N R 481049-0 1 

# syngent a

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

DAT A REQUIREMENT(S) : No t Applicabl e AUTHOR(S) : W . Thoma s Beidler , Ph.D . Charle s Breckenridge , Ph. D J . Charle s Eldridge , Ph.D . Jame s T . Stevens , Ph.D. , DABT , FAT S Jonatha n M . Akins , Ph.D. , DAB T COMPLETIO N DATE : May 20 , 

### 201 0 

### PERFORMIN G LABORATORY : Syngent aCro p Protection , Inc . 41 0 Swin g Roa d Greensboro , N C 2740 9 US A LABORATOR Y PROJEC T ID : SUBMITTER/SPONSOR : Repor t Number : TK002342 4 Tas k Number : TK0023424 Syngent aCro p Protection , Inc . 41 0 Swin g Roa d Pos t Offic e Bo x 1830

0 Greensboro , N C 27419-830 0 US A NOTE : Thi s repor t

contain s colo r

page(s) . VOLUM E J ^ O F J

_O F STUD Y PAG E i O F 6 3

### STATEMEN T O F DAT A CONFIDENTIALIT Y CLAIM S STATEMEN

T O F N O DATA CONFIDENTIALIT Y CLAIM S UNDE RSPECIFIED FIFRA PROVISION S N o clai mo f

confidentiality i s mad e fo r an y informatio n containe di n thi s study o n th e basi s o f it s fallin g withi n th e scop e o f FIFRA Sectio n 10(d)(l)(A ) (disclose s manufacturin g o r quality contro l processes) , (B ) (disclose s th e detail s o f method s fo r

testing , detectin g o r measurin g th e quantity o f an y deliberatel y adde dinert ingredien t

o f apesticide) , o r

(C ) (disclose s th e identity o r

percentag e quantity o f

an y deliberatel y adde diner t ingredien t

o f a pesticide) . Compan y Syngent aCro p Protection , Inc . Company Agent : Da

n Campbel l

Dat e Notwithstandir/ g th e marking s (CIBA-GEIG Y - CONFIDENTIAL ) foun do n page s 54-5 5 w e are makin g n o supplemental claim s o f confidentiality unde r

FIFRA §(10) ao r

(b) . Syngent ai s th e owne r

o f thi s study . Syngent aha s submitte dthi s materia l t o th e Unite d State s Environmenta

l Protectio n Agenc y specifically unde r th e provision s containe din . .. . FIFRA a s amende dand , hereby , consent s t o us e an d disclosure o f thi s material b y EPA . . * ! accordin g t o FIFRA . Notwithstandin g th e wordin g o f ou r markin g TRADE*SECRET , thi s marking , b y itself, convey s n o supplemental claim s o f confidentiality unde r

FJFJ^A ; • . * . Section s 10(a

) or 10(b) (addressin g protectio n o f trad e secret s and commercia l ari dfinancia l information) . I n submittin g thi s materia l t o EP A accordin g t o metho dan dforrg^ t ••••• • requirement s containe di n P RNotic e 86-5 , w e d o no t waiv e an y protectio n ortrigi t involvin g thi s material tha t

woul dhav e bee n claime db y th e compan y i f thi s material had'no t

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

FIFR A Sectio n 3 (concernin g dat aexclusivity an d dat acompensation ) o r FIFR A S ectio n 10(g)».... " (prohibitin g disclosur e t o foreig n an dmultinationa l pesticid e companie s o r thei r agents) .

Report Number : TK002342 4 Pag e 2 o f 6 3

### GOO D LABORATOR Y PRACTIC E COMPLIANC E STATEMENT

A s thi s volum e contain s comment s an d assessment s o f informatio n an d dat ai n report s submitte d separately , aGoo d Laborator y Practic e Complianc e Statemen t i s no t appropriate . Study Director : Ther

e i s n o GLP Stud

y Director

fo r thi s volume .

W . Thoma s Beidler , Ph.D . Dat e Autho r Representativ e o f

Submitter/Sponso r

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

Report Number : TK002342 4 Pag e 3 o f 6 3

### TABLE O F CONTENT S

STATEMEN T O F DAT A CONFIDENTIALIT Y CLAIM S 2 GOO D LABORATOR Y PRACTIC E COMPLIANC E STATEMEN T 3 TABL E O F CONTENT S 4 1. 0 EXECUTIV E SUMMAR Y 6 2. 0 INTRODUCTIO N 8 3. 0 RATIONAL E FO RACCEPTANC E O F SIMAZINE OSR I FO R SPECIFI C TIE R1 ASSAY S 9 3. 1 Amphibia n Metamorphosi s (OPPTS890.i l 00 ) .......................................... . 9 3. 2 Androge n Receptor Bindin g (OPPTS890.i l 50 ) ....................................... . 1 3 3. 3 Aromatas e -Human Recombinan t (OPPT S 890.1200 ) .............................. . 1 5 3. 4 Estroge n Recepto r Bindin g Assa y Usin g Rat Uterin e Cytoso l (ER

RUC ) (OPPT S 890 . 1250 ) ........................................................................... . 1 7 3. 5 Estroge n Recepto r Transcriptiona l Activatio n - Huma n Cel l Lin e HeLa-990 3 (OPPT S 890 . 1300 ) .................................................................. . 2 0 3. 6 Fis h Shor t TermReproductio n (OPPT S 890 . 1350 ) ................................... . 2 3 3. 7 Hershberge r - Ra t (OPPT S 890.1400 ) ....................................................... . 2 4 3. 8 Femal e Puberta l - Rat (OPPT S 890.1450 ) ................................................. . 2 6 3. 9 Mal e Puberta l - Rat (OPPT S 890.1500 ) ..................................................... . 2 8 3.1 0 Steroidogenesi s - Human Cel l Lin e H295 R(OPPT S 890.1550 ) .............. . 3 1 3.1 1 Uterotrophi c - Ra t (OPPT S 890 . 1600 ) ...................................................... . 3 3 4. 0 REPRODUCTIV E AN D DEVELOPMENTA L TOXICIT Y . • ;• ;

CHARACTERISTIC S O F SIMAZIN E I'T l " . & 5. 0 CONCLUSIO N ' ' * • • 6. 0 REFERENCE S APPENDICE S SECTIO N APPENDI X 1 ToxCast TM • • . • • • 3 7 

### ••••• • • 

## .4 0 APPENDI X 2 Assay Descriptio n Shee t fro mCalipe r

LifeScience s -Bovin e Estrogen * Receptor Bindin g Assa

y *?.?4 9 APPENDI X 3 Assay Descriptio n Shee t fro mCalipe r

LifeScience s -Human Estroge n Recepto r Bindin g Assa y 5 1 APPENDI X 4 Titl e Pag e an dMRI D Numbe r o f Previousl y Submitte dStudie s 5 3

Report Number : TK002342 4 Pag e 4 o f 6 3

### LIS T O F TABLE S

TABLE 1 Othe r

Scientificall y Relevan t

Informatio n (OSRI) : Existin g Simazin e Studie s Whic h Satisf

y EDS P Tie

r 1 Assa y Requirement s 7 TABL E 2 OSR I an dFIFR A 40CF RPart 15 8 Studie s Whic h Includ e Examinatio n o f th e Thyroi dand/o r

Thyroi dHormone s 1 1 TABL E 3 EP A Guidelin e Reproductiv e an d Developmenta l Toxicit y Studie s o n Simazin e Technical 3 6

>••• • • • • 

## : :

. . . >••• • • • • 

## • • « • • * * ••••• •

Report Number : TK002342 4 Pag e 5 o f 6 3

### 1. 0 EXECUTIV E SUMMARY

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

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

i n human s that

i s simila r t o a n effec t

produce db y anaturall y occurrin g estrogen , o r

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

"shal l provid e fo r

th e testin g o f

al l pesticid e chemicals . " USEP A develope daTier

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

th e T1 S assay s wa s published , an do n Decembe r 10,2009 , USEP A sen t

th e tes t order s (DCI#EDSP

- 080807 ) fo r th e T1 S t o registrant s o f

th e simazine . Th e technica l registrant s o f simazin e hav e forme da consortiumto addres s th e simazin e tes t order . Th e Agenc y ha s als o indicate dtha t "othe r scientificall y relevan t

information " (OSRI ) that i s functionall y equivalen t t o th e informatio n obtaine dfro mth e Tier

1 assay s may b e utilize di n lie u o f conductin g th e Tie r

1 assays . Th e objectiv e o f

thi s documen

t i s t o identif

y th e mos t relevan t OSR I that i s availabl e fo r

simazin e an dt o provid e th e justificatio n fo r waiver s o f

al l eleve n EDS P Tie r

1 assays . Th e simazin e consortiu midentifie daspecifi c se t o f OSRI that

specificall y establishe s that al l o f th e Tier 1 screen s o f

th e Endocrin e Disrupte r

Screenin g Programhav e bee n previousl y conducte dan dsatisfie dfo r simazin e (Tabl e 1) . Simazin e ha s bee n evaluate di n mos t o f th e EDS P Tie r 1 assay s and/o r

substantiall y simila

r assays ; thes e OSR

I studie s suppor

t th e registratio n an dreregistratio n o f simazine , an dsome o f

th e OSR I studie s were conducte d unde r th e directio n o f

th e EP A Offic e o f

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

o f th e ToxCas t

progra m(Judso n e t

al. , 2010 ) o r

a s par t o f th e EDS P validatio n program . Th e ToxCas t

assay s evaluate destrogen , androge n an d thyroi drecepto r bindin g an d . ., . aromatas e inhibitio n usin g th e NI H Chemica l Genomic s Center , Attagene , crvkkwascresR * • testin g facilities . Thes e assay s fulfi l th e requirement s o f th e OPPT S Series;89§.£l50 , * 890.1200 , 890.1250 , an d890.130 0 guidelines , an dprovid e aweigh t o f th e evideoc e tha f

»^ » meet th e requirement s o f

th e guidelin e 890.110 0 tes t wit h regardt o thyroi dresponse . . .

Report Number : TK002342 4 Pag e 6 o f 6 3

### TABLE 1 Othe r Scientificall y Relevan t

Informatio n (OSRI) : Existin g Simazin e Studie s Whic h Satisf

y EDS P Tie r

1 Assay Requirement s OPPT S Guidelin e

890.110 0

890.115 0

890.120 0

890.125 0

890.130 0

890.135 0 890.140 0 890.145 0 890.150 0 890.155 0

890.160 0 Tie r

I Scree n

Amphibia n Metamorphosi s

Androge n Recepto r Bindin g Assay (rat ventra l prostate) Aromatase , placenta l and/o r recombinan t Estroge n Recepto r Bindin g Assa

y (rat uterus ) Estroge n Recepto r Transcriptiona l Activatio n

Short-ter mFis h Reproductio n Assay Hershberge r

Assa y Femal e Puberta l Mal e Puberta

l Steroidogenesi s H295 R Uterotrophi c Assa

y OSR

I Reference(s ) Houck , 200 9 Zorrillaetal. , 201 0 Stoke r et al. , 200 5 Birkho j e t al. , 200 4 Judsonetal.,201 0 Bie r & Oliveira , 198 0 Taietal. , 1985 a Taietal , 1985 b McCormic k

& Green , 198 8 McCormic k

e t al. , 198 8 Fan g e t al. , 200 3 Kojimaetal.,200 4 Birkho j e t al. , 200 4 Judsonetal.,201 0 Hinfrayetal.,200 6 Judsonetal.,201 0 Blai r e t al. , 200 0 Tennan t e t al. , 1994 b O'Conno r etal. , 200 0 Judsonetal.,201 0 Conno r etal. , 199 6 Legle r etal. , 200 2 Balague r e t al. , 199 6 Kojim ae t al. , 200 4 Judsonetal.,201 0 Maye r an dSanders , 197 5 Birkho j e t al. , 200 4 Zorrillaetal. , 201 0 Stoke r et al. , 200 5 Sanderso n etal. , 200 1 Tenan t etal . 1994 a Conno r etal. , 199 6 O'Conno r e t al. , 200 0 Cite d OSR I Identica l t o Tie r

1 Scree n Supplementa l Supplemental Supplementa

l Supplementa l Supplementa l Supplementa l Supplementa

l Supplementa l Supplementa l Supplementa l Substantiall y similar Substantiall y simila

r Substantiall y similar Substantiall y similar Supplementa l Substantiall y simila r Substantiall y simila r Substantiall y similar Substantiall y similar Substantiall y simila

r Substantiall y similar Substantiall y similar Substantiall y similar Substantiall y simila

r Substantiall y similar Substantiall y similar / exceed s Requirement s Substantiall y similar Ye s Ye s Substantiall y similar Substantiall y similar Substantiall y simila

r Substantiall y simila r Comment s ToxCas t thyroi dgen e assay s EPA' s puberta l female scree n EPA' s pubertal mal e screen Hershberge r

scree n Thyroi drecepto r

gen e assay s 2 1 -da y dermal stud y i n rabbit s 90-da y feedin g study i n rat s 90-day feedin g stud y i n dog s 1-yea r feedin g study i n dog s 2-yea r chroni c toxicity / oncogenicit y feedin g stud y i n rat s Recombinan t

ra t A R hA Rexpresse di n cell s hA Rexpresse di n cell s Ra t an dhuma n A Rbindin g plu s huma n A Rreporte r

assay s Trou t (brai n & ovarian ) CPY1 9 Huma n aromatas e gene s Rat uterin e cytosoli c recepto r Ra t uterin e cytosoli c recepto r Ra t uterin e cytosoli c recepto r Bovin e an dHuma n E Rbindin g hE Rtransfecte dt o MCF- 7 cell s hE Rtransfecte dt o T47JD •eif e hE Rt(anjf$4tg dt o He*Ua»c*ell » hE Rtjmsfjctj dint o CHO . hE Rgenes , nfUltipl e fc(pe s . • • • • • • • Fis h Ful l Lif

e Cyc£«sWr

/ • • • • • * * • • Antagonis mTejftf

l •• ' • • • • EDS P Tie r

1 scffe f a, , * EDS P Tie r 1 screen H295 Rcell s Adul t ovariectomize drat s Immatur e rat s Adul t ovariectomize drat s 

### Report Number : TK002342 4 Pag e 7 of6 3A s depicte d i n th e tabl e above , simazin e ha s bee n thoroughl y teste deithe r b y usin g th e actua l Tie r 1 assay protoco l and/o r

substantiall y simila

r protocol s fo r

al l o f th e endpoint s require d b y th e Tie r

1 assay s o f

th e USEP A Endocrin e Disruptio n Screenin g Progra m(EDSP) . Simazin e ha s als o bee n teste d fo r endocrine-relate dendpoint s vi aexistin g EPA-require d guidelin e studie s (e.g. , two-generatio n reproductio n study , developmenta l toxicit y studies , fis h ful l lif

e cycl e study , etc.) . Thes e highe r tie r studie s provid e informatio n that i s "functionall y equivalent " t o tha t obtaine dfro mth e Tier

1 assay s an dca n b e utilize di n a weigh t o f evidenc e determinatio n o f endocrin e disruptio n potential . Therefore , th e simazin e consortiu mrequest s waiver s fo r th e eleve n Tie r

1 assay s i n th e EDS P tes t order . 

### 2. 0 INTRODUCTIO N Th e Federa l Foo dDru g an dCosmeti c Ac t require dth e U . S . Environmenta l Protectio n Agenc y (USEPA ) to "develop ascreenin g program , usin g appropriat e validate dtes t

system s an dother scientificall y relevan t

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

i n human s tha t

i s simila r t o a n effec t

produce db y anaturall y occurrin g estrogen , o r

other effect s a s [EPA] ma

y designate. " Sectio n 408(p)(3 ) specificall y require d that th e Administrato r

"shal l provid e fo r

th e testin g o f

al l pesticid e chemicals. " EPA develope d aTier

1 Screenin g (T1S ) batter y o f eleven separate assay s to screen fo r potentia l endocrine-modulatin g substances . USEP A publishe daserie s o f guideline s fo r

th e T1 S assays , an do n Decembe r 10 , 2009 , USEP A sen t

th e tes t order s fo r

T1 S (DC I #EDSP

080807 ) to registrant s o f

th e simazine . Th e technica l registrant s o f

simazin e hav e forme da consortiu mt o addres s th e simazin e tes t order . EPA als o indicate d that

"othe r scientificall y relevan t

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

t o th e informatio n obtaine dfro mth e Tie r 1 assay s may b e utilize di n lieu o f conductin g th e Tie r

1 assays . However , EP A indicate dth e OSR I mus t mee t specifi c an d functional equivalenc e criteri awhic h includ e th e following : Dat aar e o f

hig h quality an d conducte dusin g soun dscientifi c practises. . • I * • Study conducte di n accordanc e wit h accepte dscientifi c methodolog y er protocol . Deviation s fro mTie r

1 assa y protocol s mus t

b e identifie di n th e OSRUfiaiionale * Impac t o f an y deviation s fro mth e protoco l o n th e result s o f th e studie ^ justifyin g a * waive r reques t shoul d b e discusse d i n th e rationale . • • • • Th e objectiv e o f

thi s documen t

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

simazin e andt®» » provid e th e justificatio n fo r waiver s o f

al l o f th e eleve n EDS P Tier

1 assays . Th e simazin e consortiu midentifie dase t o f th e existin g simazin e OSRI , whic h indicat e tha t simazin e ha s bee n alread y evaluate d i n th e EDS P Tie r 1 screen s and/o r

substantiall y similar

assay s fo r

al l eleve n assays . Therefor e th e studie s cite dherei n shoul djustif y awaive r

o f th e Tie r 1 screenin g battery :

• 890.110 0 - Amphibian Metamorphosi s (Frog ) • 890.1150

- Androge n Recepto r Bindin g (Rat

Prostate ) Report Number . TK002342 4 Pag e 8 o f 6 3• 890.120 0 - Aromatas e (Human Recombinant ) • 890.125 0 - Estroge n Recepto r Bindin g • 890.130 0 - Estroge n Recepto r Transcriptiona l Activatio n (Human Cel l Lin e HeLa

9903 ) • 890.135 0 - Short-termFis h Reproductio n • 890.140 0 -Hershberge r (Rat ) • 890.145 0 - Femal e Puberta l (Rat) • 890.150 0 - Mal e Puberta l (Rat) • 890.155 0 - Steroidogenesi s (Huma n Cel l Lin e - H295R ) • 890.160 0 - Uterotrophi c (Rat ) Simazin e ha s bee n thoroughl y teste deithe r b y usin g th e actual Tie r

1 assay protoco l and/o r substantiall y simila

r protocol s fo r

al l o f th e endpoint s require db y th e Tie r 1 assay s o f

th e USEP A Endocrin e Disruptio n Screenin g Progra m(EDSP) . Simazine ha s als o bee n teste d fo r endocrine-relate d endpoint s vi aexistin g EPA-require dguidelin e studie s (e.g. , two-generatio n reproductio n study , developmental toxicit y studies , fis h ful l lif

e cycl e study , etc.) . Thes e higher tie r studie s provid e informatio n that i s "functionall y equivalent " t o that obtaine dfro m th e Tie r 1 assay s an dca n b e utilize di n aweigh t o f evidenc e determinatio n o f endocrin e disruptio n potential . Therefore , th e simazin e consortiu mreques t waiver s fo r

th e eleve n Tie

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

### 3. 0 RATIONAL E FO RACCEPTANC E O F SIMAZINE OSR I FO R SPECIFI C TIE R1 ASSAY S 3. 1 Amphibia n Metamorphosi s (OPPT S 890.1100) Purpos e o f

Assa y Th e purpos e o f

th e amphibia n metamorphosi s assa y (AMA ) i s t o scree n fo r potentia l thyroi d systemdisruptin g compounds . Throug h th e hypothalamus-pituitary-thyroi d(HPT ) glarf d•* ; axis , thyroi dhormon e regulate s man y processe s i n organism s includin g de^eicprpent , . somati c growth , metabolism , an d reproduction . Metamorphosi s mXenopu S laevi s i s ajvell« . studie dmode l fo r

thyroi dhormon e function . • • •• • . . . Assa y Methodolog y Summar y Th e OPPT S 890.110 0 protoco l involve s a21-day exposure o f tadpole s t o the*fe*s t

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

developmental'stag e 51 , whic h can b e reache dapproximatel y 1 4 day s post-fertilization . Fou r concentration * oj^* » th e tes t substanc e plu s adilutio n wate r control , an dfou r

replicat e tank s pe r

concentration , ar e employe d i n aflow-throug h system . At tes t initiation , atota

l o f 2 0 stag e 5 1 Xenop mlaevi s tadpole s ar e place d i n eac h tank . Environmenta l condition s o f 22±1°C , p H o f

6.5-8.0 , an da 1 2 hour : 1 2 hour

/ light:dar k photoperio dare employed . Fiv e tadpole s fro meac h treatmen t tan k ar e randoml y remove dat da y 7 fo r evaluatio n o f

we t weigh t an drat e o f

metamorphosi s developmenta l stage , whol e bod y length , hin dlim b length , an dsnout-ven t

length . O n Day 2 1 th e remainin g tadpole s are evaluate dfo r thes e sam

e endpoints . Histologica l evaluatio n o f thyroi d tissu e i n asub-sampl e o f fiv e tadpole s fro meac h replicat e tan k i s als o performe di n Repor t Number : TK002342 4 Pag e 9 o f 6 3orde r t o lin k an y observe d change s i n th e rat e o f metamorphosi s t o morphologica l change s i n th e primary thyroi d synthesi s organ . Othe r Scientificall y Relevan t

Informatio n Simazin e ha s arobus t

mammalian an dfis h toxicity databas e tha t provide s justificatio n fo r adequat e testin g o f

th e hypothalamic-pituitary-thyroi d(HPT ) an dshoul dprovid e th e basi s fo r a waive r o f th e AMA . Th e origina

l basi s fo r

EDSTA C (1996 ) to ad d th e AM A to th e EDS P Tier 1 batter y i s indicate d i n th e followin g quote : ". . .there ar e n o clea r marker s o f

thyroi d hormon e actio n that

coul d b e use dwithi n th e contex t o f aT1 S assay . I n contrast , tai l resorptio n i n amphibia

n metamorphosi s represent s an assay whic h utilize s specifi c thyroi d hormone-dependen t

effect s a s a n endpoin t

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

th e Amphibia n Metamorphosi s Assay : "Th e AM A i s ascreenin g assa y intende dt o empiricall y identif y substance s whic h may interfere with th e norma l functio n o f th e hypothalamic-pituitary-thyroi d(HPT ) axis . Th e AM A represent s ageneralize dvertebrat e mode l t o th e exten t that i t i s base do n th e conserve dstructur e an dfunction s o f thyroi dsystems . I t

i s no t intende dt o quantif

y o r confirmendocrin e disruption , o r to provide aquantitativ e assessment o f risk , bu t only provid e evidenc e that

thyroi dregulate dprocesse s ma y b e sufficientl y perturbe dt o warran t mor e definitiv e testing. " Wit h respec t

t o simazine , ther e hav e bee n numerou s OSR I studies , a s wel l a s FIFRA 40CF R Par t 15 8 studies , that

examine d th e thyroi da s apotential target organ , an d thes e studie s provid e extensiv e an dhig h quality dat aaddressin g th e potentia l o f thyroid-relate deffects . Du e t o th e conservatio n o f th e thyroi drecepto r

acros s specie s (W u e t

al. , 2007) , th e existin g dat ashoul d serv e a s OSR I fo r potentia l thyroi deffect s i n th e AM A tes t model . Thes e studie s (Tabl e 2 ) wer e no t

limite dt o analyzin g potentia l effec t t o thyroi dhormones ; the y als o include d examinatio n o f

more sensitiv e marker s such a s thyroi dhormon e recepto r bindin g • ; glan dweigh t

an dhistopathology . Th e OSRI studie s o f simazin e are summSfftfctCi n Tabl e 2. "

Report Number : TK002342 4 Pag e 1 0 o f 6 3

### TABL E 2 OSRI an d FIFR A 40CF RPar t 15 8 Studie s Whic h Includ e Examinatio n o f

th e Thyroi d and/o r

Thyroi dHormone s Thyroi dendpoint s T 3 an dT 4 levels , thyroi dweigh t an dhistopatholog y i n femal e rat s T 3 an dT 4 levels , thyroi dweigh t an dhistopatholog y i n mal e rat s T 4 levels , thyroi dweigh t Human an d ra

t thyroi dgenes , a an dp receptors , P agonis t

an d antagonist s Thyroi dhistopatholog y Thyroi dhistopatholog y Thyroi dhistopatholog y Thyroi dweigh t

an d histopatholog y Thyroi dhistopatholog y Reference s Zorrillaetal. , 201 0 Stoke r e t al. , 2005 ; Stoker , 201 0 Birkho j e t al , 2004 Judso n e t al. , 201 0 Bier and Oliveria , 198

0 Taietal.,1985 a TaietaL , 1985 b McCormic k

an dGreen , 198 8 McCormic k

e t al. , 198 8 Comment s

EDS P puberta l femal e stud y (complet e HP T axis ) EDS P puberta l mal e stud y (complet e HP T axis ) Hershberge r

scree n (complet e HP T axis ) EP A ToxCas t

Progra mdat a 2 1 -da y derma l study i n rabbit s (complet e HPT axis ) 90-da y feedin g stud y i n rat s (complete HP T axis ) 90-da y feedin g stud y i n dog s (complete HP T axis ) 1-yea r feedin g stud y i n dog s (complet e HP T axis ) 2-yea r Combine dchroni c toxicity / oncogenicit y stud y i n th e ra t (complet e HP T axis ) 

### Zorrill ae t

al. , 201 0 I n thi s study , twenty timed-pregnan t femal e Wista r

rat s arrive dfro mth e supplie r o n gestatio n da y 1 3 an dwer e house don e per cag e a t 22° C an do n a1 4 hour : 1 0 hour / light : dark

cycle . Th e day o f

birth wa s designate dpostnatal day (PND ) 0 . O n PN D 3 , th e litter s wer e standardize dto 8-1 0 pup s eac h to maximiz e uniformity i n postnatal growt h rates . O n RNlX* * 21 , al l femal e pup s were weane dan d weigh t ranked . Pup s wer e the n assigrtec * s o jthat . treatmen t group s ha dsimila

r bod y weigh t

mean s an dvariances . Littermate S wer £ equall y •

••••• • ' ^ * * 

### distribute damon g th e treatmen t groups , with only 1 pu p fro magiven litter injeacji i treatment group . Afte r

assignment , similarl y treate dfemale s wer e house dtw o pe r cag e ahtl weigies U I • dail y throughou t

treatment . Simazin e wa s administere dat level s o f 0 , 6.25,*?5,*7§ , 15 0 an d 30 0 mg/kg/day . Dosin g bega n o n PND 2 2 an d animal s were sacrifice do n PNET^2/4 3 (21-.; . day exposure ) o r

th e day o f

firs t estru s afte r

PN D 6 2 (41-8 2 day exposure ) fo r th e protoco l endpoin t measures . Trun k

bloo dwa s collecte dan dwa s allowe dt o clo t at roo mtemperature* * fo r 1 h an d the n centrifuged ; th e seru mwa s collecte d an d store dfroze n a t -80° C fo r subsequen t

hormon e assay s include dthyroxin e an dT4 . Thyroid s wer e weighed , fixe dan d evaluate dhistologically . Stoke r e t al. , 200 5 Simazin e wa s teste db y th e U S EP A Reproductiv e Toxicolog y Divisio n o f NHEERL , followin g th e adopte d EDS P Tie r 1 mal e ra

t pubertal screen . Group s o f intac t 2 3 da y ol d weanlin g mal e Wista

r rat s wer e administere dsimazin e at level s o f 0 , 6.25 , 25 , 75 , 15 0 an d 30 0 mg/kg/da y fo r

3 0 days . Preputia l separatio n wa s monitore dfro mpost-nata l day (PND ) Repor t Number : TK002342 4 Pag e 1 1 o f 6 3

### 3 3 onwards . A t necropsy , pituitary , testes , ventra l prostate , latera l prostate , epididymides , an dsemina

l vesicle s wit

h coagulatin g glan dwere removed , weighed , an d examine db y histology . Bloo dwa s analyze dfo r TSH , T4 , T 3 

### , luteinizin g hormon e (LH) , prolacti n (PRL ) an destrogens . Thyroid s wer e fixe dan devaluate dhistologically . Birkho j e t al. , 200 4 Simazin e wa s studie db y Birkho j e t al . (2004 ) i n aHershberge r typ e screen . Group 1 wa s th e castrate dcontro l grou p that receive dth e vehicl e peanu t oil . Group s o f

adul t rat s wer e treate d fo r seven day s wit

h 0. 5 mg/kg/da

y testosterone propionate (TP) sc , T P + flutamid e (contro l antagonist ) o r

T P + simazine , 2 5 mg/kg/day b y gavage . Animal s wer e dose dfo r aperio do f 7 day s at approximatel y th e same tim e every day . Bloo dwa s take n fro mth e anaesthetize d (COa/Oa ) animal s b y externa l heart puncture . Seru mwa s store dat -80° C an duse dfo r hormon e analyses . Th e animal s were the n decapitate dfollowe db y athoroug h necropsy . Several tissues , includin g th e thyroi dglan dwer e dissecte dan dweighed , an dT 4 wa s on e o f th e hormone s measured . Judso n e t

al. , 201 0 Th e ToxCas t

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

things , agonist/antagonis t activity fo r

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

gene s wer e evaluate di n th e presenc e o f simazine . Se e Appendi x 1 an dJudso n e t al. , 201 0 fo r

mor e detail s o n th e methodolog y use di n thes e screen s an dacces s t o th e ToxCas t result s vi athei r websit e links . Bie r an d Oliveira , 1980 Simazin e wa s evaluate d i n 21-da y dermal study i n New-Zealan dwhit e rabbits . I n thi s study , simazin e wa s moistene dwith isotoni c salin e an dapplied , unde r occlusiv e conditions , t o th e ski n (intac t

o r abraded ) o f

rabbit s at dose s o f 0,10,100 , an d1000 mg/k g bod y weight/day . Th e applicatio n wa s performe d6 hour s pe r day fo r at leas t 2 1 consecutiv e days . Thyrqjds;* ; wer e harvested , fixe dan dexamine dhistologically . J * * I * I " * Ta i et al. , 1985 a•':•* : :*'.I " I n thi s 90-day stud y i n rats , fou r groups , eac h consistin g o f 1 0 mal e an d1 0 female 1 

### Cr l jCO B f 

### C D (SD ) B Rrats , receive dsimazin e a t dietary concentration s o f 0 , 200 , 2009,;an

4 400 & pp m correspondin g t o 0,12.6 , 126.0 , 247. 0 mg/k g bod y weigh t i n male s an d0 , 1&3 » 158.9 , and* $ 

### 304. 8 mg/k g bod y weigh t i n females . Thyroid s wer e harvested , fixe dan dexamine d histologically . • • Ta i et al. , 1985 b 

### Dog s receive dsimazin e i n th e die t at concentration s o f

0,200 , 200 0 an d400 0 pp mfo r at leas t thre e months . Th e dietar y concentration s corresponde d t o 0 , 6.9 , 65. 2 an d 133. 6 mg simazin e /k g bod y weigh t fo r male s an d0 , 8.2 , 64. 3 an d136. 7 mg/k g bod y weigh t fo r females . Thyroid s wer e harvested , fixe dan dexamine dhistologically .

Report Number : TK002342 4 Pag e 1 2 o f 6 3McCormic k an d Green , 198 8 Fou r groups , eac h consistin g o f at leas t fou r mal e an dfou r

femal e beagl e dogs , receive d simazin e i n th e diet

a t concentration s o f

0 , 20,100,125 0 pp mfo r 1 year . Th e dietary concentration s corresponde dt o 0.7 , 3.4 , 4 3 mg/k g bod y weigh t i n male s an d 0.8 , 3.6 , an d 44. 9 mg/k g bod y weigh t

i n female s fo r

20,10 0 an d125 0 pp mrespectively . Thyroid s wer e harvested , weighed , fixe dan dexamine dhistologically . McCormic k

e t al. , 198 8 Simazin e wa s administere dorall y a s afee dadmixtur e t o thre e group s o f mal e an dfemal e rat s (8 0 o r 90/sex ) a t dietar y concentration s o f 10,100 , o r

100 0 pp mfo r

a t leas t 10 4 weeks . A n additiona l group o f

rat s (90/sex ) receive d untreate drodent diet an d serve da s controls . Mea n compoun dintake wa s 0.4 , 4.2 , an d45. 8 mg/kg/day fo r 10,100 , an d1000 pp mmales , an d 0.5 , 5.3 , an d63. 1 mg/kg/day fo r 10,100 , an d1000 pp mfemales , respectively . Afte r 5 2 week s o f treatment , up to 1 0 rats/sex/group were sacrificed . Th e remainin g animal s continue do n stud y an dsurvivor s wer e sacrifice dat stud y termination . Thyroid s wer e harvested , fixe dan dexamine dhistologically . Conclusion : Th e simazin e consortiu mrequest s awaive r fo r th e Amphibia n Metamorphosi s scree n base do n th e thoroug h evaluatio n o f simazin e relate dthyroi deffect s i n severa l vertebrat e model s b y anumbe r o f researc h teams . Thes e studie s include da n evaluatio n o f effect s o n th e thyroi dreceptor s i n vitr o an deffect s o n th e HP T axi s throug h thyroi dweigh t and/o r histolog y and/o r

thyroi dhormones . I n addition , simazine wa s include di n th e EPA sponsore dToxCas t

screen conducte dunde r

th e directio n o f

NHEERL . 

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

Assay Th e androge n recepto r

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

may affec t th e endocrin e syste mb y bindin g t o th e androge n receptor . Th e tes t measure s th e ability o f acompoun dt o compet

e wit h a[ 3 H]-ligan d fo r

bindin g i n acytoso l hompgenate.ffi* ; rat ventra l prostat e (VP ) tissue , whic h i s replet e wit h androge n receptors . T$e%R;bindin g . assay doe s no t

asses s functiona l o r

transcriptiona l activity o n th e part o f abfnder."l n addition , t o detectin g possibl e androge n agonist s an d antagonists , th e A Rbindin g assayj s ; • • • complementary i n supportin g an agonisti c o r antagonisti c resul t

i n th e Hershberjee r

• • • •••* • Assay Methodolog y Summar y I.. . t • Th e A Rbindin g assay i s conducte dwith aserie s o f cultur e tube s tha t

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

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

receive s dilution s o f

aknow n hig h affinit y androge n ligand , an dathir dse t receive s dilution s o f

aknow n lo w affinit y ligand . A precis e amoun t o f [ 3 H]-R-1881 , th e hig h affinit y ligand , i s the n dispense d to al l tubes , whic h are the n incubate dovernight a t 4°C . O n th e followin g day aseparatio n ste p isolate s radioactivity boun dt o A Ri n eac h tube , whic h ar e counte db y liqui dscintillation . Th e principl e o f th e assa y i s that

acompetin g ligan dabl e t o bin dt o A Rwil l displac e th e [ 3 H]-labele dligan dan d produc e anegativ e sigmoi ddose

respons e plo t

o f boun dradioactivity . A curve-fittin g compute r progra mca n determin e th e relativ e affinit y o f

th e tes t substanc e b y comparin g it s plo t parameter s versu s thos e o f th e Repor t Number : TK002342 4 Pag e 1 3 o f 6 3know n binders . Th e assa y principl e furthe r

hold s that

acompoun dwhic h displace s n o radiolabele dligan di s unabl e t o bin dt o AR ; it s dose-respons e plo t wil l res t at 100 % maximu mbindin g o f

trace r an dhav e n o downwar dslope . Othe r Scientificall y Relevan t

Informatio n Severa l studie s reporte di n th e literatur e hav e evaluate dth e potentia l o f simazin e t o compete fo r androge n receptor

binding . Thes e studie s wer e conducte do n awid e variet y o f substances , includin g simazine , t o scree n fo r potentia l A Rbinding . I n addition , simazin e wa s evaluate di n th e ToxCas t researc h program , includin g rat an dhuma n A Rbindin g plu s human A Rreporter

assays . OSR I Recombinan t

rA R hAR-linke dreporte r

i n CH O cell s hAR-linke dreporte r

i n CH O cell s Recombinan t

rA Ran dhA R Reference s Fan g e t al. , 200 3 Kojim ae t al. , 200 4 Birkho j e t al. , 200 4 Judsonetal. , 201 0 Comment s Recombinan t

rat A R hA Rexpresse di n cell s hA Rexpresse di n cell s Recombinan t

ra t an dhuma n A R Thes e studie s wer e al l conducte db y reputabl e investigator s usin g widel y accepte dmethods , an dth e result s were reporte di n peer-reviewe dpublications . Fan g e t al. , 200 3 Thi s stud y assesse dbindin g o f 20 2 chemicals , includin g simazine , t o asolubilise drat A R produce db y recombinan t technology . I t

wa s state dthat

thi s recepto r

molecul e ha s th e identica l hormon e bindin g domai n a s huma n AR . [ 3 H]-R188 1 wa s use dfo r competitio n studie s an dhydroxyapetite wa s use dfo r separation . Th e competito r concentratio n rang e wa s 4.2 8 n M t o 42 8 uMolar . Th e metho dappear s t o b e substantiall y simila r t o th e validate d EDS P Tie r

1 protocol , excep t

fo r th e sourc e o f

A R( arecombinan t

protei n versu s extractio n o f ra t prostate) . Kojim ae t

al. , 200 4 •••• • "" * ' Thi s study examine dth e abilit y o f 20 0 chemical s to induc e arespons e i n an*AR*-lsiked. . . reporte r system . Huma n A Rwa s transfecte dint o Chines e hamste r ovary (GHt>)TCell s \$itn«» * vecto r an dalinke dluciferas e reporte r gen e s o that

bindin g o f

aligan dt o A RwewT dpro^u^fj . aluminescen t

signal . Althoug h a

n AR-linke dreporte

r systemha s no t

been tafctfeie dfo r inclusio n i n th e EDS P Tier 1 scree n battery , th e principl e i s presentl y bein g «v«titate dan d . wil l likel y b e adopte di n th e futur e becaus e i t i s highl y specifi c fo r

A Rbinders . SimazhSe**** * wa s on e o f

th e 20 0 teste d chemicals , a t aconcentratio n rang e o f 1 0 nMola

r t o 1 0 uMolar, ? ••• .

• •• • Birkho j e t al. , 200 4 Thi s study wa s simila

r t o th e Kojim astudy . Chines e hamster oocyte s were transfecte dwit h hA Ran daluciferas e reporte r system . Cell s wer e incubate dwith simazin e at twelv e concentration s fro m2 5 nMola r t o 5 0 uMolar . Contro l incubation s with know n A Rligand s establishe d that

reporte d activit

y correlate dto tes t

substanc e binding .

Report Number : TK002342 4 Pag e 1 4 o f 6 3

### Judso n e t al. , 201 0 Th e ToxCas t

group identifie s an dprioritize s reliabl e HT S assay s to asses s amon g other thing s agonist/antagonis t

activity fo r

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

al. , 201 0 fo r

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

websit e links . Deviation s to OPPT S 890.115 0 Protoco l OPPT S 890.115 0 Requirement s Criteri a A Ri n Ra t V P Cytoso l

3 H-competitor

i s R

188 1 Separatio n o f

boun d an dfre e trace r

usin g hydroxyapetit e ge l Fan g e t al. , 200 3 Deviation s t o OPPT S 890.115 0 Recombinan t

Rat A R Non e

Non e Kojim ae t

al. , 200 4 Deviation s t o OPPT S 890 . 1 1 5 0 Human A Ri n CH O cell s N o radiolabel ; bindin g respons e measure dwith a linke dreporter

gene N o separatio n step ; A Rbindin g i s linke dt o areporte r Birkho j e t al. , 200 4 Deviation s to OPPT S 890.115 0 Human A Ri n CH O cell s N o radiolabel ; bindin g respons e measure dwith a linke dreporter

gene N o separatio n step ; A Rbindin g i s linke d t o areporte r Judso n et al. , 201 0 Deviation s t o OPPT S 890 . 1 1 5 0 Recombinan t

Rat A R Non e

Non e Justificatio n fo r

OSR I Binder s reac t

th e sam e wit h bot h huma n an dra t

A R Know n androgen ligand s respon di n an identica l manner Method s ar e equall y vali d 

### Conclusions : Th e simazin e consortiu mrequest s awaive r fo r th e A Rbindin g assay becaus e simazin e ha s bee n alread y teste db y multipl e researcher s usin g substantiall y simila r methodology . Al l use dcommo n method s that ar e widel y accepte da s reliabl e measure s o f A Rbinding , includin g on e that wa s extremel y clos e t o th e validate dprotocol . Eac h stud y evaluate dalarg e variety o f compounds , whic h promote s confidenc e i n al l th e result s b y allowin g compariso n o f

result s fro mother

publications . Methodologica l deviation s shauki . hav e n o impac t

o n justificatio n fo r

usin g OSRI i n consideratio n o f simazirj ? fl$ejactiorfwitl f androgen receptors . 1* 1 3. 3 Aromatas e -Huma n Recombinan t (OPPT S 890.1200) • • • • • i 

### Purpos e o f

Assa y « Th e aromatas e assay i s ascreen intende dt o identif y chemical s that

may affec t

th e endb'cHrie 1 

### syste m(e.g. , steroid o genesis ) b y inhibitin g catalyti c activity o f aromatase , th e enzyme."'* , responsibl e fo r

th e conversio n o f

androgen s t o estrogens . * * * * Assa y Methodolog y Summar y Th e assay selecte db y th e EP A fo r validatio n use s ahuma n placenta l microsomal preparatio n that contain s aromatase . Th e assay measure s th e conversio n o f androgeni c t o estrogeni c structure s i n microsome s isolate dfro mvariou s target tissue s o r cell line s containin g aromatase , o r

recombinan t

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

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

microsom e Report Number : TK002342 4 Pag e 1 5 o f 6 3

### preparations , whic h ca n b e obtaine d fro mcommercia l sources . On e cited sourc e i s manufacture db y Gentes t

(Woburn , MA ) that

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

### H]

- labele dwater . Method s usin g bot h recombinan t

an dplacenta l extract s wer e carrie dthroug h th e EDS P validatio n stage s t o demonstrate equivalence . Th e aromatas e assa

y i s conducte db y incubatin g aradioactiv e substrat e ( 3 

### H-androstenedione ) an dNADP H with th e microsom e preparation . E^ O i s release ddurin g th e conversio n o f androstenedion e t o estrone , an dresult s are reporte da s adirec t measuremen t

o f enzym e turnove r activit y pe r uni t reactio n time . Competitiv e inhibitio n o f aromatas e activit

y can b e detecte db y serial reactio n tube s containin g increasin g concentration s o f th e competito r

o f interest . Othe r Scientifically Relevant

Informatio n On e study , publishe di n apeer-reviewe djournal , reporte dresult s o f simazin e activit

y o n aromatas e with tissu e preparation s containin g th e CYP-1 9 complex . Furthermore , th e ToxCas t researc h progra

mspecificall y teste dsimazin e fo r aromatas e inhibitio n potential . OSR I Microsome s extracte dfro mtrou t brai n & ovar y Huma n CYP C 1 9 gen e Reference s Hinfray e t al. , 200 6 Judsonetal. , 201 0 Comment s Teleos t fis h aromatase , [ 3 H]-labele d substrate Novascreen cell-fre e hCYPC1 9 enzym e inhibitio n assa y 

### Hinfra y e t

al. , 200 6 Thi s study , conducte di n aFrenc h government environmenta

l researc h laboratory , examine d th e ability o f

4 3 divers e chemicals , includin g simazine , t o affec t aromatas e activity . Th e methodolog y use dmicrosome s prepare dfro mrainbo w trou t ovar y an dbrai n tissue . [ 3 

### H]-j» » androstenedion e wa s incubate dfo r 3 0 minute s i n vitro , ± variou s tes t subs$rft££;an d• • • radiolabele destron e wa s extracte dan dcounted . Th e manuscrip t include dSetail s o f

an 9 

### extensiv e validatio n o f

th e protocol . A wid e variet y o f environmentall y sef*M f

€ cheriic^s f includin g some heav y metals , were evaluated . Simazin e wa s incubate dat one«oo*ncentration > 

### (1 0 uMolar) , with brai n an dovaria n microsom e preparations . Judso n et

al . 201 0 Th e ToxCas t

grou p identif

y an dprioritiz e reliabl e HT S assay s t o asses s amon g other thiag s agonist/antagonis t

activit y fo r anumbe r o f nuclea r receptor s an dgene s involve di n sterflftM metabolism . T o examin e aromatas e inhibition , simazin e wa s evaluate dNovascreen' s ADME_hCYPC1 9 assay . Se e Appendi x 1 an dJudso n e t al. , 201 0 fo r

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

websit e links .

Repor t Number : TK0023424 Pag e 1 6 of 6 3

### Deviation s to OPPT S 890.120 0 Protoco l OPPT S 890.120

0 Requirement s Criteri a Eithe r human placenta

l extrac t o r recombinan t

huma n microsome s asCYP-19sourc e Us e [ 3 H]-androstenedion e a s enzym e substrat e Enzym e activity measure da s [ 3 H]-water , produce dfro m substrat e b y enzym e actio n Hinfra y et al. , 200 6 Deviation s t o OPPT S 890.120 0 Aromatas e i n microsome s extracte dfro mfis h brai n an d

ovary N o deviation s Enzym e activit y measure da s [ 3 H]-estrone , th e steroi d produc t o f th e enzyme actio n Judso n e t al. , 201 0 Deviation s t o OPPT S 890.120 0 N o deviations , human recombinan t

Novascree n hCYP19 t Use ddibenzylfluorescei n a s asubstrat e Use dth e fluorescen t

produc t liberate di n th e cleavag e o f oxidize dDB F Justificatio n fo r OSR I Aromatas e i s bioidentica l i n numerou s anima l specie s Substrate s use dhav e bee n validate da s substrate s fo r

CYP

- 1 9 Al l marker s hav e bee n validate da s specifi c product s o f

enzyme i n th e method s use d 

### Conclusions : Th e simazin e consortiu mrequest s awaive r fo r th e aromatas e assay becaus e simazin e ha s bee n alread y teste db y multipl e researcher s usin g substantiall y simila r methodology . Methodological deviation s shoul dhav e n o impac t o n justificatio n fo r

usin g OSR I i n consideratio n o f

simazin e interactio n with th e aromatas e complex . 3. 4 Estroge n Recepto r Bindin g Assa y Usin g Ra t Uterin e Cytoso l (ER-RUC ) (OPPT S 890.1250 ) Purpos e o f

Assay Thi s assay detect s th e potentia l o f acompoun dt o interac t

with th e estroge n receptor s (ERs ) isolate dfro mth e ra t

uterus . Hormona l activity begin s whe n asubstanc e bind s t o aspecifi c recepto r an d initiate s acascad e o f event s leadin g to aphysiologica l response . Th e estrogen recepto r (ER) i s locate dinsid e targe t cells , i n o r

nea r th e nucleus , an dhormone-boun dE R interact s with specifi c site s i n th e genom e o f th e cel l t o contro l productio n o f mRNA . Th e hormone-bindin g domai n (HBD ) o f th e estroge n recepto r

i s highl y specifi c compared.tt> I * I othe r steroi dclasses , an di s highl y conserve dacros s species . Thi s tes t o f aco*n|>ound's " ^ abilit y to bin dto E Rconstitute s adirect , simpl e evaluatio n o f it s estrogen ^ potentia

l i n . vertebrates . Environmenta l chemical s tha t compete wit h endogenou s estrogen s cna y »itn« * induc e (agonist ) hormone-dependen t transcriptiona l activity , o r bloc k (antagonist ) noijnal J . hormon e functio n b y preventin g th e endogenou s hormon e fro mbindin g to'ftJe * receptor . • ••• • Assa y Methodolog y Summar y • Th e assay measure s th e ability o f aradiolabele destroge n (17(3-estradiol ) t o interac t wirh'm'e , E Ri n th e presenc e o f

increasin g concentration s o f ates t chemical . Rat

uterin e cytoso l (RUC ) containin g ERi s incubate d i n tes t tube s wit h concentration s o f

ates t substanc e an d a

n aliquo t o f radiolabele d[ 3 

### H]-estradiol . I f th e tes t substanc e interact s with th e receptor' s HBD , les s radioligan dcan bind , thu s an activ e competito r produce s adescendin g dose-respons e plot . Similarly , compound s tha t d o no t displac e radiolabele destradio l fro mE Rca n b e presume dt o b e devoi do f receptor-bindin g activity . Th e assay ha s alon g history o f us e withi n th e researc h communit y fo r rapi dan d relativel y inexpensiv e detectio n o f chemical s with E Rbindin g capability . Repor t Number : TK002342 4 Pag e 1 7 o f 6 3Th e Tie r 1 scree n E Rbindin g assa y (ER-RUC ) consist s o f

asaturatio n bindin g experimen t an dacompetitiv e bindin g experiment . A t eac h o f 8 concentration s (o f

radiolabele destradio l i n th e saturatio n bindin g assay ; o f tes t chemica l i n th e competitiv e bindin g assay) , thre e simultaneou s replicate s ar e prepared . Eac h se t o f 8 concentration s constitute s arun . Thre e adequat e independent

run s constitut

e a n experiment . Othe r Scientificall y Relevan t

Informatio n There ar e fou r

existin g studie s that

measure dcompetitiv e estroge n recepto r bindin g with simazine , an d thes e studie s ar e substantiall y similar t o th e EDS P Tier l E Rbindin g screen . Th e mos t recent o f thes e wa s conducte db y th e EPA' s ToxcastTM researc h program . OSR I Rat Uterin e Cytosol , [ 3 H ] -estradio l Rat Uterin e Cytosol , [ 3 H]-estradio l Rat Uterin e Cytosol , [ 3 H]-estradio l Bovin e ER , [ 3 H]-estradio l Reference s Blai r e t al. , 200 0 Tennant , 1994

b O'Connor , 200 0 Judso n e t al. , 201 0 Comment s

Substantiall y similar

t o Tie r I scree n Substantiall y similar

t o Tier I scree n Substantiall y simila

r t o Tier I scree n Substantiall y similar

t o Tie r I scree n Blair et al. , 200 0 Thi s study , conducte dat

th e Nationa l Cente r

fo r Toxicologica l Researc h (NCTR ) i n Arkansas , reporte dtha t

the y use d " avalidate d(standardized ) estroge n recepto r (ER ) competitive-bindin g assay t o determin e th e E Raffinit y fo r alarge , structurall y divers e grou p o f chemicals. " Rat

uterin e cytoso l wa s prepare dan d[ 3 H]-estradio l wa s use dt o asses s competitio n fo r

binding . Approximatel y 20 0 chemical s wer e teste d fo r

abilit y to displac e radiolabele destradio l fro mER , an dsimazin e wa s teste dat concentration s u p t o 10" 4 Molar (10 0 |4.Molar) . Hydroxyapetit e ge l wa s use dfo r separatio n o f

boun dan dfre e tracer . • •• • 

### Tennan t et al. , 1994 b '."I" . '" ' ' Thi s study use dara

t uterin e cytoso l preparatio n to determin e th e potential•for'simazin&to* . displac e bindin g o f

[ 3 H]-estradio l t o ER . Concentration s o f u p t o 10" 3 MoM3[1*fliMolir7 « . * wer e tested . Th e onl y variatio n noted , i n contras t t o th e EDS P validatio n studios * wa s Jhe.ijs. e o f dextran-charcoal fo r

th e separatio n o f

boun dan d fre e tracer . It shoul db^flflted , however , tha t th e IS R(Integrate dSummary Report ) reporte do n evaluation s o f th e dextiwn'-charc.oaf

. procedur e an d conclude d"Th e RBA s obtaine di n th e differen t separatio n system s wer&"" * observe d t o b e i n th e sam e range . There di d no t appea r t o b e an y greate r

variatio n i n /€^ 8 • . value s betwee n th e tw o separatio n system s tha n tha t observe dwithi n separatio n systems"* * (USEPA , 2009 , p . 22) . O'Connor , 200 0 Thi s study , reporte d fro mHaskel l Laboratory , teste dth e potentia l fo r simazine , an d other triazine s an dmetabolites , t o interac t wit h anumbe r

o f recepto r systems . Detail s o f

th e exac t methodolog y wer e no t

include d i n th e publication ; eight differen t recepto r bindin g studie s wer e conducte d fo r

th e investigator s b y th e NOVASCREE N Corp , Hanover , MD . Th e publicatio n state s onl y tha t [ 125 I]-estradio l wa s use da s th e competin g ligand . I t wa s no t state d whether

RU C wa s th e sourc e fo r ER ; however , estroge n recepto r ca n b e extracte d Repor t Number : TK0023424 Pag e 1 8 of 6 3

### fro mthousand s o f species , includin g birds , fish , reptile s an damphibians , an dretai n virtuall y identica l bindin g characteristics . Judso n e t

al. , 201 0 Th e ToxCas t

group ha s examine dth e competitiv e bindin g profil e o f 32 0 chemicals , includin g simazine , i n th e Novascree n assay s that ar e abbreviate dNVS_NR_bE Ran d NVS_NR_hE Rwithi n thei r databas e o f

man y high-throughpu t

screens . More detail s o n th e methodolog y use di n thes e studie s a s part o f ToxCas t can b e foun di n Appendi x 1 an dJudso n e t al. , 2009 . Specifi c description s o f thes e estroge n bindin g assay s wer e obtaine dfro m Caliper Lif e Science s an dare provide di n Appendice s 2 an d3 . NVS_NR_bE Rteste d th e competitiv e bindin g o f simazin e i n estrogen receptor preparation s derive dfro mbovin e uterin e membranes . 3 

### H-Estradio l wa s use da s th e radiolabelle dligand . 17|3-Estradio l wa s use da s apositiv e control . Reaction s wer e carrie dou t i n an appropriat e buffe r solutio n a

t 0-4° C fo r

1 8 hours . Th e reactio n wa s terminate db y th e additio n o f dextran coate dcharcoal an dincubate dfo r 2 0 minute s at

0-4°C . Th e reactio n mixture s wer e centrifuge dan d th e radioactivit y boun di n th e supernatant wa s assessed an d compare dto contro l value s i n orde r

t o ascertai n an y interaction s o f tes t compoun dwith th e estradio l bindin g site . NVS_NR_hE Rteste dth e competitiv e bindin g o f simazin e i n estroge n recepto r preparation s derive dfro mhuma n primar y breas t cance r cel l lin e (MCF- 7 cells) . 3 

### H-Estradio l wa s use da s th e radiolabelle dligand . 17|3-Estradio l wa s use da s apositiv e contro l an da s amode l substrat e fo r

compariso n t o simazine . Reaction s wer e carrie dou t i n a n appropriat e buffe r solutio n a t 0-4° C fo r

1 8 hours . Th e reactio n wa s terminate db y th e additio n o f dextran coate dcharcoa l an dincubate dfo r 2 0 minute s at

0-4°C . Th e mixture s were centrifuge dan d th e radioactivit

y boun di n th e supernatant wa s assessed an d compare dto contro l value s i n orde r t o ascertai n an y interaction s o f tes t compoun dwith th e estradio l bindin g site . ••••• • • • • Fo r th e saturatio n bindin g experiments , reproducibl e K dvalue s an dBmax3/arle 2 fro mprio r run s at Calipe r Lif e Science s ar e show n i n Appendice s 2 an d3 , an dfal l wiftjiftflj e acceptabl e rang e a t th e typical substrate concentratio n that i s used . Chemical s i n ToxCafc t were initiall y screene dat

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

2£j^ M fo r

flftfE R bindin g assays . Chemicals-assay pair s that showe dsignifican t

activity were^t^n'ru n i n • concentratio n respons e format , fro mwhic h an AC5 0 valu e wa s extracted . Fo r concentfaticfi * response , 8 concentration s wer e teste di n th e range s 0.0229-5 0 /j . M fo r non-CY P assays.. . Thes e assay s wer

e ru n b y Novascreen ( apar t o f Calipe r Lif e Sciences , Hanover , MD ) under * contrac t t o EPA .

Report Number : TK002342 4 Pag e 1 9 o f 6 3Result s o f thes e assay s ar e publicl y availabl e o n th e ToxCas t websit e usin g th e followin g information : http://www.epa.gov/ncct/toxcas t #Dat aSe t

Name : Novascree n #Fil e Name : /share/server_data/data/ACToR/ToxCast/data/ToxMinerExport/ToxCast_Novascree n _20090807.tx t Deviation s t o OPPT S 890.125 0 Protoco l OPPT S 890.125 0 Requirement s Criteri a ERfro mra

t uterin e cytoso l Hydroxyapetit e ge l fo r separatio n o f boun dan dfre e Blai r

e t al. , 200 0 Deviation s to 3PPT S 890.125 0 Non e

Non e Pennan t

e t al. , 1994 b Deviation s t

o OPPTS 890.125 0 Non e

Dextran-charcoa l separatio n ste

p O'Connor , 200 0 Deviation s to JPPT S 890 . 125 0 Possibl y

Unknow n Judso n e t

al. , 201 0 Deviation s t

o OPPTS 890.125 0 Mino r (bovin e uteru s preparation )

Non e Justificatio n fo r

OSR I Non e neede d The IS Rstated tha

t hydroxyapetit e an d dextran-charcoa l gav e similar

an d acceptable result s Conclusions : Th e simazin e consortiu mrequest s awaive r fo r th e estroge n recepto r

bindin g assa y becaus e simazin e ha s bee n alread y teste db y multipl e researcher s usin g substantiall y similar methodology . Al l thre e OSRI report s use dmethodolog y tha t i s widel y accepte dfo r evaluatio n o f

E Rbinding , an dwer e mos t likel y ver y similar

t o th e Tie r

1 validate dprot»c»te

j Methodologica l deviation s hav e n o impac t o n justificatio n fo r

usin g OSR I trt

tj*£ideratf<3 n d f simazin e interactio n wit

h estrogen receptor

binding . • • 

### Estroge n Recepto r

Transcriptiona l Activatio n - Huma n Cel l Lin e He1*a» * 990 3 (OPPT S 890.1300) 3. 5 Purpos e o f

Assa y • * Th e purpos e o f

thi s i n vitr o transcriptiona l activatio n (TA ) assay i s t o evaluat e th e abilkV'd f a chemical t o functio n a s an E Rligan dan dactivat e an ER-mediate dagonis t response . TA * assay s ar e base d upo n th e productio n o f areporte r

gen e produc t

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

i n away that

promote s subsequen t

downstrea mtranscriptiona l activation . TA assay s hav e lon g been use d a s screen s to evaluate th e specifi c gene expressio n regulate db y specifi c nuclear receptors , suc h a s th e ERs , an dhav e bee n propose d b y th e EDS P fo r

th e detectio n o f

estrogen-mediate dtransactivation . Nuclear ER s exis t

a s at leas t tw o subtypes , terme daan d p . They ar e encode db y distinc t genes , hav e differen t

tissu e distribution , relativ e ligan d bindin g affinitie s an dbiologica l functions . Nuclear ER a mediate s th e classi c estrogeni c responses , therefor e model s currentl y bein g develope dt o measur e E Ractivatio n mainl y us e ERa-linke dreporters . Repor t Number : TK002342 4 Pag e 2 0 o f 6 3

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

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

regulatio n o f

cellular processes . Furthermore , thi s Tes t

Guidelin e doe s no t

addres s antagonis t

interactio n with th e hER aan d subsequen t

effec t o n transcription . Assa y Methodolog y Summar y Th e T A assa

y i s a n i n vitro assa

y that measure s signa

l bindin g o f

th e estrogen receptor

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

r expressio n o f

luciferas e enzyme . Luciferi n i s asubstrate that i s transforme db y th e enzyme t o abioluminescen t produc t that ca n b e simpl y an dquantitativel y measure dwit

h aluminometer . Th e tes t syste mprovide di n thi s guidelin e utilize s th e hERa-HeLa-990 3 cel l line , whic h i s derive dfro mahuma n cervica l tumor . Th e cell s contai n tw o stabl y inserte dconstructs : (1 ) th e hE Raexpressio n construc t (encodin g th e full-length huma n receptor) , an d(2 ) afirefl y luciferas e reporte r

construc t bearin g fiv e tande mrepeat s o f avitellogeni n Estrogen

Responsiv e Elemen

t (ERE) drive n b y amous e metallothionei n (MT) promote r TATA element . Dat ainterpretatio n fo r thi s assay i s base dupo n whethe r o r no t th e maximu m respons e leve l induce db y ates t chemica l equal s o r

exceed s an agonis t

respons e equal t o 10 % o f tha t induce db y amaximall y inducin g ( 1 nM ) concentratio n o f th e positiv e contro l 17 p estradio l (E2 ) (i.e. , th e PC10) . Othe r Scientificall y Relevan t

Informatio n Simazin e ha s bee n teste di n at leas t seve n studie s usin g th e E Rtranscriptional activatio n assa y (Balaguer , et

al. , 1996 ; Connor

et al. , 1996 ; Tra

n et al. , 1996 ; O'ComjgteUl- , 20QQ*' : Legle r e t al. , 2002 ; Kojim ae t

al. , 2004 ; Orto n e t

al. , 2009) . A t

leas t tw o stadia s specificall y teste dth e ER asubtype , an dth e other s appea r t o hav e use dth e ER asubtyp e Althoug h ittyas \ no t identifie dspecificall y i n th e publications . I n addition , Judso n e t al. , 2010lreporte d o n * * testin g simazin e usin g multipl e huma n E Rgen e transactivatio n reporte r ass3#£.unde

r tlj S • • • • umbrell ao f

th e ToxCast TM system . Methodologica l deviation s hav e n o imj^ t o h • justificatio n fo r

usin g OSR

I i n consideratio n o f

simazin e interactio n wit

h estroge n recejtfpj; . transactivatio n systems . Example s an ddiscussio n o f th e fiv e mos t

relevan t report s follow ^ OSRI E Rtransfecte dwit h reporte r E Rtransfecte dwit h reporte r E Rtransfecte dwith reporte r E Rtransfecte dwit h reporte r E Rtransfecte dwith reporte r Reference s Conno r et al. , 199 6 Legle r e t al. , 200 2 Balague r e t al. , 199 6 Kojim ae t al. , 200 4 Judso n e t al. , 201 0 Comment s

hE Rtransfecte dint o MCF- 7 cell s hER atransfecte dint o T47D.Lu c cell s hER atransfecte dint o HeLacell s hER atransfecte dint o CH O Multipl e huma n E Rgen e transactivatio n

reporte r assay s 

### Repor t Number : TK0023424 Pag e 2 1 o f 6 3

### Conno r e t al. , 199 6 Thi s stud y use dMCF- 7 cells , ahuma n breas t cancer-derive dcel l line , transientl y transfecte d with an E Rchimer aan daluciferas e reporte r gene . Cell s wer e plate dan dincubate dwit h medi acontainin g simazin e ( 1 nMola r to 1 0 uMolar ) o r

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

2 4 hr befor e luciferas e activit y wa s measured . Althoug h no t identifie dspecificall y a s a n ER aconstruct , th e manuscrip t state s that

th e chimeri c receptor include d"th e ligan dbindin g domai n o f th e ER " (p.96) , whic h i s no w wel l understoo dt o b e th e sequenc e late r

identifie da s th e LE D o f

ERa . Legle r e t al. , 200 2 Thi s study examine d th e abilit y o f approximatel y tw o doze n substance s to induc e arespons e i n an ER-linke dreporte r

system . Human ER awa s transfecte dint o aT47 D cel l lin e with a vector , an daluciferas e reporte r gen e wa s linke ds o tha t

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

signal . Simazin e (1 0 nMola r t o 2 5 uMolar ) wa s teste di n compariso n t o estradiol-17 p a

t 0.0 1 pMolar

t o 10 0 nMolar . Balague r et al. , 199 6 Thi s stud y mos t

closel y duplicate dth e EDS P validate dprotoco l methodology . HeL acell s wer e stably transfecte dwith achimeri c recepto r expressio n systemcontainin g ER aan d luciferase . Eigh t

tes t substances , includin g simazine , wer e incubate dat concentratio n range s o f 0. 1 nMola

r t o 1 0 uMolar , an dcompare dt o estradio l a t 0. 1 pMolar

t o 1 0 nMolar . Kojima , 200 4 Thi s stud y examine dth e abilit y o f 20 0 chemical s to induc e arespons e i n a n ER-linke d reporte r system . Huma n ER awa s transfecte dint o Chines e hamste r ovar y (CHO ) cell s with a vector , an daluciferas e reporte r gen e wa s linke ds o tha t

ligan dbindin g t o tb.e.r.ef$pto r ^gnfc j produc e aluminescen t

signal . Simazin e (1 0 nMola r t o 1 0 uMolar ) wa s on § ofitrf e 20 0 teste d chemical s i n compariso n to estradiol-171 3 (a t 0.0 1 pMola

r to 10 0 nMolar) . ].... ! ;* . • . Judso n e t

al. , 201 0 Th e ToxCas t

researc h progra mutilize dreporte r gen e assay s relate dt o estrog& i receptors, , including : J...J . • NI H Chemica l Genomic s Center (NCGC ) assay , fo r

hER aagonis t

an dfo r hER*a

< 

### antagonis t functio n i n HEK293 H cells , utilizin g a n Invitroge n GeneBLaze

r reptirtej ; • assay system . • Attagen e reporte r

assays , fo r

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

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

assay s i n HepG 2 cells . Simazin e wa s evaluate db y th e EP A i n thes e screens . Se e Appendi x 1 an dJudso n e t al. , 201 0 fo r mor e detail s o n th e methodolog y use di n thes e screen s an dacces s t o th e ToxCas t result s vi athei r

websit e links .

Report Number : TK002342 4 Pag e 2 2 o f 6 3

### Deviation s t o OPPT S 890.1300 Protoco l OPPT S 890.130

0 Requirement s Criteri a

HeL acel l lin e

Cell s stabl y transfecte dwith ER G linke dt o 1996

Deviation s t o OPPT S 890 . 130 0

MCF- 7 cel l lin e

Cell s transientl y transfected , probabl y wit h ER a(subtyp e no t specified ) Legle r

e t al. , 2004 Deviation s t o OPPT S 890.130 0 T47 D cel l lin e

Non e Balague r

e t al. , 199 9 Deviation s t o OPPT S 890.130 0 Non e

Non e Kojim ae t

al. , 200 4 Deviation s t o OPPT S 890.130 0 Chines e Hamste r Ovary Cell s

Non e Judso n e t

al. , 201 0 Deviation s t o OPPT S 890.130 0 HEK293 H cell s an dHepG 2 cell s

Non e Justificatio n fo r

OSR I Cel l line s i n thes e report s ar e commonl y an d reliabl y use dfo r

T A studies , particularl y fo r steroi dreceptor s Al l construct s and/o r cell s designe dt

o measur e transactivatio n b y E Rbinding , o r estroge n mediate d growt h 

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

transcriptiona l activatio n i n existin g studie s usin g substantiall y simila r methodology . Th e cite dreport s ar e fro mreputabl e an dexperience dlaboratorie s that hav e extensiv e publishe dliteratur e describin g transfecte dsteroi dreceptor-linke dcel l reporte r systems . Al l use dwidel y accepte d technolog y i n cel l line s that hav e bee n studie dfo r

man y years , an dmos t

o f th e studie s specificall y teste dth e ER asubtype . Methodologica l deviation s hav e n o impac t o n justificatio n fo r

usin g OSRI i n consideratio n o f simazin e interactio n wit

h estrogen recepto r

transactivatio n systems . 3. 6 Fis h Shor t

Ter mReproductio n (OPPT S 890.1350 ) Th e fis h short-termreproductio n assay wa s develope dt o identif y potentia l chemfca l effects * t 

### o n morpholog y (secondar y sexua l characteristics) , histopathology , spawning ; anf l specific ? • biochemica l endpoint s whic h migh t reflec t interferenc e with th e norma l functionin g of>the> t • hypothalamic-pituitary-gonada l (HPG ) axis . Assa y Methodolog y Summar y Th e OPPT S 890.135 0 guidelin e utilize s matur e fathea dminnow s (Pi m are expose dt o aminimu mo f thre e concentration s o f

tes t chemical an dappropriat e control(s ) fo r 2 1 days . Spawnin g i s establishe di n apre-exposur e perio do f a t leas t 1 4 days . I n eac h o f fou r replicat e tank s pe r

concentratio n ar e place dfou r female s an dtw o males . Assesse d endpoint s includ e survival , behavior , fecundit y measure da s numbe r o f spawn s an dnumbe r o f egg s pe r femal e reproductiv e day , fertilizatio n succes s measure da s numbe r o f fertil e egg s per femal e reproductiv e da

y an d percent

fertil e eggs , gonada

l histopathology , gonadosomati c index , secondary se x characteristic s measure dprimaril y a s tubercl e numbe r bu t coul dals o includ e bod y coloration , fatpa dindex , an doviposito r size , an dbiochemica l measure s o f vitellogenin , estradiol , an d testosterone .

Repor t Number : TK002342 4 Pag e 2 3 o f 6 3Othe r Scientificall y Relevant

Informatio n Simazin e a s Aquazin e (80 % formulation ) ha s bee n previousl y teste di n tw o fis h ful l lifecycl e studie s (Mayer

an dSanders , 1975) . On e stud y employe dconstan t exposure t o simazine , whil e th e othe

r wa s designe dto asses s th e potential fo r effect s du e to degradatio n o f simazine . OSR I Fis h ful l lifecycl e test s Referenc e Maye r an d Sanders , 197 5 Comment s

Fathea dminno w ful l lifecycl e study (120-day exposure ) with formulate d simazin e (80%) . Assessmen t o f effect s o n th e F O generatio n fro madul t t o eg g o f th e F l generatio n (30-day fry) . Maye r an d Sanders , 197 5 Fathea dminno w (Pimephale s promelas ) wer e continuousl y expose dt o fiv e concentration s o f Aquazin e (80 % simazine ) an dawate r contro l fro madul t

fis h (1 0 month s old) , h i asecon d stud y exposure wa s modifie dt o includ e expecte ddegradation . Th e effect s o f exposur e o n survival , growt

h an dreproductiv e succes s o f firs t generatio n (FO ) fis h an dth e hatchin g success , surviva l an d growt h o f thei r progen y (Fl ) wer e assessed . Deviation s to OPPT S 890.135 0 Protoco l OPPT S 890.135 0 Requirement s Criteri a Mayeran dSanders , 197 5 Deviation s t o OPPT S 890.135 0 Justificatio n fo r OSR I

Fathea dminno w shor

t ter m reproductio n assa

y Th e stud y presente di s ahighe r tie r fis h reproductio n assa

y i n tha t i t involve s exposure o f

al l fis h lifestages . Th e ful l lifecycl e tes t

doe s no t includ e secondar

y sexua l characters , vitellogeni n o r gonada l histopatholog y (mandatory endpoints ) o r bloo dplasm aE o r

T litre s (optional endpoints) . Th e tes t

ite mwa s no t

technica l grad e materia l bu t

ahig h concentratio n formulate dproduc t

(80%) . Th e ke y apica l endpoint s o f development , growt h an dreproductio n ar e include di n th e study . Th e uncertaint y o f

no t includin g endocrin e biomarker

response s ca

n b e addresse db y aweigh t

o f evidence * • • evaluatio n o f

al l th » wattin g (toxicologicaf t Sata i 1 , Conclusions : Th e simazin e consortiu mrequest s awaive r fo r th e fis h shor

t tfirm ! . reproductio n assay base do n th e availability o f fis h ful l lifecycl e data . •••• • ;...; , 

### 3. 7 Hershberge r

- Ra t (OPPT S 890.1400

) 'IIII ' Purpos e o f

Assa y Th e Hershberger

Bioassay i s ashort-termi n viv o screenin g tes t usin g accessor

y tissue s o f

th e roden t mal e reproductiv e tract . Th e bioassa y serve s a s amechanisti c assa y fo r androge n agonists , androge n antagonist s an d 5a-reductas e inhibitors . I t i s intende dt o b e include di n a batter y o f i n vitr o an di n viv o test s t o identif y substance s with potentia l t o interac t with th e endocrin e system . Assa y Methodolog y Summar y Th e OPPT S protoco l fo r

th e Hershberge r

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

o f fiv e androgen-dependen t tissue s i n th e castrate-peripuberta l mal e rat . I t evaluate s th e ability Report Number : TK002342 4 Pag e 2 4 o f 6 3

### o f

achemica

l t o elici t biological activitie s consisten t with androge n agonists , androge n antagonist s or

3.55a-reductas e inhibitors . Th e fiv e androgen-dependent tissue s include di n thi s Tes t Guidelin e ar e th e ventra l prostat e (VP) , semina l vesicl e (SV ) (plu s fluid s an d coagulatin g glands) , levato r ani-bulbocavernosu s (LABC ) muscle , paire dCowper' s gland s (COW ) an dth e glan s peni s (GP) . h i th e castrate-peripuberta l mal e rat , thes e fiv e tissue s al l respon dt o androgen s wit h an increas e i n absolut e weight . Whe n thes e sam e tissue s ar e stimulate db y administratio n o f apotent referenc e androgen , the y al l respon d to antiandrogen s with adecreas e i n absolut e weight . Th e scree n i s conducte db y dail y administratio n (gavag e o r subcutaneou s injection) , fo r a perio dofte n consecutiv e days , t o aminimu mo f tw o treatmen t

group s o f

experimenta l animal s a t on e dos e leve l pe

r group . Th e animal s ar

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

mor e targe t

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

androgeni c activity . Certai n androgens , suc h a s trenbolone , that canno t b e Set-reduced , hav e amor e pronounce deffec t o n th e LAB C an dG P versu s testosteron e propionate (TP) , but al l responsiv e tissue s ar

e expecte d to displa

y increase d growt h i n th e presenc e o f

atru e androgen . Whe n screenin g fo r

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

subcutaneou s (sc ) injectio n fo r aperio dofte n consecutiv e days , i n concer t wit h daily s c injection s o f

testosteron e propionate (0. 2 or 0. 4 mg/kg/d) . Th e validatio n programdetermine dthat

eithe r dos e o f T P coul db e used . Graduate ddose s o f th e tes t substanc e ar e administere dt o aminimu mo f thre e treatmen t

group s o f

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

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

organ weight s o f

th e tes t substanc e plu s T P groups , compare dt o th e T P onl y contro l group , suggest s that th e substanc e i s positiv e fo r antiandrogeni c act^ijy ^ i • • • 

### Othe r Scientificall y Relevan t

Informatio n Simazin e ha s bee n previousl y teste di n astud y whic h utilize dsubstantiall y methodolog y a s th e EDS P Tie r 1 Hershberge r

(Birkho j e t al. , 2004) . . . . " \ OSR I Hershberge

r Referenc e

Birkho j et al. , 200 4 Comment s

Hershberger

protoco l 

### Birkho j e t al. , 200 4 Thi s study wa s designe dt o asses s th e "anti-androgeni c effects " o f severa l pesticides , includin g simazine . Group s o f adul t rat s wer e treate dfo r

seve n day s wit

h 0. 5 mg/kg/day testosteron e propionat e (TP ) sc , T P + flutamid e (contro l antagonist ) o r T P + simazine , 2 5 mg/kg/da y b y gavage . Group mea n body weight

wa s no t significantl y affecte di n th e simazin e group . A t

necrops y 4 androgen-dependen t tissue s wer e dissecte dan dweighed , thre e o f whic h ar e amon g th e fiv e tissue s examine db y th e EDS P Tie r 1 Hershberge r

screen .

Repor t Number : TK0023424 Pag e 2 5 o f 6 3Deviation s t o OPPT S 890.1400 Protoco l OPPT S 890.140

0 Requirement s Criteri a Castrat e peripuberta

l mal e rat s Tes t substanc e administere d1 0 days , eithe r

gavag e o r s c Tes t fo r agonis t propertie s b y direc t administratio n to castrat

e rat s Tes t fo r antagonis musin g concomitan t T P administratio n Weig h 5 tissues : VP , SV , LABC , COW , GP Birkho j et al , 200 4 Deviation s to OPPT S 890.140 0

Non e Animal s treate dfo r

7 day s b y gavag e Non e Non e Onl y VP , S V an dLAB C weighed ; CO W & G P no t

weighe d Justificatio n fo r OSR I Sam e anima l mode l a s EDS P

assa y EDS P protoco l call s fo r

eithe r gavag e o r s c administratio n EDS P protoco l include s bot h agonis t an dantagonis t evaluatio n Same evaluatio n desig n a s EDS P protoco l Man y androgen-dependen t parameter s include d Conclusions : Th e simazin e consortiu mrequest s awaive r fo r th e EDS P Tie r

1 Hershberge r scree n becaus e simazin e ha s alread y bee n adequatel y teste di n an existin g study usin g substantiall y similar

methodology . Ther e appea r t o b e n o methodologica l deviation s that woul dimpac t

th e justificatio n fo r

usin g OSR

I i n consideratio n o f

simazin e activit

y i n th e Hershberge r

screen . 

### 3. 8 Femal e Puberta l - Rat (OPPT S 890.1450) Purpos e o f

Assa y Th e purpos e o f

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

mixture s tha t

have , th e potentia l t o interac t

with th e endocrin e system , b y evaluatin g effect s oncubarta l *•• " developmen t

an dthyroi dfunctio n i n th e intac t juvenile/peripuberta l femal e J"at*T{ii s assa y i s capabl e o f

detectin g anti-thyroid , estrogeni c o r anti-estrogeni c chemical s (i»»kidi» g aglfit e * whic h ac t

vi aalteration s i n receptor bindin g o r

steroidogenesis) , o r

agent s whft^ i Ca

n atyer . puberta l developmen t

vi achange s i n luteinizin g hormone , follicl e stimulati»gd»»rmone f prolacti n o r

growt h hormon e level s o r

vi aalteration s i n hypothalami c Assay Methodolog y Summar y ... , Th e femal e puberta l scree n use s fiftee n femal e rat s i n eac h o f thre e treatmen t

group s (onc»» i administere dvehicl e an dtw o give n ates t substanc e at

differen t dos e levels) . Th e Sprague

Dawle y rat

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

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

b e pregnan t

fo r th e firs t tim e an dtime dt o delive r o n th e same day . T o maximiz e uniformit y i n growt h rates , th e litter s ar e standardize dt o 8 t o 1 0 pup s pe r litte r betwee n post-nata l day s (PND ) 3 an d5 . Th e pup s ar e weane do n PN D 2 1 an dtreatmen t i s initiate dfro mPND 2 2 throug h PND 42 . Tes t chemical s are administere db y ora l gavag e i n 2. 5 t o 5. 0 m l vehicle/k g bod y weigh t at 0700-090 0 daily . Th e followin g observation s ar e made : Report Number : TK002342 4 Pag e 2 6 o f 6 3Vagina l Openin g . Beginnin g o n PN D 22 , female s ar e examine ddail y fo r vagina l openin g (VO) . Estrou s Cyclicity . Beginnin g o n th e day o f vagina l opening , dail y vagina l smear s ar e obtaine dan dclassifie da s diestrus , proestrus , o r estrus . Metestru s i s classifie da s (an earl y part of ) diestrus . A t

th e en do f

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

- to 5-da y cycles) , irregula

r cyclin g (som

e regula r cyclin g bu t with aperio do f

diestru s longe r

tha n 3 day s o r

aperio do f

cornificatio n longe r

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

leukocyti c smears) . Th e preferre dmetho d o f

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

t o obtai n asufficien t

volum e o f

bloo dfo r th e hormon e an dchemistrie s measurements . Trun k

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

h attache dportio n o f

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

eac h excep t

th e thyroid/trache aan d uteru s recorde d (kidneys , adrenals , an dovarie s ar e weighe da s pairs. ) Th e ovaries , uterus , kidney , an dthyroi dtissu e ar e place d i n 10 % buffere dformali n fo r at leas t 2 4 hour s an dthe n store di n 70 % ethano l unti l embedde d i n paraffin . Section s are cu t an dstaine dwit

h hematoxyli n an deosi n (H&E) fo r subsequen t histologica l evaluations . Hormonal Assays . Immunoassay s o f thyroxin e (T4 ) an dTS H can b e conducte dusin g radioimmunoassa y (RIA) , immunoradiometri c assa y (IRMA) , enzyme-linke d immunosorbent

assa y (ELISA) , or

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

follicular cel l heigh t

an d colloi dare ausin g afiv e poin t grading 5 gc,al e ( 1 j = . shortest/smallest ; 5 = tallest/largest) , an d an y abnormalities/lesion s noted . •Ovaria n histolog y shoul dinclud e a

n evaluatio n o f

follicula r development

(includin g presencey^feehc e o f • tertiary/antra l follicles , presence/absenc e o f corpor alutea , change s i n corpu s luteu m••••• • development , an dchange s i n numbe r o f bot h primar y an datreti c follicles ) i n additio n to,an, y abnormalities/lesions , suc h a s ovaria n atrophy . Uterin e histolog y shoul ddocumen t cas*e*»o £* uterin e hyper

- o r

hypotroph y a s characterize db y change s i n uterin e horn diamete r an d myometrial , stromal , o r

endometria

l glan ddevelopment . Othe r Scientificall y Relevan t

Informatio n Simazin e wa s recentl y teste di n th e femal e puberta l assa y usin g substantiall y similar methodolog y a s th e EDS P Tie r 1 scree n guidelines . Th e stud y wa s conducte db y th e USEP A toward s th e development o f th e EDS P protoco l an d assesse d th e majority o f th e targete d parameter s o f

thi s apical test . Report Number : TK002342 4 Pag e 2 7 o f 6 3OSR I Femal e puberta l assa y Referenc e

Zorrill ae t al. , 201 0 Comment s Tes t o f Tie r 1 Scree n Assa y Zorrilla , et

al. , 201 0 Thi s study , conducte db y aUSEP A laboratory researc h team , wa s conducte da s aprototyp e fo r th e EDS P Tie

r 1 pubertal ra

t screen , an d i t

include d simazin e a s ates t compound . Femal e rat s wer e administere dsimazin e b y oral gavag e at 5 dose s fro m12. 5 t o 20 0 mg/kg/day , plu s avehicle-onl y group , fro mPN D 2 2 throug h 42 . A secon dse t o f anima l group s wa s treate d with simazin e throug h PN D 62-6 5 i n orde r t o examin e estrou s cyclin g pattern s i n greate r detail . Date o f

vagina l openin g wa s recorded , a s wer e estrou s cyclin g pattern s followin g VO . A t necrops y seru mwa s prepare dfro mbloo dan danalyze dfo r T 4 an dTS H content , amon g othe r hormones . Uterus , ovaries , thyroid , liver , kidney , pituitary , an dadrenal s fro meac h animal were al l dissecte dan dweighed , an duter i an dovarie s wer e processe dfo r histolog y an d examined . Deviation s t o OPPT S 890.145 0 Protoco l

OPPT S 890.145 0 Requirement s Criteri a Jus t weane dfemal e rat s Treate dfro mPN D 22-4 2 2 tes t dose s b y gavage , includin g 1 near

MT D Evaluat e VO , E cycl e patterns, seru mT4 , TSH , variou s chemistrie s Histologica l exa mo f

ovaries , uterus , thyroid , kidne y Zorrilla , e t al. , 201 0 Deviation s t o OPPT S 890.145 0

Non e Non e 6 doses ; 2 highes t

dose s suppresse dB W Non e except

non-endocrin e seru mchemistr y measure s no t

don e Histologica l evaluatio n conducte do f

ovarie s an duter i Justificatio n fo r OSR I Non e neede d Non e neede d Exceed s T l S protoco l requirement s Al l o f th e targete dendocrin e parameter s o f

thi s apica l tes t

wer e assesse d. • J '

• • • Essentiall y i n *in $ wit h T l S * protocol , excejrtforthynJB ^ ' Sistolog j • • ' Conclusions : Th e simazin e consortiu mrequest s awaive r fo r th e EDS P Tiert l iSemal e 1 ••• • • Puberta l assay becaus e simazin e ha s previousl y bee n teste db y th e EP A usin g substantiall y similar

t o th e Serie s 890.145 0 guideline . Therefore , simazin e ha s bee n thoroughl y evaluate di n th e EDS P Tie r 1 femal e puberta l rat

screen . *.... ' 

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

Assa y Th e purpos e o f

th e mal e puberta l assa

y i s t o identif

y chemical s that

hav e th e potentia l t o interac t wit h th e endocrin e system , b y identifyin g effect s o n puberta l developmen t an d thyroi dfunctio n i n th e intac t juvenile/peripuberta l mal e rat . Thi s assay i s capabl e o f detectin g anti-thyroid , androgeni c o r anti-androgeni c chemical s (includin g agent s whic h ac t Repor t Number : TK002342 4 Pag e 2 8 o f 6 3vi aalteration s i n recepto r bindin g o r

steroidogenesis) , o r

agent s whic h alte r

puberta l developmen t

vi achange s i n luteinizin g hormone , follicl e stimulatin g hormone , prolacti n o r growt h hormon e level s o r

vi aalteration s i n hypothalami c function . Assa y Methodolog y Summar y Th e OPPT S protoco l require s fiftee n mal e rat s i n eac h treatmen t grou p ( 1 vehicle-treate dan d 2 tes t substance-treate ddoses) . Th e Sprague-Dawle y o r Wista r strain s ar e preferred . Th e highes t dos e leve l shoul db e a

t o r jus t belo w th e Maximu mTolerate dDos e (MTD) , an d th e lower dos e at approximatel y one-hal f

th e highe r

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

tha n PN D 23 . Tes t

substance s ar e administere db y oral gavag e i n 2. 5 t o 5. 0 m l vehicle/k

g body weight

a t 0700-090 0 daily , fro mPND 2 3 throug h PND 53 . Th e followin g observation s ar e made : Preputia l Separation . Beginnin g o n PN D 30 , male s ar e examine ddail y fo r preputia l separatio n (PPS) . Th e appearanc e o f partia l an dcomplet e preputia l separatio n o r apersisten t threa do f tissu e betwee n th e glan s an d prepuc e are al l recorde do n th e day s the y are observed . Th e day o f

complet e preputia l separatio n i s th e designate dendpoin t fo r th e parameter . Necropsy . Subject s ar e kille do n PN D 53 . Trun k bloo di s collecte dimmediatel y b y inversio n ove r afunnel , i n seru mseparatio n tube s (n o anticoagulant) . Th e seru mi s store dat -2 0 ° C o r colde r fo r subsequen t

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

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

eac h excep t

th e thyroid/trache arecorded . Th e thyroi d(with attache dportio n » £ • th e trachea ) an d asingl e testi s an d epididymi s fro meac h anima l ar e prepare dfe ; histo'logica l examination . Testi s an depididymi s ar e place di n Bouin' s fixativ e overnigh t andlstore din * 70 % ethano l unti l embedde di n paraffin . The y ar e the n sectione dstaine dwitk^iejnato^yKn. * an deosi n (H&E ) fo r

subsequen t

histologica l evaluations . Th e thyroid , with aUadhe dtrachea^ i s fixe di n 10 % buffere dformali n fo r at leas t 2 4 hours . The n th e thyroi d(wi^pqrathyfoids ) i s dissecte dfro mth e trachea , blotte dan dweighed , place di n 70 % ethano l unti l efnbedded*i n paraffin , sectioned , staine dwit h H&E , an dhistologicall y evaluated . Kidne y i s als o fix$d*ih* * 10 % buffere dformali n fo r a t leas t 2 4 hours , the n place di n 70 % ethano l unti l embedde d » • paraffin , sectioned , staine dwith H&E , an dhistologicall y evaluated . * • • • • Hormona l Assays . Hormona

l measurement s ca

n b e conducte dusin g radioimmunoassa y (RIA) , immunoradiometri c assay (IRMA) , enzyme-linke dimmunosorben t assay (ELISA) , o r time-resolve dimmunofluorescen t procedures . Seru mhormon e level s t o b e measure dar e testosterone , thyroxin e (T4 ) an d thyroi dstimulatin g hormon e (TSH) .

Report Number : TK002342 4 Pag e 2 9 o f 6 3

### Bloo dChemistry . An y standar dpane l o f bloo dchemistr

y test s that

include s creatinin e an d bloo d ure anitroge n (BUN ) ma y b e use da s lon g a s th e measurement s are calibrate d fo r rat s an dth e norma l range s fo r

control s ar e reported . Histology . Testis , epididymis , thyroid , an don e kidne y ar e evaluate dfo r pathologi c abnormalitie s an dpotentia l treatment-relate deffects . Thyroi dsection s ar e subjectivel y evaluate dfo r

follicula r cel l height

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

Informatio n A s with th e puberta l femal e EDS P Tie r 1 screen , th e USEP A Reproductiv e Toxicolog y Divisio n o f

NHEER L conducte d th e pubertal mal e EDS P Tie r 1 screen a

t thei r laborator y i n Researc h Triangl e Park , Nort h Carolin a(Stoke r e t al. , 2005) . OSRI Mal e puberta l assa

y Referenc e

Stoke r e t al. , 200 5 Comment s Tes t o f Tier I Screen Assay 

### Stoke r e t al. , 200 5 (Stoker , 2010 , persona l communication ) Th e study desig n fo r

thi s mal e rat

puberta l scree n wa s consisten t wit h th e study desig n that wa s eventuall y adopte db y th e EDS P a s OPPT S 890.1500 . Th e result s o f thi s scree n were reporte dat

th e Societ

y fo r Study o f Reproductio n (SSR ) i n Quebe c City , Canad ai n 2005 . Th e ful l pape r

reportin g o n th e detail s o f th e scree n desig n i s i n preparatio n (Stoker , 2010 , persona l communication) . Group s o f intac t 2 3 day ol dweanlin g mal e Wista r rat s wer e administere dsimazin e a

t dos e level s o f

0 , 6.25 , 25 , 75,150 , an d30 0 mg/kg/day fo r 3 0 days . Preputia l separatio n wa s monitore dfro mpost-nata l day (PND ) 3 3 onward s an dth e animal s wer e necropsie do n PN D 53 . Th e pituitary , testes , ventra l prostate , latera l prostate , epididymides , an dsemina l vesicle s with coagulatin g glan dwere remove dan dweighed . Tissu e sample s wer e examine db y histology . Bloo dwa s analyze dfo r TSH , T4 , TS , . .. . luteinizin g hormon e (LH) , prolacti n (PRL) , androge n an d estrogens . Addikionalljy , Lr f an d • PR L wer e determine di n th e anterio r pituitaries . 1* 1 *

Report Number : TK002342 4 Pag e 3Oof6 3

### Deviation s to OPPT S 890.150 0 Protoco l OPPT S 890.150

0 Requirement s Criteri a S D o r Wistar ra t strai n recommende d Dosin g PN D 23-5 3 2 hig h doses , nea

r MT D Weig h th e testes , epididymides , ventra l prostate , dorsolateral prostate , semina l vesicl e wit h coagulatin g gland s an dfluid , levato r an i plu s bulbocavernosu s muscles , liver , kidneys ,

pituitary , an dadrenal s Histologi c evaluatio n o f

testis , epididymis , thyroid , an dkidne y fro mal l treate dsubject s Measur e thyroxine , TSH , testosterone , creatinine , BUN i n seru m Stoke r et al. , 200 5 Deviation s t

o OPPT S 890.150

0 Non e Non e 5 doses , includin g 3 a

t MT D o r abov e Weighe dpituitary , testes , ventra l prostate , dorsolatera l prostate , seminal vesicl e with coagulatin g gland s an dflui d Histologi c evaluatio n o f

testis , epididymi s an dthyroi di n high-dos e

grou p onl y Measure dseru mthyroxine , TSH , testosterone , an dman y othe r hormona l parameter s bu t

no t seru mchemistrie s Justificatio n fo r OSR I Non e neede d Non e neede d Exceede drequirement s Nearl y al l androge n target s weighe d Specifi c scree n target s were evaluate dhistologicall y at highes t treatmen t

dos e Measure s fo r

EDS P screen relate dtarget s were conducte d 

### Conclusions : Th e simazin e consortiu mrequest s awaiver fo r th e EDS P Tie

r 1 Mal e Pubertal assa y becaus e simazin e ha s alread y bee n thoroughl y teste dh i an existin g stud y usin g identical methodolog y t o OPPT S Serie s 890.150 0 Protocol . N o methodologica l deviation s wer e identifie dthat

woul dimpac t

th e justificatio n fo r

usin g OSR I i n consideratio n o f simazin e activity i n th e mal e rat prepuberta l screen . 3.1 0 Steroidogenesi s - Huma n Cel l Lin e H295 R(OPPT S 890.1550) Purpos e o f

Assay • •• • Th e H295 RSteroidogenesi s Assay i s designe dt o identif y th e potentia l fo r

jcopljiotic s t o • affec t th e steroidogeni c pathway , beginnin g wit h reaction s occurrin g afte r th e goSiadotrop m o r corticotrophi n receptor s are occupied . Thi s i n vitr o assa y i s no t intended^ndpntify ; •.. * substance s that

ma y affec t

Steroidogenesi s du e t o actio n o n th e hypothalamus'aBpituitary^j ^ gland . ••;•* . " Assa y Methodolog y Summar y Th e purpos e o f

th e OPPT S i n vitr o assay i s t o tes t th e potentia l o f

xenobiotic s t o affec t

Jthe * • t 

### synthesi s o f

th e steroi dhormone s b y inducin g o r inhibitin g enzyme s i n th e steroi dhormMife * synthesi s pathway . Th e assay use s H295 Rcell s (ATCC-2128 ) that wer e originall y derive d fro mahuma n adrenocortica l carcinoma . Thes e cell s synthesiz e an dsecrete principa l androgens , estrogens , glucocorticoids , mineralocorticoids , an dprogestin s normall y produce d b y bot h adrenocortica

l an d gonada

l tissues . Thi s cell typ e represent s adistinctio n versu s non-malignan t

adrenocortica l tissu e that normall y secrete s ver y littl e estroge n o r testosterone , an dwhic h require s ACT H t o initiat e Steroidogenesis . Report Number : TK002342 4 Pag e 3 1 o f 6 3Thi s EDS P assay i s conducte d b y incubatin g H295 Rcell s with 7 concentration s o f th e tes t chemical i n 24-wel l tissu e cultur e plates . Contro l incubation s us e forskoli n ( astimulator ) an dprochlora z (an inhibitor ) o f steroidogenesis . Measure s o f 17|3-estradio l an d testosteron e conten t o f th e incubatio n medi aar e performe dusin g appropriat e steroi dhormon e assays . Cel l viability , an importan t inde x o f potentia l toxicit

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

Informatio n Chlorotriazines , includin g simazine , hav e bee n teste di n anumbe r o f studie s o f

steroidogeni c cel l system s (extensivel y reviewe db y Y i e t al. , 2010) . Aromatas e i s th e on e steroidogeni c enzym e that

convert s androgen s t o estrogens . Level s o f thi s enzyme , typicall y assesse db y measurin g th e amount

o f 3 H-wate r produce d fro m3 H-androstenedion e substrate , an d als o b y measurin g level s o f

mRN A fo r

th e enzym e itself, hav e bee n show n t o increas e i n cel l culture s incubate d with chlorotriazines . Th e EDS P Tie r

1 scree n that

use s H295 Rcell s i s designate da s a"Steroidogenesi s Assay. " Th e protoco l doe s no t

cal l fo r measure s o f

eithe r aromatas e activity pe r

s e (e.g. , 3 H-wate r production ) o r

quantit y (e.g. , mRN A fo r

th e enzyme) . Th e targete d measure s fo r th e screen ar e level s o f

testosteron e an destradio l release dint o th e incubatio n media , a s representative s o f th e entir e steroidogeni c pathway . B y thes e criteria , on e repor t present s result s appropriat e to OSR I consideratio n fo r

simazin e (Sanderso n e t

al. , 2001) . However , thi s stud y include d measure s o f

estron e outpu t

amon g th e result s presented , bu t no t estradio l o r

testosterone . Nevertheless , ther e appea r t o b e n o methodologica l deviation s that woul dimpac t

th e justificatio n fo r

usin g OSR

I i n consideratio n o f

simazin e activit

y i n th e H295 R steroidogenesi s screen .

OSRI Steroidogenesi s H295 R Referenc e Sanderso n etal , 200 1 Comment s H295 Rcells ; measure destron e outpu t • •• • ••••• • Sanderso n e t

al. , 200 1 • • • Thes e experiment s were focusedprimaril y o n th e enzym e aromatas e i n H2&& & cells ; enzym e i s no t

th e specifi c targe t

o f th e EDS P Tie r

1 scree n assa y that

use s H f 9,$ Rcells . However , incubation s with simazin e wer e conducte dfo r thi s repor t

an dthe.kw9stigators*cfid * measur e estron e outpu t

int o th e incubatio n media . Whil e estron e itsel f i s no j ^.sjJecifie d• produc t t o b e measure d i n th e Tie r 1 scree n test , th e hormon e i s nevertheles s atermina l •••• • produc t o f steroidogenesis . Th e rang e o f tes t substanc e concentration s wa s muc h narroxvet . tha n th e recommende d scree n bu t wa s near th e highes t

level s calle dfo r

i n th e protocol. * G<j H viabilit y wa s evaluated .

Repor t Number : TK002342 4 Pag e 3 2 o f 6 3

### Deviation s t o OPPT S 890.155 0 Protoco l OPPT S 890.155 0 Requirement s Criteri a H295 Rcell s Po s + Ne g control s Chec k fo r cel l viabilit y Measur e testosteron e an destradio l outpu t i n medi a Sanderson , e t al. , 200 1 Deviation s t o OPPT S 890.155 0

Non e Non e Don e bu t use dadifferen t

metho dthan EDS P T1 S protoco l Estron e measured ; testosteron e an destradio l

no t measure d Justificatio n fo r OSRI Non e neede d Non e neede d Bot h method s see mreliabl e Estron e i s als o aproduc t

o f steroidogenesi s 

### Conclusions : Th e simazin e consortiu mrequest s awaive r fo r th e EDS P Tie r

1 H295 R Steroidogenesi s assa y becaus e simazin e ha s alread y bee n teste di n existin g studie s usin g substantiall y similar

methodology . Ther e appea r t o b e n o methodologica l deviation s that woul dimpac t

th e justificatio n fo r

usin g OSR I i n consideratio n o f simazin e activity i n th e steroidogenesi s assay . 3.1 1 Uterotrophi c - Ra t (OPPT S 890.1600 ) Purpos e o f

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

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

thi s activity begin s wit h bindin g o f estroge n t o th e estroge n recepto r (ER) . Thus , th e tes t

i s acomplet

e assessment

o f ates t substance' s abilit

y to bin d to E Ran dt o promot e well-know n an deasil y measure dresponse s t o E Rbindin g i n alivin g animal . 

### • • • Assa y Methodolog y Summar y •**;* ? *" • Th e Uterotrophi c assay i s designe dt o detec t th e potentia l fo r

axenobioti c tf

t expres s . . . estrogeni c effects . Approve dmethod s permi t us e o f eithe r rat s (Sprague-EPawfe y o r \\Asfar * strain s preferred ) o r

mice . Tes t

subject s ca n b e intac t

immatur e (testin g initiate dduringJJiJl D 21-24 ) o r ovariectomize d(OVX ) adults . Tes t material s can b e administereJfU y oral gavag e o r injecte dsubcutaneously . There shoul db e at leas t tw o dos e groups , with*thfe»highe r dos e at o r near th e animals ' maximu mtolerate ddose , plu s agrou p administere dvehicl e only . «f^er e shoul db e at

leas t 6 animals/dos e groups . Administratio n o f areferenc e hormone , suducfs** * • 17a-ethiny l estradio l i s considere dhelpfu l bu t i s no t mandatory . Treatmen t

shoul db e administere ddail y fo r thre e consecutiv e days , followe db y sacrific e o n day 4 . Th e complet e uterin e organ i s dissecte dat necropsy , carefull y cleane do f al l fat an d extraneou s connectiv e tissue , cu t open to releas e intraluminal fluid , blotte d bu t no t dried , an d the n weighed . Anima l weigh t shoul dals o b e take n at

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

uni t B W may b e importan t

i f th e administere dcompoun daffecte dbod y weights . Report Number : TK002342 4 Pag e 3 3 o f 6 3Othe r Scientificall y Relevan t

Informatio n Simazin e ha s bee n previousl y teste di n uterotrophi c assay s usin g substantiall y simila r methodolog y a s th e EDS P Tie r 1 scree n guidelines . Th e cite dstudie s evaluate dth e importan t endocrine-relate d targe t

identifie di n th e OPPT S Serie s 890.160 0 protocol . Th e anima l strain , low-estroge n conditio n o f th e tes t animals , an d metho do f

administratio n wer e acceptabl e a s describe di n th e OPPT S protocol . There appear t o b e n o methodologica l deviation s that

woul dimpac t

th e justificatio n fo r

usin g OSR

I i n consideratio n o f

simazin e activity i n th e femal e rat

prepuberta l screen . OSR I Uterotrophi c assa y Uterotrophi c assa y Uterotrophi c assa y Reference(s )

Tenan t etal. , 1994 a Conno r etal. , 199 6 O'Conno r etal. , 200 0 Comment s Adul t ovariectomize drat s Intac t immatur e rat s Adul t ovariectomize drat s Tennan t e t al. , 1994 a I n thi s study , adul t

S D rat s were purchase dafte r ovariectomy an dwer e administere d simazin e b y gavag e fo r

thre e day s at

dose s u p t o 30 0 mg/kg . Grou p mea n B W at th e highes t dos e wa s 12 % belo w th e vehicl e group weight . Tes t group s wer e 5- 6 pe r

dose . A positiv e contro l grou p receive d estradio l injecte ds.c . Uter i wer e dissected , cleane dan dweighed . Conno r et al. , 199 6 Thi s study , fro madifferen t university laboratory , ca n b e viewe da s a n extensio n o f th e Tennan t e t al. , 1994 areport . Group s o f 4- 5 immatur e intac t

S D rat s (PND 21 ) wer e administere d 3 dose s o f

simazin e b y gavag e at

aslightl y lowe r

dosin g range . A positiv e contro l grou p receive d estradio l b y i p injection . Uter i wer e dissected , cleane dan dweighed . O'Conno r

e t al. , 200 0 . . Thi s study , conducte d at

th e Haskel l Laboratory , use d42-da y ol d ovariectomize dCrl:C P B r rats . Tes t compound s wer e administere db y intraperitonea l (ip ) injectio n SXMa'il ^ fo r 4 days * Total dail y dos e o f

simazin e wa s 100 , 200 , o r 30 0 mg/kg/day . A positiv e contro l grou g j^aj . injecte dwith 5 0 |ig/kg/da y estradiol . Grou p mea n B W at th e highest

simazlfifeMas e wa s reduce d25 % at

sacrifice . Uteri wer e dissected , cleane dan dweighed .

Report Number : TK002342 4 Pag e 3 4 o f 6 3Deviation s t o OPPT S 890.1600 Protoco l OPPT S 890. 1 60 0 Requirement s Criteri a S D o r Wistar rats , intac t OV X adul t

o r immature , o r mic e 2 dose s plu s vehicle , at

o r near MT D Gavag e o r injecte ds c 3 day s treatment , sa c o n day 4 Dissect , clean , weig h uteru s Tennan t

e t al. , 1994 a Deviation s t o OPPT S 890.160 0 None : use dS D OV X adul t rat s None : 3 dose s plu s vehicle ; MT D exceede d None : gavag e Non e Non e Conno r

et al , 199 6 Deviation s t o OPPT S 890.1600 None : use dimmatur e S D rats , treatmen t starte da t PND 2 1 None : 3 dose s plu s vehicle , MT D exceede d None : gavag e Non e Non e 3'Conno r

e t al. , 200 0 Deviation s t o OPPT S 890.160 0 None : use dS D OV X adul t rat s None : 3 dose s plu s vehicle ; MT D exceede d None : injecte di.p . None ; 4 day s treatmen t Non e Justificatio n fo r OSR I

Non e neede d

Non e neede d Non e neede d Non e neede d Non e neede d Conclusions : Th e simazin e consortiu mrequest s awaive r fo r th e EDS P Tie r

1 Uterotrophi c assay becaus e simazin e ha s alread y bee n thoroughl y teste di n existin g studie s usin g substantiall y simila

r methodology . 

### 4. 0 REPRODUCTIV E AN D DEVELOPMENTA L TOXICIT Y CHARACTERISTIC S O F SIMAZIN E A s discusse dabove , simazin e ha s bee n teste dusin g stud y protocol s whic h ar e identica l t o o r substantiall y similar

t o th e OPPT S Serie s 89 0 stud y guidelines . I n additio n ther e i s awealth o f dat afro mhighe r

tie r studie s (Tabl e 3 ) availabl e fo r simazin e whic h evaluate s apical endpoint s i n intac t

animals . Change s i n thes e endpoint s upo n exposure t o ates t substanc e woul dindicat e th e potentia l fo r th e substanc e t o interferenc e wit h endocrin e systems . (Se e USEPA , 2006 , fo r

mor e details ) Base do n result s o f

studie s i n rat s an drabbit s i t wa s conclude dthat

simazin e doe s an y specifi c teratogeni c o r fetotoxi c effect s whe n administere dt o pregnan t daigi £ durin g th e perio do f organogenesis . Fetotoxi c effect s wer e reporte donl y at extremel y hig h *doses;ti^t * . wer e significantl y toxi c t o th e dams , i.e . >10 0 mg/k g fo r th e rat an d>7 5 mgAl^ g if

l rabbits . * * Likewis e simazin e di d no t caus e an y impairmen t

i n reproductiv e performanc e i n rat s fij d' * * * simazin e a

t dietar y level s up t o 50 0 pp mfo r tw o successiv e generations . Pubfcshfe d• reproductio n an ddevelopmenta l studie s i n mic e an drat s usin g simazin e i n a'mixtur e wtfc.S * othe r pesticide s at

concentration s u p t o 10 0 time s th e maximu mpermissibl e drinkin g wa^r, . leve l confirme dth e absenc e o f advers e effect s o n reproductiv e o r developmental parametar«» * (USEPA , 2006) .

Report Number : TK002342 4 Pag e 3 5 o f 6 3

### TABL E 3 EP A Guidelin e Reproductiv e an dDevelopmenta l Toxicit y Studie s o n Simazin e Technica

l Study Developmental Toxicity i n Rat , Mainier o e t

al. , 198 6 Developmenta l Toxicity i n Rabbit , Arthur , 198

4 2-Generatio n Reproductio n Stud y i n Rats , Epstei n e t al. , 199 1 MRI D

Numbe r 4061440 3

16140 7

4180360 1 Mod e o f

Dosin g Gavag e

Gavag e

Dietary Dos e level s

30 , 300 , 60 0 mg/kg/da y

5 , 75 , 20 0 mg/kg/day

0.6 , 6 , 3 2 mg/kg/da y NO(A)E L

(mg/kg/day ) 300 Fetal-30 0

Materna l - 5 Fetal - 5

Parenta l - 0 6 Offsprin g - 0 6 LOAE L

(mg/kg/day ) Materna l -60 0 Fetal-60 0

Materna l -7 5 Fetal -7 5

Parenta l - 6 Offsprin g - 6 Effect s

Severe materna l toxicit y accompanie d b y mino r feta l skeletal effect s Death ; abortion ; tremors ; stool s (little/no/soft) ; decrease dfee d consumption , body weight , an dbod y weigh t gain s Reductio n i n body

weight , body-weigh t gai n an dfoo d consumptio n 

### 5. 0 CONCLUSIO N Simazin e ha s bee n use da s ates t substanc e i n th e development/validatio n o f tw o o f th e existin g eleven Tie

r 1 assay s (mal e an dfemal e pubertals) , s o th e protoco l followe di n thes e case s wa s essentiall y identica l t o th e final OPPT S Serie s 89 0 protocols . I n mos t o f th e other assays , simazin e wa s teste db y substantiall y simila r methods . Althoug h an amphibian metamorphosi s study ha s no t bee n conducte do n simazine , numerou s studie s hav e bee n cite d i n rats , dog s an drabbit s whic h include dan evaluatio n o f effect s o n th e thyroi dreceptor s i n vitr o an deffect s o n th e HP T axi s throug h thyroi dweight and/o r histolog y and/o r

thyroi d hormones . I n addition , simazin e ha s bee n teste dvi ath e standar dhighe r tie r roden t studie s require dfo r

registratio n o f

apesticid e i n th e US ; thes e studie s includ e a n evaluatio n o f a numbe r o f additional endpoint s whic h ar e functionall y equivalen t t o th e specifi c endpoint s teste di n th e Tie r

1 screenin g battery . Furthermore , afis h ful l lif e cycl e study ha s bee n conducte dwit

h simazin e formulate dfo r

us e a s an aquati c herbicide . Overall , th e weight of. , evidenc e indicate s tha t

th e simazin e toxicity databas e i s complet e with respect.tp.th e •.. * I endocrin e relate dendpoint s measure db y th e Tie r 1 assays . Il l • I n conclusion , simazin e ha s bee n evaluate di n th e EDS P Tie r 1 screen s and/orjsuljstanti'all y * simila r assays . Th e simazin e consortiu mrequest s awaive r fo r al l o f th e Tier 1 assays . * 9

TK0023424.do c sbh : 5/19/1 0



### Report Number : TK002342 4 Pag e 3 6 o f 6 3

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### Judso n RS , Houc k KA , Kavloc k

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## ••;•• : t • • Sanderso n JT , Letche r

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### Annua l Mtg . Jul y 24-27 , 2005 . Quebe c City , Quebec , Canada . Stoke r T . 2010 . Persona l communicatio n with Dr . Jame s Stevens . Repor t Number : TK002342 4 Pag e 3 8 o f 6 3Tai C , Breckenridg e C , Gree n J . 1985a . Simazin e Technical : Subacut e Ora l 13-week

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Mediate d Response s b y Chloro-S-Triazine-Derive dCompound s i s Dependen t o n Estradio l concentratio n i n yeast . Bioche mBiophy s Re s Commun . 227:140-6 . USEPA . 2006 . Reregistratio n Eligibility Decisio n fo r Simazine . Special Revie w an d Reregistratio n Division . Unite dState s Environmenta l Protectio n Agency . Report N° . EP A 738-R-06-008 : Cas e No . 0070 . Apri l 2006 . USEPA . 2009 . Integrate d Summar y Repor t fo r Validatio n o f

an Estroge n Recepto r

Assa y usin g Rat Uterin e Cytoso l a s Sourc e o f Recepto r a s aPotentia l Scree n i n th e Endocrin e Disrupte r Screenin g Progra mTie r 1 Battery . March , 2009 . Wu W , Nile s EG , LoVerde PT . 2007 . Thyroi dhormon e recepto r orthologue s fro m invertebrat e specie s with emphasi s o n Schistosom amansoni . BM C Evol . Biol . 7:150 . Y i KD , Simpkin s JW , Breckenridg e CB . 2010 . Evaluatio n o f th e Potentia l Mechanisme'o,? " • Action s Underlyin g th e Effec t o f Triazine s o n Aromatas e Expressio n i n v/"z5"o ? jRjlevanc e t o Ma n an dth e Environment . Revie w submitte dt o th e U S Environmenta l Prfltectid n an dals o t o Rev . Environ . Contam . Toxicol . (h i press) . MRI D No . 47972809".; * * • • • Zorrill aLM , Gibso n EK , Stoker TE . 2010 . Th e Effect s o f Simazine , aChlorojtrTajin e Herbicide , o n Puberta l Developmen t i n th e Femal e Wistar

Rat . Repro .

Report Number : TK002342 4 Pag e 3 9 o f 6 3

### APPENDICE S SECTIO N



## • • • • * . ••;•• ; * *• •

Repor t Number : TK002342 4 Pag e 4 0 o f 6 3

### APPENDI X 1 ToxCast TM

ToxCast TM i s aU S EP A programthat include s abattery o f

i n vitro , high-throughpu t screenin g (HTS ) assay s t o scree n chemical s fo r potential huma n toxicity . Over 30 0 chemical s hav e undergon e HT S unde r Phas e I o f

th e ToxCast TM program , th e majorit y o f thes e bein g pesticides . A number o f thes e chemicals , includin g simazine , hav e current o r forthcomin g requirement s fo r Endocrin e Disruptio n Screenin g Program(EDSP ) Tie r 1 screening . Potentia l redundancie s exis t betwee n som e o f

th e ToxCast TM assay s an dth e EDS P Tie r 1 screening objectives ; thus , ToxCastTM datamayb e usefu l OSP J (Othe r Scientificall y Relevan t Information ) t o submi t

i n lie u o f o r t o supplemen t

Tie r 1 require dtests . Informatio n regardin g th e ToxCas t Progra mresult s becam

e generally availabl e to th e publi c throug h Environmenta l Healt h Perspective s (EHP ) an d th e EP A ToxCast TM Program websit e (www.epa.gov/ncct/toxcast / index.html ) i n mid-Decembe r 2009 , with th e ra w dat a fo r man y o f th e assay s poste d o n th e ToxCast TM websit e i n mid-February 2010 . Th e dat a presente d i n th e EH P articl e (Judso n e t al . 2010 ) an dsupplementar y material s (Judson_ehp.0901392.s001.pdf) , an d o n th e ToxCast TM Progra

mwebsit e i s fairl y opaque , with individua l chemical dat atabulate dan dmuc h o f th e detai l o n assa

y methodologie s provide d onl y throug h websit e links . Thi s revie w wa s develope d t o evaluat e th e dat amos t relevan t t o potentia l endocrin e modulatio n an d th e result s specifi c t o simazine . Thi s revie w describe s briefl y th e HT S assay technologie s incorporate d i n ToxCast TM relevan t t o th e EDS P Tie r

1 screenin g requirements , provide s a

n overvie w o f

th e HT S assay s mos t

relevan t to potential estrogen/androgen/thyroi d hormon e (EAT ) endocrin e modulation , an ddiscusse s th e EDS P Tier 1 screenin g assay s tha t coul db e potentiall y fulfilled . 1 . ToxCast TM HT S Tes t Batter y an d Relevanc e t o Potentia l Endocrin e Modulatio n Th e ToxCast TM HT S tes t battery include s nin e separat e i n vitr o assay technologies : Cell-fre e HT S assay s t o measur e bindin g constant s an denzym e inhibitio n Cell-base d HT S assay s t o measur e bindin g constant s an d enzym e intibftio n values* . Hig h conten t

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

phenotypfe s i n eithe r

^ th e huma n hepatom acel l lin e HepG 2 o r rat primary hepatocytes ; • Gen e expressio n i n huma n primar y hepatocytes , focuse do n Phas e I anc l T I metarJaU«S « enzyme s an dtransporters ; Multiple x transcriptio n reporte r assay s t o evaluat e 4 8 transcriptio n facto r bindin g sites ; Biologicall y multiplexe d activity profilin g (BioMAP ) t o characteriz e effect s potentiall y relevan t

t o huma n tissu e an dinflammator y diseas e biology ; Report Number : TK002342 4 Pag e 4 1 o f 6 3

### • Phas e I & I I xenobioti c metabolizin g enzyme (XME ) cytotoxicit y assay s base do n microarra y technology ;

• High-throughpu t

genotoxicit

y screenin g base do n th e GreenScreen hig h conten t GADD45A-GFP HT S genotoxicit y assay ; an d

• Real-time cel l electroni c sensin g (RT-CES ) t o measur e cellula r change s i n respons e t o chemicals . O f thes e assay technologies , thos e that provid e informatio n mos t directl y relevan t

t o th e issu e o f potentia l EA T endocrin e modulatio n includ e th e cell-fre e HT S assays , th e cell-base dHT S assays , an dth e multiple x transcriptio n reporte r assays . Eac h o f

thes e assay s an dthei r

dat a relevan t t o potentia l endocrin e modulatio n i s describe dbelow . 1. 1 Cell-Fre e HT S Assay s Thes e assay s were ru n unde r contrac t t o EP A b y Calipe r

Lif e Science s (formall y NovaScreen ; no w Calipe r Lif e Sciences) . O f

th e 23 9 endpoint s examined , bindin g t o th e followin g receptor

protein s relevant

to th e endocrin e syste

mwa s evaluated : • Bovin e estroge n recepto r fro muterin e membran e (bER) ; • Huma n estrogen receptor fro mbreas t

cance r cell s (hER) ; • Bovin e progesteron e recepto r fro muterin e membran e (bPR) ; • Human progesteron e recepto r fro mT47-D 1 

### cell s (hPR) ; • Rat androge n recepto r

(rAR ; recombinant) ; • Huma n androge n recepto r

fro mLnCAP cell s (hAR) ; • Rat thyrotropin-releasin g hormon e recepto r fro mforebrai n membrane s (rTRH) ; an d • Huma n thyroi dhormon e receptor-alph a(hTRa ; recombinant) . Al l o f thes e protein s are 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 dreceptor . Additionally , al l o f these , wit h th e exceptio n o f

th e hTRa , wer e evaluate di n competitiv e bindin g assay s that measure d th e displacemen t

o f radiolabele dligan dfro mth e recepto r proteins . Th e hTR aassay , i n • ;• ; contrast , examine drecepto r activatio n b y measurin g th e luminescen t outpu t ftcgnja n hTRa

responsiv e reporte r

gene . Inhibitio n o f

th e aromatas e enzym e (CYP19A 1 o t CYB C 19 ) als o evaluated . Assa y parameter s specifi c t o eac h o f thes e endpoint s ar e detaile d j n Appendi x Tabl e 1 . *. .

1 EP A indicate dtha t

th e huma n progesteron e recepto r wa s derive dfro mMCF- 7 cells ; however , accordin g t o Calipe r Lif e Sciences , th e recepto r

protei n wa s derive dfro mT-47 D cells . 

### Report Number . TK002342 4 Pag e 4 2 o f 6 3

### Appendi x 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

eron e recepto r Androge n recepto r Thyroi d Relate d Aromatas e bE R

hE R bP R hP R rA R hA R rTRH hTRa c hCY P C1 9 Assay typ e Competitiv e bindin g Competitiv e bindin g Competitiv e bindin g Competitiv e bindin g Competitiv e bindin g Competitiv e bindin g Competitiv e bindin g Recepto r activatio n Enzym e inhibitio n Referenc e

compoun d N/A a 17B-estradiol promegeston e promegeston e methyl

trienolon e methyl

trienolon e thyroi dreleasin g hormon e (TRH ) Bisphenol- A Ketoconazol e Positiv e

contro l compoun d 17B-estradio l 176-estradio l promegeston e promegeston e methyl

trienolon e methyl

trienolon e TRH N/ A N/ A Ligan d

(cone. , M ) 3 H-estradio l (7xlO-'° ) 3 H-estradio l ( 1 x 10- 10 ) 3 H-promegeston e ( 7 x Iff 10 ) 3 H-promegeston e (7xlO-'°) b 3 H-methyl

trienolon e ( 1 x ID' 9 ) 3 H-methyl

trienolon e ( 5 x ID' 10 ) 3 H-3-meHis 2 - TRH (2xlO' 9 ) Triiodo-thyronin e (T 3 ) (l.SxlO' 8 ) Di(benzyloxy

methoxy ) fluorescein d (2. 5 x Iff 6 ) Kd(M )

7 x 10-' ° 5x10- " 6x10- " 3 x 10- 1 U 4. 6 xlO' 9 3 x 10- 1 U 2.3x10- ' 3.5x10- * N/ A Bma x

(fmol/m g protein ) 40 6 3,84 4 15 2 15 2 103,92 8 27 0 3 4 N/ A N/ A

aN/A = no t

applicable . b EP A list s th e ligan dconcentratio n a s 3 x 10' 1 0 M ; however , accordin g t o Calipe r Lif e Sciences , th e ligan dconcentratio n wa s 7xlO-'°M . c Assa y o f antagonis mt o ligand-dependen t bindin g o f co-facto r t o th e receptor . dAromatas e substrat e bindin g affinit y = 4. 4 x 10" 6 M an dvma x = 0.0 3 pmo l product/pmo l CYPC19/min . 

### I n al l cases , th e dat awer e normalize dt o thos e o f th e solven t

control s fro mth e sam e plat e a s th e teste dchemical s an dresult s expresse da s th e percen t o f neutra l control . Fo r

th e parameter s above , chemical s wer e initiall y screene dat asingl e concentratio n o f

2 5 (i M (1 0 ji M fo r aromatase ) i n duplicat e wells . Thos e chemical s tha t wer e positiv e i n th e initial . .. . scree n wer e the n ru n usin g eigh t concentration s i n th e rang e o f 0.0229-5 0 \$ & (QJ)0914-^ 0 • |o.M fo r aromatase ) t o determin e th e ACs o (i.e. , th e hal f maxima l activit y cobceHt»ation)Cerl

Base dHT S Assay s " " " " 1. 2 Cell-base dHT S Assay s Thes e assay s were conducte db y th e NI H Chemica l Genomic s Cente

r (NCGC) . Bot

h ag9nisi g 

### an dantagonis t

activity at

variou s nuclear

receptors , includin g th e huma n androge n receptor . • (hAR) , huma n ER a(hERa) , an dhuma n thyroi dhormon e receptor- p (hTRp) , wer e examined * usin g areporter

gene assa

y syste m(misidentifie da s Invitrogen TM GeneGLAze r i n EPA materials ; mos t

likely , th e Invitrogen TM GeneBLAzer ® reporte r assa y system) . Accordin g t o th e Invitrogen TM websit e (http://www.invitrogen.com/) , th e GeneBLAzer ® technolog y use s a beta-lactamas e reporter

system , bu t

specific s abou t

ho w th e systemwork s are no t provided . Report Number : TK002342 4 Pag e 4 3 o f 6 3Th e nuclea r receptor source s an d th e assa

y parameter s relate d to eac h o f th e examine d endpoints , a s provide d b y th e EPA , ar e detaile di n Appendi x Tabl e 2 . Appendi x Table2 : Paramete r Specific s Regardin g Endocrine-Relate d Endpoint s o f th e Cell-Base d HT S Assay s Androge n Relate d Estroge n relate d Thyroi d relate d Activit

y hA Ragonis t hA Rantagonis t hERc t agonis t hER aantagonis t hTRp agonis t hTR p antagonis t Targe t sourc e HEK293 H cell s HEK293 H cell s HEK293 H cell s HEK293 H cell s HEK293 T cell s HEK293 T cell s Referenc e

compoun d R188 1 N/A 17(3-estradio l 4-hydroxy-tamoxife n Triiodothyronin e (T s ) N/ A Positiv e contro l

compoun d 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 lO' 8 ) N/ A 17p-estradio l ( 5 x lO' 10 ) N/ A T 3 ( 4 x ID' 10 ) aN/A = no t

applicable . Exac t concentration s o f

chemical s teste di n thi s assay syste mar e no t clear . A recen t publicatio n indicate s that chemical s wer e teste dat fiftee n concentration s i n th e rang e o f 0.0012-9 2 u M (Judso n e t al . 2010) . However , th e summary dat afil e fo r thes e assay s provide do n th e ToxCast TM websit e (http://www.epa.gov/ncct/toxcast/index.html ) indicate s tha t chemical s wer e teste d t o amaximu mconcentratio n o f 20 0 (iM . h i th e cas e o f

simazine , concentration s i n th e rang e o f 0.001 0 - 76. 6 jo 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 achemical t o b e active . Th e supplementa l informatio n t o Judso n e t al . (2010 ) indicate s that , "value s wher e onl y th e highes t concentratio n exceede d 50 % activity wer e exclude dt o eliminat e weak o r fals e positives. " Cel l viabilit y wa s als o evaluate di n thi s assa y system ; however , th e criteri afo r excludin g concentration s t o b e teste di n th e assa y systembase do n cytotoxicity ar e no t clear . 1. 3 Multiple x Transcriptio n Reporte r Assay s Thes e assay s wer e performe 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 dint o Hepp2;* J cell s (Marti n e t

al . 2010) . Cis-actin g reporte r gene s examine dth e activitiesjo f Jfiftiscripfio n * factor s endogenou s t o th e HepG 2 cells . Tran s gene s evaluate dth e activitie s of*e»ogengvs , • chimeri c proteins . Eac h chimer awa s comprise do f th e ligand-bindin g domafrjbf & nuclear * • " recepto r an dayeas t

Gal 4 DNA-bindin g domain . Whe n activated , thes e chimeM e protefos,.! . activate dtranscriptio n o f areporter

gen e expresse ddownstreamo f aSX-U/fSvFAJ A promoter . I n al l cases , th e reporte r gen e wa s identica l i n sequenc e an dinclude&a'restrigtion , enzym e cleavag e site ; th e positio n o f th e restrictio n site , however , wa s uniqu e t o eac h • * * * reporter gene . Th e syste mallowe dfo r reporte r gen e product s t o b e identifie dthroug h ,**** • sequencin g an dseparatio n vi ahig h resolutio n electrophoresis . Usin g thi s system , th e followin g endpoint s relevan t t o endocrin e modulatio n wer e evaluate d(liste di n Appendi x Tabl e 3 , alon g wit

h thei r respectiv e referenc e compounds , a s provide d i n EPA materials) .

Report Number : TK002342 4 Pag e 4 4 o f 6 3Appendi 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 Androge n relate d Estroge n relate d Thyroi drelate d Endpoin t Huma n androge n recepto r Huma n ER- a Human estroge n respons e element 3 Huma n estrogen-relate dreceptor- a Huma n estrogen-relate dreceptor- y Human thyroi d hormon e receptor- a Referenc e Compound s Dioxin , FI C 17p-Estradiol , diethylstilbestro l Estrogen s N/A b 4-Hydroxytamoxife n Triiodothyronin e (Ts ) aTh e human estrogen respons e elemen t wa s teste d a s aci s element ; al l other endpoint s relevan

t to endocrine modulatio n were teste da s tran s factors . b No t applicable . Cytotoxicity 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 exposure t o fiv e tes t concentrations , with an uppe r concentratio n o f 5 0 u M an d10-fol d seria l dilutions . Th e maximu mtolerate dconcentratio n (MTC ) wa s derive da s one-thirdth e cytotoxi c ICso , o r i f n o ICs o valu e wa s determined , the n th e MT C wa s se t at 10 0 uM . Tes t

compound s were the n assaye dat seve n differen t

concentration s i n th e rang e o f

0.0014-10 0 uM , startin g a t th e MTC , followe db y three-fol dseria l dilutions . Afte r a24-hou r

exposure , cell s were harvested , reporter gen e product s isolate dan dreverse transcribe d usin g fluorescent-tagge dprimers , an dresolve db y hig h resolutio n ge l electrophoresis . Fro mth e resultin g data , th e lowes t effectiv e concentratio n tha t activate da cis-actin g respons e elemen t o r transcriptio n facto r

wa s determine d(LEL ; lowes t concentratio n a t

whic h ther e wa s astatisticall y significan t chang e fro mth e concurren t negativ e control) . 2 . Compariso n t o EDS P Tie r 1 I n Vitro Screenin g Requirement s Th e ToxCast TM dat asupplement , an di n some cases , may fulfil l th e i n vitr o dat arequirement s o f th e EDS P Tie r

1 E Rbindin g assay , A Rbindin g assay , E Rtransactivatio n assay an d aromatas e inhibitio n assay . I n addition , thes e dat aprovid e important OSR I that shoul d b e considere di n aweight-of-the-evidenc e evaluatio n o f functionall y equivalen t

informatio n supportin g th e fulfillmen t o f severa l EDS P Tier

1 mammalia n i n viv o screenin g tests . E Rbindin g assay : Th e EDS P Tie r 1 E Rbindin g assay require s that binding»l» » evaluate d * usin g rat E Rderive d fro muterin e tissu e take n fro movariectomize dSpragu p D&viley rat s (EPA 2009d) . I n th e ToxCastTM cell-fre e HT S assays , bindin g to bovin e andh«s»an , rat , E Rwa s evaluated . I n th e EDS P Tie r 1 screenin g assay , th e concentrationt&T:!

- estradio l ligan d use di n th e assay i s 1 n M an dth e tes t chemical s ar e evaluat«d«t » 0 concentration s o f

1 x 10' 1 0 t o 1 x 10'3 M (EP A 2009d) . I n th e ToxCast TM asgai.tB e concentratio n o f

radiolabele destradio l varie ddependin g o n th e E Rsourc e (bER : 7 x hER : 1 x 1 0" 1 0 M) , bu t

wa s generall y simila

r t o tha t recommende di n th e EDS P Tier 1 E R• • • bindin g assay ; however , compound s wer e teste da t an initial concentratio n o f 2. 5 x 10" 5 M* " an di f positiv e fo r

bindin g activity , wer e furthe r evaluate dat

concentration s o f

2. 3 x 10" 8 - 5 x 10" 5 M . Thi s concentratio n rang e i s narrowe r than th e rang e recommende di n th e EDS P Tie r 1 screenin g assay , bu t

stil l shoul db e adequat e t o detec t activity i n amoderat e concentratio n range . Report Number : TK002342 4 Pag e 4 5 o f 6 3Androge n receptor bindin g assay : Th e EDS P Tier 1 scree n A Rbindin g assa

y require s that bindin g b e evaluate dusin g rat A Rderive dfro mprostat e tissu e take n fro mcastrate dSpragu e Dawle y rat s (EP A 2009b) . Th e concentratio n o f 3 H-R188 1 ligan duse di n th e assa y i s 1 n M an dth e tes t

chemical s ar e evaluate dat concentration s o f

1 x 10~ 9 t o 1 x 10" 4 M . I n th e ToxCast TM cell-fre e HT S assay , bindin g t o both rat an dhuma n androge n receptor s wa s evaluated , s o i t

provide s amore comprehensiv e scree n tha n doe s th e EDS P Tie r 1 battery . Radiolabele d R188 1 wa s use da s th e radioligan di n thi s assa y an dconcentration s varie d dependin g o n th e A Rsourc e (rAR : 4.5 9 x 10" 9 M ; hAR : 3 x 10~ 1 0 M) , bu t wer e 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 di n th e ToxCast TM cell-fre e HT S assa

y a t a n initia l concentratio n o f

2. 5 x 10" 5 M , an d i f

positiv e for bindin g activity , furthe r

evaluate dat

concentration s o f

2. 3 x 10" 8 - 5 x 10" 5 M . Thi s concentratio n rang e i s slightly narrower than that recommende di n th e EDS P Tie r 1 screen A Rbindin g assay guidelin e ( 1 x 10~ 9 - 1 x 10" 4 M ; EP A 2009b) , bu t stil l shoul db e adequat e t o detec t activity i n amoderat e concentratio n range . E Rtransactivatio n assay : Th e EDS P Tie r 1 scree n E Rtransactivatio n assay require s tha t th e assay b e conducte di n hER-HELA-990 2 cells , whic h expres s th e huma n ER- aan dhav e bee n stabl y transfecte d with aluciferas e reporte r gen e linke dt o a n upstrea mvitellogeni n estrogen responsiv e element . Tes t compound s are evaluate da t concentration s o f

1 x 10~ 9 t o 1 x 10" 3 M . Finally , ates t

chemica l i s considere dpositiv e fo r E Rtransactivatio n i n th e EDS P Tier 1 assa y i f th e maximu mrespons e induce di s a t leas t 10 % o f tha t induce db y 1 nM l?p-estradio l (EPA2009a) . I n th e cas e o f

th e ToxCast TM cell-base dHT S assays , detail s regardin g th e gen e reporte r syste mwere no t

available ; however , activit y at th e huma n ER- awa s assesse dan dtes 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" 4 M ) a s recommende di n th e EDS P Tie r 1 screen . Th e dat aoutput s fro mth e ToxCast TM cell-base d HT S assay s (ACs o values ) ar e differen t tha n thos e require di n Tie r 1 screening , an di n mos t cases , positiv e contro l compound s wer e no t tested . However , i f

achemical elicite dalo w leve l o f activit y a t th e highes t

concentratio n teste di n th e cell-base dassay , i t can b e assume d tha t i t woul d b e negativ e accordin g t o th e EDS P Tie r 1 screenin g criteria . I t

shoul db e note d tha t th e ToxCastTM program' s cell-base d HT S assa y provide s mor

e robus t dat athan that require d i n th e EDS P Tie r 1 E Rtransactivatio n assa y becaus e i t als o examine dth e tes t

. * I* ! chemical' s abilit

y t o ac t a s an antagonis t

at th e ER-a . J**t* J ** . • * • • • • . Th e multiple x transcriptio n reporte r assay als o examine dtransactivatio n o f fifiihulnan I?R-a ? I n thi s case , th e assay i s conducte di n HepG 2 cell s (instea do f HELA-990 2 cell»)

j an d tHa..! » outpu t fro mth e reporte r

gen e an d ho w i t

i s measure di s differen t

fro mtha t

irWfc'a'W di n m e . EDS P Tie

r 1 screenin g assay . HepG 2 i s aperpetua l cel l line , whic h wa s derived»fro mth e • live r tissu e o f a15-yea r ol dCaucasia n America n mal e with awel l differentiate d• hepatocellula

r carcinoma . Th e HepG 2 cell s an d it s derivative s are ofte n use d a s amodei " * * • syste mfo r

studie s o f

live r metabolis man dxenobiotic s toxicit y becaus e o f thei r metabolic * capabilities . Th e rang e o f tes t chemica l concentration s teste d (1. 4 x 10" 9 - 1 x 10" 4 M ) i s similar t o tha t recommende di n th e EDS P Tier 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 di n EDS P Tie r 1 screening , an dpositiv e contro l compound s wer e no t tested . However , i f

achemica l Report Number : TK002342 4 Pag e 4 6 o f 6 3elicite dalo w leve l o f activity at

th e highes t

concentratio n teste di n th e multiple x transcriptio n reporter

assay , i t can b e assume dtha t

i t woul db e negativ e accordin g t o th e EDS P Tier

1 screenin g criteria . Arotnatas e assay : Th e EDS P Tie r 1 scree n aromatas e assay require s that enzym e activity b e evaluate d usin g human recombinant microsome s containin g CYP1 9 plu s reductase . Radio

labele dandrost-4-ene-3,17-dion e at aconcentratio n o f

1 x 10~ 8 M i s use da s th e enzym e substrat e an dtes t

compound s ar e evaluate da t concentration s o f

1 x 10~ 1 0 M - 1 x 10~ 3 M (EPA . 2009c) . I n th e ToxCast TM cell-fre e HT S assay , huma n aromatas e activit y wa s evaluate d usin g di(benzyloxymethoxy ) fluorescein , whic h produce s afluorescen t produc t a s th e enzyme substrate . Compound s were initially screene d a t asingle concentratio n o f 1 x 10~ 5 M , an dthos e chemical s that were positiv e i n th e initial scree n wer e the n ru n usin g eigh t concentration s i n th e rang e o f 9.1 4 x 10~ 9 M - 2 x 10~ 5 M . Thi s concentratio n rang e i s narrowe r than th e rang e recommende di n th e EDS P Tie r 1 screenin g assay , bu t

stil l adequat e t o detec t activity i n amoderat e concentratio n range . I t shoul db e note dthat

th e ToxCast TM HT S dat ase t include s informatio n that shoul db e usefu l to submit

a s OSRI , an d i n fact , goe s beyon dth e i n vitr o testin g require d i n th e EDS P Tie r 1 screenin g progra mt o provid e additiona l informatio n o n th e potentia l t o interac t with th e EA T systems , includin g thyroid-relate dreceptors , a s wel l a s progesteron e receptors . I t doe s not , however , directl y addres s EDS P Tie r 1 steroidogenesi s i n vitr o tes t requirements .

• •



## .... .

Repor t Number : TK002342 4 Pag e 4 7 o f 6 3

### Appendi x 1 - Reference s

Environmenta l Protectio n Agenc y (EPA) . (2009a) . Endocrin e Disrupto r Screenin g Progra

m Test Guidelines . OPPTS 890.1300 : Estroge n Recepto r

Transcriptiona

l Activatio n (Human Cell Lin e (HeLa-9903)) . EPA-740-C-09-006 . October 2009 . Environmenta l Protectio n Agenc y (EPA) . (2009b) . Endocrin e Disrupto r Screenin g Program Tes t Guidelines . OPPT S 890.1150 : Androge n Recepto r Bindin g (Rat

Prostate Cytosol) . EPA-640-C-09-003 . Octobe r 2009 . Environmenta

l Protectio n Agenc y (EPA) . (2009c) . Endocrin e Disrupto r Screening Progra

m Tes t 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 Progra

m Tes t Guidelines . OPPT S 890.1250 : Estroge n Recepto r Bindin g Assa

y Usin g Ra

t Uterin e Cytoso l (ER-RUC) . EPA-740-C-09-005 . October 2009 . Environmental Protectio n Agenc y (EPA) . (2010) . ToxCastTM Progra mwebsite . Availabl e a

t www.epa.gov/ncct/toxcast / index.html . Judso n R.S . e t

al . (2009) . I n vitr o Screenin g o f Environmenta l Chemical s fo r Targe t Testin g Prioritization-Th e ToxCas t Projects ; Supplementa l Information . Se e http://www.epa.gov/ncct/toxcast / Supplementa l dat aan dfigure s fo r Judso n e t

al . Judson , R.S. , K.A . Houck , R.J . Kavlock , T.B . Knudsen , M.T . Martin , H.M . Mortensen , D.M . Reif , D.M . Rotroff , I . Shah , A.M . Richard , an dD.J . Dix . (2010) . I n vitr o screenin g o f environmenta l chemical s fo r targete dtestin g prioritizatio n - th e ToxCas t Project . Enviro n Healt h Perspectives . 118:485-492 . Martin , M.T. , D.J . Dix , R.S . Judson , R.J . Kavlock , D.M . Reif , A.M . Richard , D.M . Rotroff , S . Romanov , A . Medvedev , N . Poltoratskaya , M . Gambarian , M . Moeser , S.S . Makarov , an d K.A . Houck . (2010) . Impac t o f environmenta

l chemical s o n key transcriptio n regulator s an d correlatio n t o toxicity endpoint s withi n EPA' s ToxCast TM Program . ChemicalResearchtin**' \ Toxicology 23:578-590 . "

• • » • « •



### Report Number : TK0023424 Pag e 4 8 o f 6 3

### APPENDI X 2 Assa y Descriptio n Shee t fro mCalipe r

LifeScience s -Bovin e Estroge n Recepto r

Bindin g Assa y

ESTROGE N (BOVINE ) BINDIN G ASSA Y

1 u 1 C ' 5 o > Q . (/ ) 5 S 110- ] 100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 - 0 - -10 - -13. 0 -10. 5 -8. 0 -5. 5

lo g [drug ] (M ) Referenc e Compoun d K i (nM ) 1 7 , p Estradio l Romogestron e 0. 2 N/ A

Cat# : Assay Characteristics : K D (bindin g affinity) : B ma x (receptor

number) : Material s an dMethods : Recepto r Source : Radioligand : Non-specifi c Determinant : Referenc e Compound : Positiv e Control : Incubatio n Conditions : 100-012 6
0. 7 n M 40 6 finol/mg protei n Bovin e uterin e membrane s [ 3 H ] Estradio l Fina l ligan dconcentratio n - [0. 7 nM ] 17P , Estradio l -[10nM ] 17P , Estradio l Reaction s are carrie dou t

i n 1 0 m M TRIS-HC I (p H 7. 4 containin g l.SmMEDT A l.OmMDTT , an d2 5 m M odiu mmolybdat e a t 0-4°Cforl 8 hours . Th e reactio n i s terminate db y th e additio n o f dextran coate dcharcoa l an dincubate dfo r 2 0 minute s at

0-4°C . 

### Repor t Number : TK002342 4 Pag e 4 9 o f 6 3Th e reactio n mixture s are centrifuge dan dth e radioactivit y boun di n th e supernatan t

i s assesse dan dcompare dt o contro l value s i n orde

r to ascertai n any interaction s o f tes t compoun dwit

h th e estradio l bindin g site . Literatur e Reference : Haji , etal . Age-Relate dChange s i n th e Concentration s o f Cytoso l Receptor s fo r

Se x Steroid s i n th e Hypothalamu s an dPituitar y Glan do f th e Rat . Brai n Research . 204 : 373-38 6 (1980 ) with modifications . O'Keefe , J.A . an dHanda , R.J . Transien t Elevatio n o f

Estrogen Receptor s i n th e Neonata l Ra t Hippocampus . Develop . Brai n Res . 57 : 119-127(1990) .

• • ••*•• •

Report Number : TK002342 4 Pag e 5 0 o f 6 3

### APPENDI X 3 Assay Descriptio n Shee t fro mCalipe r

LifeScience s -Huma n Estroge n Recepto r

Bindin g Assa y ESTROGE N (HUMAN ) BINDIN G ASSA Y

-1 0 -13. 0 -10. 5

lo g [drug ] (M ) Referenc e Compoun d -5. 5 Ki(nM ) 17,pEstradio l Methyltrienolon e 0.02 5 N/ A Cat# : Assa y Characteristics : KD (bindin g affinity) : B ma x (recepto r

number) : Material s an dMethods : Recepto r Source : Radioligand : Non-specifi c Determinant : Referenc e Compound : Positiv e Control : Incubatio n Conditions : 100-012 7

0.0 5 nM 384 4 fmol/m g protei n

MCF- 7 cell s [ 3 H ] Estradio l Fina l ligan dconcentratio n - [0. 1 nM ] < 170 , Estradiol-[30 0 nM ] 17P , Estradio l 17p , Estradio l Reaction s are carrie dou t i n 1 0 mM TRIS-HC I (p H 7. 4 containin g 1. 5 mM EDTA , 1. 0 mM DTT , an d2 5 mM sodiu m molybdat e atO-4° C fo r

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

dextra n coate dcharcoa l an dincubate dfo r 2 0 minute s at

0-4°C . Th e reactio n mixture s ar e centrifuge dan dth e radioactivit

y boun di n th e supernatant i s assessed an d compare d to contro l value s i n orde r

t o ascertai n an y interaction s o f tes t compoun dwit h th e estradio l bindin g site. 

### Report Number : TK002342 4 Pag e 5 1 o f 6 3Literatur e Reference : Haji , e t al . Age-Relate dChange s i n th e Concentration s o f Cytoso l Receptor s fo r

Se x Steroid s i n th e Hypothalamu s an dPituitar y Glan do f th e Rat . Brai n Research . 204 : 373-38 6 (1980 ) wit h modifications . O'Keefe , J.A . an dHanda , R.J . Transien t Elevatio n o f

Estroge n Receptor s i n th e Neonata l Rat Hippocampus . Develop . Brai n Res . 57 : 119-127(1990) .

• • 4

• • •



### Report Number : TK0023424 Pag e 5 2 o f 6 3

### APPENDI X 4 Titl e Pag e an d MRI D Numbe r o f Previousl y Submitte d Studie s

• * • • • e * *



### Repor t Number : TK0023424 Pag e 5 3 o f 6 3MRI D No . 0016140 7 EP ACor e Grad e Guidelin e 

## CIBA-GE1G Y CORPORATIO N PHARMACEUTICAL S DIVISIO

N "RESEARC H DEPARTMEN T

Rabbi t - Segmen t I I Dos e Rang e Teratolog

yStud y Pilo t (P-1-) ,

SIMAZIN E TECHNICA

L <MI 8 832021 ) (SE F #021 )

= , 1983 ; Ffey'ise d ilfeircf

c 

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CONFI D

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### Repor t Number : TK002342 4 Pag e 5 4 o f 6 3RESEARCH ; ;R;EPQ:R T

;MRiD ; : N6 ; . ,

21-DA' Y SUBACUT E DERMA L TOX1CIT Y STUD Y I N ALBIN O RABBIT S ADMINISTERE D TES T

ARTICL E SIHAZIN E

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### Report Number : TK002342 4 Pag e 5 5 o f 6 3-MRI D No . 4180360 1 EP A Cor e -Grad e Minimu m

O F O F SUBMISSIO N SI M A2IN E TECHNICA L

TOXIOOLOG * STUD

Y I N EP A GUIDELINE S NO . 83- 4



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; eiM^iG^COJR^ORATIO N Suflatit , Ke w Jerse y

t f PROVED: : SAC: : 86- ^

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## VOL.UWE3 0 O F12 3 VOLUM E J L O F M O F SUBMISSIO N SIMAZIN E TECHNICA L CHRONI C TQXieitY STUD

Y I ND06 S EP AfiUIDELlNE S NO , 83r l AUTHO R - 6 , C , MCCORMIC K STUD * COMPLETE D O

NMARC H 28 * 198 8 CONDUCTE D B

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N PHARMACEUTICA L DIVISIO N 55 6 MORRI S AVENU E SUMMIT ; m0790 1 LABORATOR Y STUD

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N CIBA-GEI6 Y CORPORATION POST OFFIC E BO

X 1830 0 GREENSBORO , N

C 2741 9

WW L NUMBE RO F PPQB S 1 5 PflG E Report Number : TK002342 4 Pag e 5 9 o f 6 36h/<«-1 9 RA Acce^sioi v Noi, : Volum e iof i



## VOLUM E± t L ECHNICA L COHMNE D CHRONI K

TOX-ICIfWONEOSEMICIT Y SIUD Y I N RAT S EPA SUIDEL1HE S W k

83-5 5 AUTHOR

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### Report Number : TK002342 4 Pag e 6 2 o f 6 3Atrazin e Evaluatio n o f

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o f Triazine s o n Aromatas e Expressio n I n Vitr o - Relevanc e t o Man an d th e

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### Report Number : TK002342 4 Pag e 6 3 o f 6 3