Sakamoto , an d K Yamasaki . (2003) . I n utero throug h lactationa

l exposur e to ethiny l estradio l induce s clef t phallu s an d delaye d ovarian dysfunctio n in the offspring . Toxicol Sc i 75:402-411 . Shin , JH , H S Kim , HJ Moon , I H Rang , T S Kim , J H Seok , I Y Kim , K L Park , S Y Han , an d S Y Nam . (2002) . Effect s o f

flutamid e o n pubert

y i n mal e rats : An evaluatio n o f th e protoco l fo r

th e assessmen

t o f pubertal developmen

t an d thyroi d function . J Toxico l Enviro n Health , Pt

A 65(5-6):433-445 . Stoker , T.E. , Louis e G . Parks , L . Ear l Gray , Ralp h L . Coope

r (2000) . Endocrine-Disruptin g Chemicals : Prepuberta

l Exposure s an d Effect s o n Sexua l Maturatio n an d Thyroi d Functio n i n th e Mal e Rat . A Focu s o n th e EDSTAC Recommendations . Critica l Review s i n Toxicology , Ja n 2000 , Vol . 30 , No . 2 , Page s 197-252 . Terry , KK , GD Cappon , M E Hurtt , M S Tassinari , an d U Gupta . (2004) . Reproductiv e toxicit y assessmen t o f lasofoxifene , aselectiv e estrogen receptor modulato r

(SERM) , i n femal e rats . Birt h Defect s Re s B De v Repro d Toxicol 71(3):150-60 . Vanag e GR , B Dao , X- J Li , C W Bardi n an d S S Koide . (1997) . Effect s o f Anordiol , a

n Antiestrogen , o n th e Reproductiv e Organ s o f th e Mal e Rat . System s Biology i n Reproductiv e Medicine Vol . 38 , No . 1 , Page s 13-2 1 Va n der School , P . (1992) . Disturbe d testicula r descen t i n th e ra t afte r prenata l exposur e to th e antiandroge n flutamide . J Repro d Per t 96:483-496 . York , RG . (2006) . Merlaborator y Validatio n o f

th e Puberta

l Mal e Assay . Fina

l Report . 3 January 2006 . USEPA (2009) . EPA' s Approac h fo r Considerin g Other

Scientificall y Relevan t

Informatio n (OSRI ) unde r

th e Endocrin e Disrupte r Screenin g Program .

### Do w AgroScience s LLC Repor t ID : 101762 Pag e 4 5 o f

5 5 

### 8. 0 Appendi x A - Triflurali n Literatur e Searc h Strateg y Searche s t o identif

y publishe d literatur e o n triflurali n wer e ru n against : Agricola , Biosis , CA B Abstracts , Chemica l Abstracts , Embase , Esbiobase , Lifesci , Medline , Pascal , an d Toxcenter . Th e followin g searc h term s wer e used : 1 ) estradiol , estrogen* , oestradiol , oestrogen* , antiestrogen* , antioestrogen* , antiestradiol , antioestradio l o r

estroge n receptor s 2 ) androgen * o r

antiandrogen * o r

androge n receptor s o r

testosteron e 3 ) steroidogen * o r

steroi d synthesi s o r

steroi d metaboli c pathwa y 4 ) aromatas e o r

cyp! 9 o r cytochrom e p45 0 o r

cytochrom e p-45 0 5 ) uterotroph ? O r

(uterin e o r

uteru s o r femal e genitalia ) an d (siz e o r weigh t o r hypertroph ? O r enlarg? ) 6 ) Herschberge r

o r (testi s o r teste s o r epididymi s o r

mal e genitalia ) an d (siz e o r weigh t o r hypertroph ? O r

hypotroph ? O r

enlarg? ) 7 ) (rat o r rattus ) an d (pubert y o r

sex * matur * o r

pre-putia l o r

vagina l open* ) 8 ) (fro g o r

amphib * o r

zenopu s o r

ran ao r ranida e o r

anura ) - retrieve d anythin g o n frog s 9 ) (fis h o r minno w o r

gupp y o r zebrafis h o r

pimephale s promela s o r

oryzia s latipe s o r

cyprinodo n o r poecili ao r

dani o reri o o r

brachidani o reri o o r

cyprinu s rerio ) - retrieve d anythin g o n fis h Fo r th e returne d searc h results , an evaluatio n wa s performe d o n th e adequac y o f th e stud y desig n an d method s a s wel l a s genera l concordanc e t o th e Tier I assays/guidelines . Sectio n 4. 0 o f th e rational e contain s summarie s o f th e literatur e studie s an d document s ho w eac h compare s wit h th e Tie r IEDS P assays . Studie s that differe d significantl y fro mth e EDS P guideline s wer e categorize d a s supplementa l whil e thos e wit h ahighe r degre e o f relevanc e t o th e guideline s were classifie d a s functionall y equivalen t o r substantiall y similar .

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### 9. 0 Appendi x B - Summar y of Triflurali n Reproductio n Toxicity Studie s Tabl e 8.1 ; Summary o f

Triflurali n Reproductio n Toxicit y Studie s Stud y Design Species/Strai

n Dose-level s Bod y Weigh t

Orga n weight

Haematolog y Fertilit y an d reproductio n parameter s

Histopatholog y Hoyt , J.A . 1986 . Becker , H . 1984

. 2 generatio n 2-generatio n reproductiv e toxicolog y Rat : Crl:CD(SD ) Rat : Wista r KFM

Ha n 0 , 200 , 630 , 200

0 0,200 , 650 , 200

0 pp mpp m BW s decrease

d a t Datano t

availabl e 200 0 pp m(9 % lower fo r parental males ; 15 % lower for parenta l females ; 10

- 1 2 % lowe r fo r F l females ) No t assesse d Datano t

availabl e

No t assesse d Datano t

availabl e N o treatment-relate d Datano t availabl e effect s o n fertility , matin g performanc e or pre-coita l periods , gestatio n length , progeny surviva

l

Tissue s examine d included : vagina , uterus , ovaries , mammar y glan d o f females , testes , epididymus , semina

l vesicles , prostate , an d kidneys , an d any grossly apparent lesions ; n o apparent treatment-relate d effect s i n eithe

r generatio n Rubin , \.eta L 198 7 2 generatio n Rat : C D 0,50 , 450 , 400

0 pp m BW G lowe

r i n bot h parental sexes ; a

t HDL , u p t o 35 % lowe r i n female s durin g pre-matin g perio d whil e B W wa s 11 . 6 % lowe

r F O Absolute an d relativ e ovarian weight decreased i n high-dos e females ; testicula r weight unaffected . Simila

r finding s i n F l generatio n Sign s o f anemi aa t high-dos e N o treatment-relate d effect s o n estrou s cyclicity , fertility , matin g performance , pre-coita l intervals ; effec t o n implantatio n los s du e to materna

l toxicit y a t hig h dos e >MT D Tissue s examine d included : testes , epididymus , semina

l vesicles , prostate , ovaries , uteru s an d vagina . Uterin e atrophy observe d a

t high-dos e (400

0 ppm ) secondar

y t o decreas e BW ; considere d to hav e exceeded MTD . Simila r effec t i n F l generation . N o Wort h rfa i 196 6 3 generatio n Rat : Harla n 0 , 200 , 200

0 ppm Apparent decreas e i n BW G a t 200 0 pp m

N o effect s o n Thyroid , adrenals , ovary , uterus , prostate , teste s weight s

N o effect s o n fertilit y or reproductiv e parameter s

Thyroid , adrenal , uteru s an d ovaries , o r prostate , semina

l vesicles , and teste s examined . N o apparent effects .Do w AgroScience s LLC Repor t ID : 101762 Pag e 4 7 o f

5 5 Hoyt,J.A.1986 . Becker , H . 1984. Rubin , \.eta l 198 7 Worth eta L 196

6 histopatholog y finding s i n other organ s examine d

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### 10. 0 Appendi x C - ToxCas t Progra m ToxCastTM i s aU S EPA progra mtha t include s abatter

y o f i n vitro , high-throughpu t

screening (HTS ) assay s t o scree n chemical s for

potentia l human toxicity . Ove

r 30 0 chemical s hav e undergon e HT S unde r Phas e I o f

th e ToxCastTM program , th e majorit y o f thes e bein g pesticides . A number o f thes e chemicals , includin g trifluralin , hav e current o r forthcomin g requirement s fo r Endocrin e Disruptio n Screenin g Progra

m(EDSP ) Tie

r 1 screening . Potential redundancie s exis t between som

e o f th e ToxCast TM assay s an d th e EDS P Tie r 1 screenin g objectives ; thus , ToxCastTM dat ama y b e usefu l OSRI (Othe

r Scientificall y Relevan t

Information ) t o submi t i n lie u o f o r to supplemen

t Tie r 1 require d tests . Informatio n regardin g th e ToxCast TM Progra mresult s becam

e generally availabl e to th e publi c throug h Environmental Healt h Perspective s (EHP ) an d th e EPA ToxCastTM Progra mwebsite (www.epa.gov/ncct/toxcast / index.html ) i n mid

Decembe r 2009 , with th e ra

w dat afo r man y o f th e assay s poste d o n th e ToxCast TM websit e i n mid

Februar y 2010 . Th e dat apresente d i n th e EHP articl e (Judso n et al . 2010) an d supplementar y material s (Judson_ehp.0901392.s001.pdf) , an d o n th e ToxCast TM Progra mwebsite i s fairl y opaque , with individua l chemica

l dat atabulate d an d muc h o f th e detai l o n assa

y methodologie s provide d onl y throug h website links . Thi s revie w wa s develope d to evaluate th e dat amos t relevan t to potentia

l endocrine modulatio n an d th e result s specifi c to trifluralin . Thi s revie w describe s briefl y th e HT S assa y technologie s incorporate d i n ToxCastTM relevant to th e EDS P Tie

r 1 screening requirements , provide s a n overvie w o f the HT S assay s mos t

relevan t t o potentia l estrogen/androgen/thyroi d hormon e (EAT) endocrine modulation , an d discusse s th e EDS P Tie r 1 screening assay s that

coul d b e potentiall y fulfilled . 1 . ToxCastTM HT S Tes t Batter y an d Relevanc e to Potential Endocrin e Modulatio n Th e ToxCastTM HT S tes t

batter y include s nin e separat

e i n vitro assa

y technologies :

• Cell-fre e HT S assay s to measur e bindin g constant s an d enzym e inhibitio n values ;

• Cell-based HT S assay s to measur e bindin g constant s an d enzym e inhibitio n values ;

• Hig h conten

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

phenotype s i n eithe

r th e human hepatom acel

l line HepG2 or

ra t primar y hepatocytes ;

• Gene expressio n i n human primar y hepatocytes , focuse d o n Phas e I an d n metaboli c enzyme s an d transporters ;

• Multiple x transcriptio n reporter assay s to evaluate 4 8 transcriptio n facto r bindin g sites ;

• Biologicall y multiplexe d activit y profilin g (BioMAP ) t o characteriz e effect s potentiall y relevan t to human tissu e an d inflammator y diseas e biology ;

• Phas e I & n xenobioti c metabolizin g enzyme (XME ) cytotoxicit y assay s base d o n microarra y technology ;Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 4 9 of

5 5

• High-throughpu t

genotoxicit

y screenin g base d o n th e GreenScree n hig h conten

t GADD45A

GF P HT S genotoxicit

y assay ; an d

• Real-tim e cell electroni c sensin g (RT-CES ) to measur e cellula r change s i n respons e to chemicals . O f thes e assa

y technologies , thos e that provid e informatio n mos t directly relevan

t t o the issue o f potentia l EAT endocrine modulatio n include th e cell-fre e HT S assays , th e cell-base d HT S assays , an d th e multiple x transcriptio n reporte r assays . Each o f

thes e assay s an d thei r

dat arelevan t

to potentia

l endocrin e modulatio n i s describe d below . 1. 1 Cell-Fre e HT S Assay s Thes e assay s wer

e ru n under

contrac t to EPA b y Calipe

r Lif e Science s (formall y NovaScreen ; no w Caliper Lif e Sciences) . O f

th e 23 9 endpoint s examined , bindin g to th e followin g receptor protein s relevant t o th e endocrine syste

mwa s evaluated : • Bovin e estrogen recepto r fro muterin e membran e (bER) ; • Huma

n estrogen recepto r

fro mbreas t

cance r cell s (hER) ; • Bovin e progesterone recepto r fro muterin e membran e (bPR) ;

f \ • Huma

n progesterone receptor

fro mT47- D cell s (hPR) ; • Ra t androgen receptor

(rAR ; recombinant) ; • Human androge n receptor fro mLnCAP cell s (hAR) ;
• Ra t thyrotropin-releasin g hormon e recepto r fro mforebrai n membrane s (rTRH) ; an d
• Human thyroi d hormon e receptor-alph a(hTRa ; recombinant) . Al l o f thes e protein s ar

e nuclea r receptors , with th e exceptio n o f th e ra t thyrotropin-releasin g hormon e recepto r (rTRH) , whic h i s aG-protei n couple d receptor . Additionally , al l o f these , with th e exceptio n o f th e hTRa , were evaluate d i n competitiv e bindin g assay s that measure d th e displacement o f radiolabele d ligan d fro mth e recepto r proteins . Th e hTR aassay , i n contrast , examine d recepto 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 enzyme (CYP19A 1 or CYPC19 ) wa s als o evaluated . Assa y parameter s specifi c t

o each o f thes e endpoint s ar

e detaile d in Appendi x Tabl e 1 .

2 EPA indicate dtha t

th e human progesteron e receptor wa s derive dfro mMCF- 7 cells ; however , accordin g to Calipe r Lif e Sciences , the receptor

protei n wa s derive dfro mT-47 D cells .Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 5 0 o f

5 5 Appendix Tabl e 1 : Paramete r Specific s Regardin g Endocrine-Relate d Endpoint s o f th e Cell-Fre e HT S Assay s Receptor/enzym

e Estroge n Recepto r Progest

erone recepto r Androge n recepto r Thyroi d Relate d Arom atas e bE R

HER 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 Receptor activatio n Enzyme inhibitio n Referenc e

compound N/A " 17B-estradio l protnegeston e promegeston e methyi-trienolon e methyl-trienolon e thyroi dreleasin g hormon e (TRH ) Bisphenol- A Ketoconazol e Positive contro l

compound 1713-estradiol 17B-estradio l promegeston e promegeston e methyl-trienolon e methyl-trienolon e TR H N/ A N/A Ligan d

(cone. , M ) 3 H-estradio l ( 7 x Iff 10 ) 3 H-estradio l ( 1 x 10' 10 ) 3 H-promegeston e ( 7 x 10' 10 ) 3 H-promegeston e ( 7 x lO' 10 ) " ^-methyl

trienolon e ( 1 x 10' 9 ) 3 H-methyl

trienolon e ( 5 x 10' 10 ) 3 H-3-meHisr-TR

H ( 2 x 10" 9 ) Triiodo-thyronin e (T3 ) (1. 5 xlO' 8 ) Di(benzyloxy

methoxy ) fluorescein d (2. 5 x 10" 6 ) Kd(M ) 7 x IO' 1 0 SxlO' 1 1 6xlO' 9 3x10-' " 4. 6 x IO' 9 3 x 10-' ° 2. 3 x 10'

9 3. 5 xlO' 9 N/ A Bma

x (frnol/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 7 x 10' 1 0 M . c Assa y o f antagonis mto ligand-dependent bindin g o f co-factor to th e receptor . dAromatas e substrate bindin g affinit y = 4. 4 x 10" 6 M an dvma x = 0.03 pmo l product/pmo l CYPC19/min . I n al l cases , th e dat awer

e normalize d to thos e o f

th e solvent

control s fro mth e sam

e plate a s th e teste d chemical s an d result s expresse d a s th e percen t o f neutral control . For

th e parameter s above , chemical s wer e initiall y screened a

t asingl e concentratio n o f

2 5 \i M (1 0 fi M fo r

aromatase ) i n duplicate wells . Thos e chemical s that

wer e positiv e i n th e initia

l scree n were the n ru n usin g eight concentration s i n th e rang e o f 0.0229-5 0 ^ M (0.00914-2 0 |i M fo r aromatase ) to determin e th e AC 5 0 (i.e. , th e hal f maxima l activit y concentration)Cell-Base d HT S Assay s 1. 2 Cell-base d HT S Assay s Thes e assay s wer

e conducte d b y th e NI H Chemica l Genomic s Cente

r (NCGC) . Bot

h agonis t an d antagonis t activit y a t variou s nuclea

r receptors , includin g th e human androgen receptor (hAR) , human ER a(hERa) , an d huma n thyroi d hormon e receptor- p (hTRp) , wer e examine d usin g areporte r gene assa y syste m(misidentifie d a s Invitrogen TM GeneGLAze r i n EPA materials ; mos t

likely , th e TM , Invitroge n GeneBLAzer® reporte r assa y system) . Accordin g to th e Invitroge n website (http://www.invitrogen.com/) , th e GeneBLAzer® technolog y use s abeta-lactamas e reporter system , bu t specific s abou t

ho w th e syste mwork s are no t provided . Th e nuclea

r recepto r source s an d th e assa y parameter s relate d to each o f th e examine d endpoints , a s provide d b y th e EPA , ar e detaile d i n Appendi x Tabl e 2 .

### Do w AgroScience s LLC Repor t ED : 10176 2 Pag e 5 1 o f

5 5 Appendi x Table2 : Paramete r Specific s Regardin g Endocrine-Relate d Endpoint s o f th e Cell-Base d HT S Assay s Androge n Relate d Estrogen relate d Thyroi d relate d Activit

y hA Ragonis t hA Rantagonis t hER aagonis t hER aantagonis t hTR 3 agonis t hTR p antagonis t Target sourc e HEK293 H cell s HEK293 H ceil s HEK293 H cell s HEK293 H cell s HEK293 T cell s HEK293 T cell s Referenc e compound RI88 I N/A l?P-estradio l 4-hydroxy-tamoxife n Triiodothyronin e (T3 ) N/A Positive contro l

compound N/A a 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 17|3-estradio l (SxlO' 10 ) N/ A T 3 (4xlO-'° ) aN/A = no t

applicable . 

### Exac t concentration s o f

chemical s teste d i n thi s assa y syste mar

e no t clear . A recent

publicatio n indicate s tha t

chemical s wer

e teste d a t 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 d o n th e ToxCast TM website (http://www.epa.gov/ncct/toxcast/index.html ) indicate s that chemical s wer

e teste d to amaximu m concentratio n o f

20 0 uM . In th e cas e o f

trifluralin , concentration s i n th e rang e o f 0.001 0 - 76. 6 ^ M wer e tested . A minimu mo f

25 % o f th e contro l efficac y wa s use d a s th e cut-of f fo r considerin g a chemica l to b e active . Th e supplementa l informatio n to Judso n et

al . (2010 ) indicate s that , "value s where onl y th e highes t

concentratio n exceede d 50 % activit y wer e exclude d to eliminat

e wea k o r fals e positives. " Cel l viabilit y wa s als o evaluate d i n thi s assa y system ; however , th e criteri afo r excludin g concentration s to b e teste d i n th e assa y systembase d o n cytotoxicit y ar e no t clear . 1. 3 Multiple x Transcriptio n Reporte r Assay s Thes e assay s wer

e performe d b 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 d into HepG 2 cell s (Marti n et al . 2010) . Cis-actin g reporte

r gene s examine d the activitie s o f transcriptio n factor s endogenou s t o the HepG 2 cells . Tran s gene s evaluate d the activitie s o f exogenous , chimeri c proteins . Each chimerawa s comprise d o f

th e ligand-bindin g domai n o f anuclea r recepto r an d ayeas t

Gal4 DNA-bindin g domain . Whe n activated , thes e chimeri c protein s activate d transcriptio n o f areporte r gene expresse d downstrea mo f a5X-UAS-TAT

A promoter . In al l cases , th e reporte r gen e wa s identica

l i n sequenc e an d include d arestrictio n enzyme cleavag e site ; th e positio n o f th e restrictio n site , however , wa s uniqu e to each reporte

r gene . Th e syste mallowe d fo r reporte r gene product s to b e identifie d throug h sequencin g an d separatio n vi ahig h resolutio n electrophoresis . Usin g thi s system , th e followin g endpoint s relevant

to endocrine modulatio n wer e evaluate d (liste d i n Appendi x Tabl e 3 , alon g with their respectiv e referenc e compounds , a s provide d in EP A materials) . Appendi x Tabl e 3 : Endocrine-Relate d Endpoint s an d Referenc e Compound s Teste d i n th e Multiple x Transcriptio n Reporte r Assay s Androgen relate d Estroge n relate d Endpoin t Human androge n recepto

r Huma n ER- a Huma n estroge

n respons e element3 Huma n estrogen-relate d receptor- a Huma n estrogen-relate d receptor- ? Referenc e Compound s Dioxin , FI C 17p-Estradiol , diethylstilbestro l Estrogen s N/A b 4-Hydroxytamoxife n

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 5 2 of

5 5 IThyroi d relate d | Human thyroi dhormon e receptor- a| Triiodothyronin e (T 3 ) | aTh e huma n estroge n respons e element wa s teste da s aci s element ; al l other endpoint s relevant

to endocrin e modulatio n were teste da s tran s factors . " No t applicable . 

### Cytotoxicit

y wa s firs t

determine d usin g th e Dimethy l thiazoly l dipheny l tetrazoliu msal t assa y an d 24

- hou r chemica l exposur e to fiv e tes t

concentrations , wit

h a n upper concentratio n o f

5 0 |j M an d 10-fol d seria l dilutions . Th e maximu mtolerate d concentratio n (MTC ) wa s derive d a s one-thir d th e cytotoxi c ICso , or i f n o ICs o valu e wa s determined , the n th e MT C wa s set a t 10 0 uM . Tes t

compound s wer

e the n assaye d a

t seve n differen t

concentration s i n th e rang e o f 0.0014-10

0 |aM , startin g a

t th e MTC , followe d b y three-fol d seria l dilutions . Afte r

a24-hou r exposure , cell s wer

e harvested , reporte

r gen e product s isolate d an d reverse-transcribe d usin g fluorescent-tagge d primers , an d resolve d b y hig h resolutio n ge l electrophoresis . Fro mth e resultin g data , th e lowes t effectiv e concentratio n that activate d acis-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 significant chang e fro mth e concurrent negativ e control) . 2 . Compariso n to EDS P Tie r 1 I n Vitro Screenin g Requirement s Th e ToxCastTM dat asupplement , an d i n som e cases , ma

y fulfil l th e i n vitr o dat arequirement s o f th e EDS P Tie r 1 ER bindin g assay , A R bindin g assay , ER transactivatio n assa y an d aromatas e inhibitio n assay . I n addition , thes e dat aprovid e important OSR I tha t shoul d b e considere d i n aweight-of-the

evidenc e evaluatio n o f

functionall y equivalent

informatio n supportin g th e fulfillmen t o f severa l EDS P Tie r 1 mammalia

n i n viv o screening tests . E Rbindin g assay : Th e EDS P Tie r 1 E R bindin g assa

y require s that

bindin g b e evaluate d usin g ra t E R derive d fro muterin e tissu e take n fro movariectomize d Spragu e Dawle y rat s (EP A 2009d) . I n th e ToxCast TM cell-fre e HT S assays , bindin g to bovin e an d human , bu t no t rat , E Rwa s evaluated . I n th e EDS P Tie r 1 screening assay , th e concentratio n o f 3 

### H-l?p-estradio l ligan d use d i n th e assa y i s 1 nM an d th e tes t

chemical s ar

e evaluate d a

t concentration s o f

1 x 10"' ° to 1 x 10" 3 

### M (EP A 2009d) . I n th e ToxCastTM assay , th e concentratio n o f radiolabele d estradio l varie d dependin g o n th e ER sourc e (bER : 7 x 10" 1 0 

### M ; hER : 1 x 10"' ° M) , bu t wa s generally simila

r to that recommende d i n th e EDS P Tie r 1 ERbindin g assay ; however , compound s were teste d a t a n initia l concentratio n o f

2. 5 x 10" 5 

### M , an d i f positiv e fo r

bindin g activity , were furthe r evaluate d a

t concentration s o f

2. 3 x 10" 8 

### - 5 x 10" 5 

### M . Thi s concentratio n rang e i s narrower tha n th e rang e recommende d i n th e EDS P Tie r 1 screenin g assay , bu t stil l shoul d b e adequate to detec t activit y i n amoderat

e concentratio n range . Androge n recepto r

bindin g assay : Th e EDS P Tie r 1 scree n A R bindin g assa

y require s tha t

bindin g b e evaluate d usin g ra

t A Rderive d fro mprostat

e tissu e take n fro mcastrate d Spragu e Dawle y rat s (EP A 2009b) . Th e concentratio n o f 3 

### H-R188 1 ligan d use d i n th e assa y i s 1 nM an d th e tes t

chemical s ar

e evaluate d a

t concentration s o f

1 x 10~

9 

### to 1 x 10 ^ M . I n th e ToxCast TM cell-fre e HT S assay , bindin g to bot h ra t an d human androge n receptor s wa s evaluated , s o i t provide s amor

e comprehensiv e scree

n tha n doe s th e EDS P Tie

r 1 battery . Radiolabele d R188 1 wa s use d a s th e radioligan d i n thi s assa y an d concentration s varie d dependin g o n th e A Rsourc e (rAR : 4.5 9 x 10" 9 

### M ; hAR : 3 x 10" 1 0 

### M) , bu t wer e

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 5 3 of

5 5 simila r to that recommende d i n th e EDS P Tie r 1 A Rbindin g assay . Finally , compound s wer e teste d i n th e ToxCastTM cell-fre e HT S assa y a t a n initial concentratio n o f

2. 5 x 10~ 5 

### M , an d i f

positiv e fo r 8 ^

— 

### bindin g activity , furthe r

evaluate d a

t concentration s o f

2. 3 x 10 " - 5 x 1 0 M . Thi s concentratio n rang e i s slightl y narrower

than tha t recommende d i n th e EDS P Tie r 1 scree n A Rbindin g assa

y guidelin e ( 1 x 10" 9 

### - 1 x 10"4 

### M ; EPA 2009b) , bu t stil l shoul d b e adequate to detec t activit y i n a moderat e concentratio n range . ERtransactivatio n assay : Th e EDS P Tie r 1 scree n ERtransactivatio n assa

y require s that

th e assa y b e conducte d i n hER-HELA-990 2 cells , whic h expres s th e huma n ER- aan d hav e been stabl y transfecte d with aluciferas e reporter

gen e linke d to a

n upstrea mvitellogeni n estrogen-responsiv e element . Tes t compound s ar

e evaluate d a

t concentration s o f

1 x 10"

9 

### t o 1 x 10" 3 

### M . Finally , ates t chemica l i s considere d positiv e fo r

ERtransactivatio n i n th e EDS P Tie r 1 assa y i f th e maximu mrespons e induce d i s a t leas t 10 % o f that induce d b y 1 nM 17p-estradio l (EP A 2009a) . In th e cas e o f

th e ToxCastTM cell-base d HT S assays , detail s regardin g th e gen e reporter syste mwere no t available ; however , activit y a t th e huma n ER- awa s assessed an d tes t chemical s wer

e evaluate d a

t asimila r rang e o f concentration s (1. 2 x 10" 9 

### - 0. 9 x 10 ^ M ) a s recommende d i 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 d i n Tie

r 1 screening , an d i n mos t

cases , positiv e contro l compound s wer e no t tested . However , i f

achemica l elicite d alo w level o f

activit y a t th e highes t

concentratio n teste d i n th e cell

base d assay , i t

ca n b e assume d that

i t woul d b e negativ e accordin g to th e EDS P Tie r 1 screening criteria . It shoul d b e note d that

th e ToxCastTM program' s cell-based HT S assa y provide s mor

e robus t dat athan that

require d i n th e EDS P Tie

r 1 ERtransactivatio n assa

y becaus e i t

als o examine d th e tes t chemical' s abilit

y to ac t a s a n antagonis t

a t th e ER-a . Th e multiple x transcriptio n reporte r assa y als o examine d transactivatio n o f th e human ER-a . In thi s case , th e assa

y i s conducte d i n HepG 2 cell s (instea d o f HELA-990 2 cells) , an d th e outpu t fro mth e reporte r gene an d ho w i t

i s measure d i s differen t

fro mthat indicate d i n th e EDS P Tie

r 1 screenin g assay . HepG 2 i s aperpetua l cell line , whic h wa s derive d fro mth e liver tissu e o f a15-yea r ol d Caucasia n America n mal e wit

h awel l differentiate d hepatocellula r carcinoma . Th e HepG 2 cell s an d it s derivative s ar

e ofte n use d a s amode l syste mfo r studie s o f live r metabolis man d xenobiotic s toxicity becaus e o f

their metaboli c capabilities . The 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 to that recommende d i n th e EDS P Tie r 1 screen . Finally , a s wit

h th e ToxCast TM cell-base d HT S assay , th e dat aoutput s (LE L values ) ar

e differen t tha n thos e require d i n EDS P Tie r 1 screening , an d positiv e contro l compound s wer e no t tested . However , i f

achemica l elicite d alo w level o f

activit y a t th e highes t

concentratio n teste d i n th e multiple x transcriptio n reporte r assay , i t ca n b e assume d tha t

i t woul d b e negativ e accordin g to th e EDS P Tie r 1 screening criteria . Aromatas e assay : Th e EDS P Tie r 1 scree n aromatas e assa

y require s that

enzyme activit

y b e evaluate d usin g human recombinan

t microsome s containin g CYP1 9 plu s reductase . Radio-labele d androst-4

- ene-3,17-dione a

t aconcentratio n o f

1 x 10" 8 

### M i s use d a s th e enzyme substrate an d tes t compound s are evaluate d a

t concentration s o f

1 x 10" 1 0 

### M - 1 x 10" 3 

### M (EPA . 2009c) . I n th e ToxCastTM cell-fre e HT S assay , huma n aromatas e activit y wa s evaluate d usin g di(benzyloxymethoxy ) fluorescein , whic h produce s afluorescent

produc t a s th e enzym

e substrate . Compound s wer e initiall y screened a

t a

### Do w AgroScience s LL C Repor t ID : 10176

2 Pag e 5 4 of

5 5 singl e concentratio n o f

1 x 10" 5 

### M , an d thos e chemical s that were positiv e i n th e initia

l scree n wer e the n ru n usin g eight

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

than th e rang e recommende d i n th e EDS P Tier 1 screenin g assay , bu t

stil l adequat

e to detec t activit y i n amoderat

e concentratio n range . It shoul d b e note d that

th e ToxCastTM HT S dat ase t include s informatio n that shoul d b e usefu l to submi t a s OSRI , an d i n fact , goe s beyon d th e i n vitr o testin g require d i n th e EDS P Tie r 1 screenin g progra mto provid e additiona

l informatio n o n th e potentia l to interac t

wit h th e EA T systems , includin g thyroid-relate d receptors , a s wel l a s progesterone receptors . It doe s not , however , directl y addres s EDS P Tie r 1 steroidogenesi s i n vitr o tes t requirements .

### Do w AgroScience s LLC Repor t ID : 10176

2 Pag e 5 5 of

5 5 Appendi x C - Reference s Environmenta

l Protectio n Agenc y (EPA) . (2009a) . Endocrin e Disrupter Screenin g Progra

mTes t Guidelines . OPPTS 890.1300 : Estrogen Receptor Transcriptiona

l Activatio n (Human Cel

l Lin e (HeLa-9903)) . EPA-740-C-09-006 . Octobe r 2009 . Environmental Protectio n Agenc y (EPA) . (2009b) . Endocrin e Disrupto r Screenin g Progra

mTes t Guidelines . OPPT S 890.1150 : Androge n Recepto r Bindin g (Ra

t Prostat e Cytosol) . EPA-640-C-09

- 003 . Octobe

r 2009 . Environmenta

l Protectio n Agenc y (EPA) . (2009c) . Endocrin e Disrupto r Screening Progra

mTest Guidelines . OPPT S 890.1200 : Aromatas e (Huma n Recombinant) . EPA-740-C-09-004 . Octobe r 2009 . Environmenta

l Protectio n Agenc y (EPA) . (2009d) . Endocrin e Disrupto r Screenin g ProgramTest Guidelines . OPPT S 890.1250 : Estrogen Receptor Bindin g Assa

y Usin g Ra

t Uterin e Cytosol (ER

RUC) . EPA-740-C-09-005 . Octobe r 2009 . Environmenta

l Protectio n Agenc y (EPA) . (2010) . ToxCastTM Progra mwebsite . Availabl e a

t www.epa.gov/ncct/toxcast / index.html . Judso n R.S . et

al . (2009) . In vitr o Screening o f Environmenta

l Chemical s for

Target Testin g Prioritization-Th e ToxCas t Projects ; Supplementa

l Information . Se e http://www.epa.gov/ncct/toxcast / Supplementa

l dat aan d figure 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 d D.J . Dix . (2010) . In vitr o screening o f environmenta

l chemical s fo r

targete d testin 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 environmental chemical s o n key transcriptio n regulator s an d correlatio n t o toxicit y endpoint s withi n EPA' s ToxCast TM Program . Chemica

l Research i n Toxicolog

y 23:578-590 .