# syngenta Syngent aCro p Protection , Inc . P.O . Box 1830

0 Greensboro , N C 27419-830 0 www.syngenta.co m Te l 33 6 63 2 600 0

I • O C u • April? , 201 0 Documen t

Processin g Des k

(PRD-EDSP ) Offic e o f Pesticid e Program s (7504P ) U.S . Environmenta l Protectio n Agenc y On e Potoma c Yar d(South Bldg ) 277 7 S . Crysta l Driv e Arlington , V A 22202-450 1 SUBJECT : ATRAZIN E EDSP-080803-# : SUBMISSIO N O F"INITIA L RESPONS E

FOR M FO RCONSORTIUM TAS K FORCE" , AND OTHE R SCIENTIFICALL Y RELEVAN T INFORMATION . Dear Si r o r Madam , O n Octobe r

29 , 2009 , EP A sen t

th e Endocrin e Disrupte r Screenin g Progra mtes t

orde r fo r th e Tie r 1 assay s t o registrant s o f th e atrazin e (DC I #EDSP-080803-#)' . Th e technica l registrant s o f atrazin e hav e forme daconsortiu m(referre dt o herei n a s "atrazin e EDS P consortium ) t o addres s th e atrazin e tes t

order . EP A als o indicate dtha t "othe r scientificall y relevan t

information " (OSRI ) tha t

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

conductin g th e Tie r

1 assays . Th e enclose dinformatio n i s th e "Consorti aDocumentatio n & Consorti aInitia l Response " submitte dt o addres s Tes t Orde r / DC I #' s EDSP-080803-1 , EDSP-080803-2 , EDSP-080803-3 , EDSP-080803-4 . Include dar e th e followin g documents :

• Complete dInitia l Respons e Formfo r Consortium/Tas k

Forc e • Volum e 1 : Transmitta l Documen t • Volum e 2 : Study Volum e containin g Othe r Scientificall y Relevan t

Informatio n A s conclude di n th e enclose dOSR I stud y volume , atrazin e ha s bee n thoroughl y teste deithe r b y usin g th e actua l Tie r

1 assay protocol s and/o r functionall y equivalen t

protocol s fo r

al l o f th e endpoint s require db y th e Tie r 1 assays . Therefore , wit h thi s submission , th e atrazin e EDS P consortiu mrequest s EP A gran t waiver s fo r

al l eleve n o f

th e Tie r 1 battery o f

test s fo r atrazine . Th e studie s cite di n th e enclose dstud y volum e ar e availabl e t o EP A eithe r throug h previou s submission s and/o r

th e publishe dliterature , an dth e atrazin e EDS P consortiu mwil l sen d courtes y copie s o f

cite ddocument s t o EP A upo n request .

' Chemica l Commo n Name : Atrazine , Order/DC I #'s : EDSP-080803- 1 (Syngent acro p Protection , Inc.) , EDSP-080803- 2 (Aga n Che mMF G LTD) , EDSP-080803- 3 (Drexe l Chemica l Company) , EDSP-080803

- 4 (Oxo n Itali aS.P. A c/ o Sipca mAgr o USA , Inc.) , signe db y R . Keigwin , Director , Pesticid e Re

evaluatio n Division , OPP/EPA , 10/29/09 .

# syngenta Apri l 7 , 2010-Atrazin e EDS P 080803- * Pag e 2 o f 2 Pleas e not e tha t

th e atrazin e EDS P consortiu magreement , thoug h no t enclosed , i s i n th e fina l stage s o f completio n an dwil l b e sen t t o EP A unde r

separat e cover . Than k yo u fo r you r consideratio n o f

thi s dat aan dreques t

fo r waiver . Pleas e cal l m e a t (336 ) 632-762 7 i f

ther e ar e an y question s concernin g thi s submission . Sincerely ,

Da n Campbel l Regulator y TeamLead , Herbicide s Syngent aCro p Protection , Inc . Atrazin e EDS P Consortiu mRepresentativ e

cc : Ro y Olson , Drexe l Chemica l Compan y Jo e Burle y an dNicol aGelmetti , Oxo n Itali aS.P. A c/ o Sipca mAgr o USA , In c Kri s Venkatesh , Aga n Che mMF G LT D Ric k Keigwin , EPA/OPP/PRD Melani e Biscoe , EPA/OPP/PR DVOLUM E 1 O F 2 O F SUBMISSIO N (TRANSMITTA L DOCUMENT ) Nam e an d Addres s o f Submitte r Syngent a Cro p Protection , Inc . P.O . Box1830 0 Greensboro , N C 2741 9 Regulator

y Actio n i n Suppor

t o f whic h thi s Packag e i s Submitte d Atrazin e EDSP-080803 - # : Submissio n o f "Initia l Respons e For m Fo r Consortiu m Tas k

Force" , an d Othe rScientificall y Relevan t Information . Transmitta l Dat e 04/07/201 0 Lis t o f Submitte d Studie s

MRI D NUMBE R VOLUM E

NUMBE R STUD Y

TITL E EP A GUIDELIN E NUMBE R 1 O F 2 Transmitta l Documen t N/ A 2 O F 2 Atrazin e - Reques t fo rWaive ro f Endocrin e No t Applicabl e

Disruptio n Screenin g Progra m Assays, , (G030027_50142 )

COMPAN Y OFFICIAL : DA N CAMPBEL L (NAME ) COMPAN Y NAME : SYNGENTA CRO P PROTECTIO N (SIGNATURE )

COMPAN Y CONTACT : DA N CAMPBEL L (NAME ) (336 ) 632-762 7 (PHONE )

Page : 1VOLUM E O F O FSUBMISSIO N



# syngenta

Atrazin e Reques t fo r Waive r o f Endocrin e Disruptio n Screenin g Progra mAssay s Assessmen t DAT A REQUIREMENT(S) : AUTHOR(S) :

COMPLETIO N DATE : PERFORMIN G LABORATORY :

LABORATOR Y PROJEC T ID : No t Applicabl e Charle s B . Breckenridge , Ph.D . J . Charle s Eldridge , Ph.D . Jame s T . Stevens , Ph.D . Jonatha n M . Akins , Ph.D . W . Thoma s Beidler , Ph.D . Apri l 6 , 201 0 Syngent aCro p Protectio n 41 0 Swin g Roa d Greensboro , N C 2740 9 US A Report Number : TK002342 2 Tas k Number : TK002342 2 SUBMITTER/SPONSOR : Syngent aCro p Protection , Inc . 41 0 Swin g Road 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 Fl

_O FSTUD Y PAGE 1 O F 5 8

### STATEMEN T O FDAT A CONFIDENTIALIT Y CLAIM S

STATEMEN

T O FN O DAT A CONFIDENTIALIT Y CLAIM S UNDERSPECIFIED FIFRA PROVISION S N o clai mo f

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

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

o f apesticide) , o r

(C ) (disclose s th e identity o r

percentag e quantit y o f

an y deliberatel y adde diner t ingredien t

o f a pesticide) . Compan y Syngent aCrop Protection , Inc . Compan y Agent : Dan Campbel l 

# u I i f & U . S . Produc t

Registratio n tyfanager Date 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 specificall y unde r th e provision s containe di n FIFRA a s amende dand , hereby , consent s to us e an ddisclosur e o f thi s material b y EPA accordin g t o FIFRA . Notwithstandin g th e wordin g o f our markin g TRAD E SECRET , thi s marking , b y itself

, convey s n o supplementa l claim s o f

confidentialit

y under FIFR A Section s 10(a

) o r 10(b ) (addressin g protectio n o f trad e secret s an dcommercial an dfinancia l information) . I n submittin g thi s material t o EP A accordin g t o metho dan dforma t requirement s containe di n P RNotic e 86-5 , w e d o no t waiv e an y protectio n o r

righ 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 ha dno t

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

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

e to foreig n an dmultinational pesticide companie s or their agents) .

Report Number : TK002342 2 Pag e 2 o f 5 8

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

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

Charle s Breckenridge , Ph.D . / 7 Date Representativ e o f

Submitter/Sponso r Performing Laboratory : SyngentaCrop Protection , Inc . 41 0 Swin g Road Greensboro , N C 2740 9 US A

flpr. l (<, , 20I O

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

### TABLE O FCONTENT S

STATEMEN T O FDAT A CONFIDENTIALIT Y CLAIM S 2 GOO D LABORATOR Y PRACTIC E COMPLIANC E STATEMEN T 3 TABL E O FCONTENT S 4 1. 0 EXECUTIVE SUMMAR Y 6 2. 0 INTRODUCTIO N 8 3. 0 RATIONAL E FO RACCEPTANC E O FATRAZIN E OSR I FO R SPECIFIC TIER1 ASSAY S 9 3. 1 Amphibia n Metamorphosi s Assay (OPPTS890.il00 ) 9 3. 2 Androge n Recepto r Bindin g Assay (OPPTS890.il50 ) 1 4 3. 3 Aromatas e Assay-Human Recombinan t (OPPT S 890.1200 ) 1 6 3. 4 Estroge n Recepto r Bindin g Assa y Usin g Rat Uterin e Cytoso l (ER

RUC ) (OPPT S 890.1250 ) 1 9 3. 5 Estroge n Recepto r Transcriptiona l Activatio n Assay - Huma n Cel l Lin e HeLa-990 3 (OPPT S 890.1300 ) 2 1 3. 6 Fis h Shor t TermReproductio n Assa y (OPPT S 890.1350 ) ...2 3 3. 7 Hershberge r Assa y - Rat

(OPPT S 890.1400 ) 2 6 3. 8 Femal e Pubertal Assa y - Ra t (OPPT S 890.1450 ) 2 8 3. 9 Mal e Pubertal Assa y - Rat (OPPT S 890.1500 ) 3 0 3.1 0 Steroidogenesi s Assa y - Huma n Cel l Lin e H295 R(OPPT S 890.1550 ) 3 3 3.1 1 Uterotrophi c Assay-Ra t (OPPT S 890.1600 ) 3 5 4. 0 REPRODUCTIV E AN D DEVELOPMENTA L TOXICIT Y

CHARACTERISTIC S O FATRAZIN E 3 7 5. 0 CONCLUSIO N 3 8 6. 0 REFERENCE S 3 9 APPENDICE S SECTIO N 4 3 APPENDI X 1 Titl e Page s o f Cite dStudie s 4 4

Repor t Number : TK002342 2 Pag e 4 o f 5 8

### LIS T O FTABLE S

TABL E 1 Othe r

Scientificall y Relevan t

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

y EDS P Tie r

1 Assay Requirement s 7 TABL E 2 EP A Guidelin e Reproductiv e an dDevelopmenta l Toxicity Studie s o n

Atrazin e Technica l 3 8



### LIS T O FFIGURE S FIGURE 1 Kloa s e t

al. , 200 9 1 2

Report Number : TK002342 2 Pag e 5 o f 5 8

### 1. 0 EXECUTIV E SUMMAR Y

Th e Federa l Foo dDru g an dCosmeti c Act , a s amende db y th e Foo dQualit y Protectio n Ac t o f 1996 , require dth e U . S . Environmenta l Protectio n Agenc y (EPA ) t o "develo p ascreenin g program , usin g appropriat e validate dtes t system s an dothe r

scientificall y relevan t informatio n t o determin e whethe r certai n substance s ma y hav e a n effec t i n human s that

i s simila r t o an effec t

produce db y anaturall y occurrin g estrogen , o r othe r effect s a s [EPA ] may designat e [2 1 U.S.C . 346a(p)]" . Sectio n 408(p)(3 ) specificall y require dthat th e Administrato r

"shall provid e fo r

th e testin g o f

al l pesticid e chemical s [2 1 U.S.C . 346a(p)(3)]. " EP A develope daTier

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

th e T1 S assays , an do n Octobe r

29 , 2009 , EP A sent

th e tes t order s fo r th e T1 S (DC I #EDSP-080803

- 1 ) t o registrant s o f

th e atrazine . Th e technica l registrant s o f atrazin e hav e forme da consortiu mt o addres s th e atrazin e tes t order . EP A als o indicate dtha t "other scientificall y relevan t information " (OSRI ) that i s functionall y equivalen t

t o th e informatio n t o b e obtaine dfro mth e Tie r

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

1 assays . Th e objectiv e o f

thi s documen t

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

atrazin e an dt o obtai n waiver s fo r

th e eleve n EDS P Tier

1 assays :

• 890.1100 - Amphibian Metamorphosi s (Frog ) • 890.115 0 - Androge n Recepto r Bindin g (Ra

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

9903 ) • 890.135 0 - Fis h Short-TermReproductio 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 ) Th e atrazin e consortiu midentifie daspecifi c se t o f th e existin g OSRI o n atrazin e whic h establishe s tha t

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 atrazin e (Tabl e 1) . Atrazin e ha s bee n evaluate d b y th e specifie dTie r

1 assay s and/o r

substantiall y simila

r assay s fo r

eac h o f th e eleve n test s i n th e EDSP .

Report Number : TK002342 2 Pag e 6 o f 5 8TABL E 1 Othe r Scientificall y Relevan t

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

y EDS P Tie r

1 Assay Requirement s OPPT S Guidelin e 890.1100

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 ! and/o r recombinan t Estroge n Recepto r bindin g Assay (ra t uterin e cytosol ) Estroge n Recepto r Transcriptiona l Activatio n Assa y Fis h Shor t Term Reproductio n

Hershberger

Assa y Femal e Puberta l Mal e Puberta l Steroidogenesi s H295 R Uterotrophi c Assay OSR I Reference(s ) Kloa s et al. , 200 9 Solomo n e t

al. , 200 8

Yamasak i e t

al. , 200 4 Fan g e t al. , 200 3 Kojim ae t al. , 200 4 USEPA , 2008 a USEPA , 2008 b Hinfrayetal. , 200 6 Hollowa y e t

al. , 200 8 USEPA , 200 9 Tennan t e t al. , 1994b Blai r e t al. , 200 0 Conno r etal. , 199 6 Kojim ae t al. , 200 4 Balague r e t al. , 199 9 Battelle , 200 5 Bringolf, 200 4 Dionne , 199 2 Maceketal. , 197 6 Ashby , 200 4 Yamasak i e t

al. , 200 4 Law s e t al. , 200 0 Ashb y e t al. , 200 2 Stoke r e t al. , 200 0 USEPA , 2008 c Sanderso n etal. , 200 1 Yietal. , 2010 a Tennan t e t al. , 1994 a Connor etal. , 199 6 Ashb y e t al. , 200 2 Stud y wa s Par t o f EDS P Tie r

1 Scree n Developmen t Supplementa

l Supplementa l
Substantiall y simila r Substantiall y simila r Substantiall y simila r Ye s Ye s Supplementa l Differen t protoco l Ye s Substantiall y simila r Substantiall y simila r Substantiall y simila r Substantiall y simila r Substantiall y simila r Ye s Substantiall y simila r Supplementa l Supplementa l Substantiall y simila r Substantiall y simila r Ye s Substantiall y simila r Ye s Ye s Substantiall y simila r Substantiall y simila r Substantiall y simila r Substantiall y simila

r Substantiall y simila r Comment s Exceed s requirement s Comprehensiv e literatur e revie w o f effect s o f atrazin e o n amphibia n metamorphosi s Recombinan t

huma n A R Recombinan t

rat A R hA Rexpresse di n cell s

Trou t (brai n & ovarian ) CPY1 9 Huma n Recombinan t

hE Rtransfecte dt o yeas t hE Rtransfecte dt o CH O

Fis h ful l lif

e cycl e stud y Lon g termlifecycl e studie s i n bluegil l sunfish , broo k

trou t an d fathea dminno w Hershberger

protoco l Hershberge r

protoco l

H295 R H295 R Adul t ovariectomize d Immatur e rat s Immatur e rat s Report Number : TK002342 2 Pag e 7 o f 5 8

### A s depicte di n th e tabl e above , atrazin e ha s bee n thoroughl y teste deithe r b y usin g th e actual Tie r 1 assa y protoco l and/o r

substantiall y similar

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 EP A Endocrin e Disruptio n Screenin g Program(EDSP) . Atrazin e ha s als o bee n teste dfo r

endocrine-relate dendpoint s vi aexistin g EPA-require dguidelin e studie s (e.g. , two-generatio n reproductio n study , developmenta l toxicit y studies , etc.) . Thes e highe r 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 atrazin 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 . 

### 2. 0 INTRODUCTIO N Th e Federa l Foo dDru g an dCosmeti c Act , a s amende db y th e Foo dQuality Protectio n Ac t o f 1996 , require dth e U . S . Environmenta l Protectio n Agenc y (EPA ) 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 similar t o a n effec t

produce db y anaturall y occurrin g estrogen , o r othe r effect s a s [EPA ] ma y designat e [2 1 U.S.C . 346a(p)]" . Sectio n 408(p)(3 ) specificall y require dthat th e Administrato r

"shal l provid e fo r

th e testin g o f

al l pesticid e chemical s [2 1 U.S.C . 346a(p)(3)]" . EP A develope daTier

1 Screenin g (T1S ) batter y o f eleve n separat e assay s t o scree n fo r potential endocrine-modulatin g substances . EPA publishe daserie s o f guideline s fo r

th e T1 S assays , an do n Octobe r

29 , 2009 , EP A sent

th e tes t order s fo r

T1 S (DC I #EDSP-080803-1 ) t o registrant s o f

th e atrazine . Th e technica l registrant s o f atrazin e hav e forme daconsortiu m t o addres s th e atrazin e tes t order . EP A als o indicate dtha t

"other scientificall y relevan t

information " (OSRJ ) tha t

i s functionall y equivalen t t o th e informatio n t o b e obtaine dfro mth e Tie r 1 assay s may b e utilize di n lie u o f conductin g th e Tier

1 assays . However , EP A indicate dth e OSRJ mus t mee t specifi c qualit y an dfunctiona l equivalenc e criteri awhic h includ e th e following : Dat aar e o f

hig h quality an dconducte dusin g soun dscientifi c practice s • Study conducte di n accordanc e wit h accepte dscientifi c methodolog y o r protoco l Deviation s fro mTier

1 assay protocol s identifie di n th e OSRJ rational e Impac t o f an y deviation s fro mth e protoco l o n th e result s o f th e study relatin g t o th e studie s valu e i n justifyin g awaive r reques t shoul db e discusse di n th e rational e Th e objectiv e o f

thi s documen t

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

atrazin e an dt o obtai n waiver s fo r

th e eleve n EDS P Tie r

1 assays :

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

Prostate ) • 890.120 0 - Aromatas e (Human Recombinant ) • 890.125 0 - Estroge n Recepto r Bindin g Report Number : TK002342 2 Pag e 8 o f 5 8

### • 890.130 0 - Estroge n Recepto r Transcriptiona l Activatio n (Human Cel l Lin e HeLa

9903 ) • 890.135 0 - Fis h Short-TermReproductio n • 890.140 0 - Hershberge r (Rat) • 890.1450-Femal e Puberta l (Rat ) • 890.150 0 - Mal e Puberta l (Rat ) • 890.155 0 - Steroidogenesi s (Human Cel l Lin e - H295R ) • 890.160 0 - Uterotrophi c (Rat ) 

### 3. 0 RATIONAL E FO RACCEPTANC E O FATRAZIN E OSRI FO R SPECIFI C TIE R1 ASSAY S 3. 1 Amphibia n Metamorphosi s Assa y (OPPT S 890.1100) Purpos e o f

Assa y Th e purpos e o f

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

potentia l thyroi dsyste m disruptin g compounds . Throug h th e hypothalamus-pituitary-thyroi d(HPT ) glan daxis , thyroi dhormon e regulate s man y processe s i n organism s includin g development , somati c growth , metabolism , an dreproduction . Metamorphosi s mXenopu s laevi s i s awell-studie d mode l fo r thyroi dhormon e function . Assay Methodolog y Summar y Th e OPPT S 890.110 0 protoco l involve s a21-da y exposur e o f tadpole s t o th e tes t

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

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

replicat e tank s pe r

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

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

treatmen t group . Environmenta l condition s o f

22±1°C , p H o f

betwee n 6.5-8.0 , an da12:1 2 light:dar k photoperio dar e employed . Fiv e tadpole s fro meac h treatmen t tank ar e randoml y remove dat

day 7 fo r evaluatio n o f

we t weigh t an drat e o f

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

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

Informatio n Th e potentia l associatio n o f atrazin e wit h endocrin e effect s o n amphibian s ha s bee n th e subjec t o f considerabl e investigatio n b y academi c researchers , USEP A an dSyngent a scientist s fo r

anumbe r o f years . Th e primar y focu s ha s bee n o n allege deffect s o f atrazin e o n sexual differentiatio n an ddevelopmen t i n amphibians , an dth e USEP A require dSyngent at o develo p study method s an dconduc t additiona l investigation s t o resolv e uncertainties . I n response , Syngent adevelope dstandardized , in-lif e an dhistologica l method s fo r testin g thi s Report Number : TK002342 2 Pag e 9 o f 5 8

### hypothesi s (Lut z e t al. , 2008) . Tw o experiments , on e i n th e Unite dState s (WLI ) an dth e othe r i n Germany( IGB) , wer e conducte dunde r Goo dLaboratory Practic e (GLP) , with inspectio n b y th e USEP A an dGerma n GL P Federa l Bureau , Institut e fo r Ris k Assessment . N o impac t o f atrazin e (0.01-10 0 ug/L ) o n metamorphosi s o r gonada l developmen t

(Fi g 1 ) wa s detecte d(Hosme r

e t al. , 2007) . Base dupo n thes e studies , whic h mee t th e mos t rigorou s desig n an dconduc t

requirements, th e USEP A "ha s conclude dtha t atrazin e doe s no t adversel y affec t

amphibia n gonadal development " (USEPA , 2007) . A FIFR A Scientifi c Advisor y Pane l (SAP ) o f

independen t

expert scientist s convene db y th e USEP A likewis e conclude dtha t

"study endpoint s tha t

ar e mor e likel y t o impac t

reproductiv e fitnes s (se x ratio , interse x condition ) wer e unaffecte di n animal s expose di n 10 0 jig/ L atrazine " an d"there i s currentl y n o availabl e proo f fo r th e hypothesi s regardin g th e purporte dactio n o f atrazin e o n th e inductio n o f

aromatase " (FIFRA SAP , 2007) . Thi s stud y an dal l supporting dat awer e submitte dt o th e USEP A (Hosme r e t al. , 2007 ) an dth e resultin g pee r reviewe dpublicatio n (Kloa s e t al. , 2009 ) i s attached . Tie r I scree n Amphibia n Metamorphosi s Amphibia n Metamorphosi s OSRI Reference(s )

Kloa s e t al. , 200 9 Solomo n e t

al. , 200 8 Comment s Negativ e fo r

steroi deffect s Comprehensiv e literatur e revie w o f effect s o f atrazin e o n amphibia n metamorphosi s 

### N o compellin g evidenc e o f impairmen t

o f metamorphosi s o r

thyroidogeni c effect s exists , an d th e focu s o f

Kloa s e t al. , 200 9 wa s o n sexual development . Thes e robus t an dreplicate d studies , whic h wer e reviewe db y th e EP A an dexterna l pee r reviewers , showe dn o evidenc e o f thyroi deffect s base do n metamorphi c success , rat e o f metamorphosi s o r

bod y length an d weigh t over arang e o f

atrazin e concentration s o f 4 order s o f

magnitude . Th e followin g table , (whic h i s take n directl y fro mth e Kloa s e t al. , 200 9 publication) , provide s specifi c detail s fo r th e wate r quality , anima l husbandr y an dexperimenta l desig n fo r th e studies . Briefl y stated , Xenopu s laevi s tadpole s wer e expose dfro mN FStag e 47-4 8 ( 8 dp f :day s pos t

fertilization ) t o 8 3 dp f

o r th e tim e t o metamorphosi s (N FStag e 66) . Treatment s include datrazin e (0.01 , 0.1 , 1.0 , 2 5 an d10 0 |ig/L) , estradio l positiv e contro l (0. 2 (ig/L ) an danegativ e control . Ther e wer e 8 replicate s an d20 0 frog s pe r treatmen t excep t fo r negativ e control s whic h wer e 1 6 replicate s an d40 0 frogs . Gros s biologica l metric s wer e collecte dat

th e tim e o f

metamorphosi s includin g length , weight , tim e t o metamorphosi s a s wel l a s gros s examinatio n o f genera l healt

h an dgonadal se x an dgonadal appearance . Al l frog s wer e examine dhistologicall y fo r gonadal se x an dpathology .

Report Number : TK002342 2 Pag e 1 0 o f 5 8Detail s o f th e Experimenta l Desig n Specification s i n th e Kloa s e t al. , 200 9 Stud y TABL Ei Experimenta l Desig n Specification s Parameter Characteristic s Wate r qualit y (AST

M standard ) Suppl y wate

r f i tiered , contaminan t

fre e Dissolve doxyge n P H Ammoni a Nitrat e Hardnes s Alkalinit y Specifi c conductanc e Supplementa

l aeratio n Anima l husbandry an dexposure condition s Anima l suppl y Shippe dfro msupplier Acclimatio n perio d Treatment perio d feed : ser u micro n feedin g rale—acclimatio n perio d feedin g rale—exposure perio d Tan k volum e Tank flo w rat e Numbe r o f larva e per

group Larva l loadin g densit y Exposur e lan k cleanin g Exposure tan k

replacemen t Temperature Ligh t intensity Lighkdark

cycle Experimental desig n an dqualit y contro l Negativ e contro l Positiv e contro l (I7p-cstradio l (E2] > Alrazin e concentration s HPLC/M S M S verificatio n for E 2 an datrazin e Stud y complianc e wit h GL Fstandard s Color code d(blind ) observation s Randomizatio n o f

tadpole s Randomizatio n o f

lan k cluster s Statistical procedure s Uni t o f analysi s Dat atransformed ? Protectiv e screenin g test s fo r alrazin e (E 2 group excluded ) Compariso n to negativ e contro l Tren dtest s (negativ e contro l an datrazin e groups ) Filtere d(0.4 5 ^imVU V sterilized ; teste dfo r chemica l contaminant s > 60 % o f

saturatio n 7.9-8. 3 0.0-0.3 5 mg/ 1 0.04-2.2 9 mg/ 1 90-130mgCaC<V I 133-17 2 mg CaCO,/ ! 726-81 7 M-S/cm Ye s XENOPU S I , Dexter . M I dp f 3 dpf4- 7 dpf 8-8 3 Ser amicro n (contaminant s an deslrogeni c potentia l evaluated) . Contents : 50 % protein , 8 % f

aI ; 4 % fiber ; 1 1 % as h dp f 4-5 , pe r 30 0 larvae: 20 0 mg ; dp f 6-8 , pe r 30 0 larvae : 20 0 m

g twic e dail y dp f 8-83 , per

2 5 larvae : thre e lime s daily , 30 0 mgAan k a t 8 dpf, increasing l o 90 0 mg per

tan k a t 3 3 dpf , the n adjuste ddownwar dproportionatel y a s frog s were remove dfro mtank s 7 1 5 0 I/da y 20 0 larvae per

treatmen t

group ; negativ e contro l group : 40 0 larva e < I g/l/day ; 2 5 larva e per lank Twic e dail y A s needed 2 2 ± I° C 100-50 0 lu x 1 2 h:! 2 h ; 3 0 mi n transitio n betwee n lighl:dark:ligh l Wate r (16 replicate s (tanks) , 4 clusters ) 0. 2 ug/ 1 (eight

replicates , tw o clusters ) 0.01,0.1 , 1 , 25 , 10 0 Mg/ 1 (eigh t replicates , tw o cluster s for each group ) dp f 8 an d1 4 al l tanks ; dp f

21-8 3 alternatin g tank s Complian t Ye s Ye s Rando mplacemen t Tank (cluster effect s were no t

significant) ; tadpol e (E 2 male s only , tank effect s were no t significant ) Onl y fo r continuou s endpoint s whe n ther e wa s evidenc e o f

substantial non-normalit y (Shapiro-Wil

k test , p < 0.01 ) o r

varianc e heterogeneit

y (Levin e test , p < 0.01) , fo r tadpol e (incidenc e dat afo r E 2 male s b y Fisher' s Exac t Test ) Analysi s o f

varianc e ^-tes t

(continuou s endpoints) ; Kruskal-Walli s lest (incidenc e endpoints ) Pair-wis e ANOV A contrast s (continuous , alrazine) ; r-test s (continuou s E") ; Wilcoxon-Mann-Whitne y (incidence , alrazine , nonmal e E2) ; Fisher' s Exac t tes t (incidence , mal e E2 ) Step-dow n Jonckheerc-Terpstr atest s fo r monotoni c tren d

Report Number : TK002342 2 Pag e 1 1 o f 5 8FIGUR E 1 Kloa s e t al. , 200 9 38 0 KLOA S ET AL . IG B

Atrazln e (vgIL) *Significan t difference s fro m negativ e contro l p < 0.05 . Atrazln e (iig'L )

Atrazln e (M9/L ) ^Significan t difference s fro m negativ e contro l p £ 0.05 . Atrazin e (M9"- | IG B

Atrazin e (M9'L) ^Significan t difference s fro m negativ e contro l p < 0.05 . Atrazin e ((Jg/L ) FIG . 1 . (a ) Snoul-to-ven l length (mean ± SE ) i n experimen t 1 (WL O (lef

t panels ) an d2 (IGB ) (right panels) . Statistica

l .significanc e i s marke db y asteris k I f < 0.05) . (b ) Weight

(mea n ± SE ) i n experimen t I (WLI ) (lef

t panels ) an d2 (IGB ) (righ t panels) . Statistical significanc e i s marke db y asteris k ( p < 0.05) . (c ) Tim e t o metamorphosi s (mea n ± SE ) i n experimen t

1 (WLI ) (lef

t panels ) an d2 (IGB ) (righ t panels) . Statistical significanc e i s marke db y asterisk ( p < 0.05) .

Report Number : TK002342 2 Pag e 1 2 o f 5 8Deviation s t o OPPT S 890.1100 Protoco l OPPT S 890.110

0 Requirement s Criteri a Xenopu s laevi s tadpole s Stud y initiatio n t o b e N FStag e 5 1 Minimu mo f

3 concentration s t o b e teste d Minimu m2 1 da y stud y duratio n Larva l densit y t o b e 2 0 tadpole s pe r tank o f betwee n 4 to 10 L volume Ligh t intensit y 60 0 t o 200 0 lu x Iodid e concentration s i n th e tes t wate r shoul db e reporte d Al l individual s use dshoul doriginat e fro masingl e spawn .

Th e hig h tes t

concentratio n woul db e se t b y th e solubilit y limit , th e MT C o r 1 0 0 mg/L , whicheve r

i s lowest . Da y 7 measurement s o f

hin dlimb ength , SVL , an ddevelopmenta l stag e collecte do n 5 tadpole s per replicate . Day 2 1 measurement s o f developmenta l stage , SVL , hin dlim b lengt h an dbod y weigh t

t o b e collecte dat

day 2 1 tes t

termination . Thyroi dhistolog y t o b e examine d Kloa s e t al. , 200 9 Deviation s t o OPPT S 890.110 0 non e Stud y initiatio n wa s at Stag e 47-48 ; 8 dp f Stud y use d5 concentration s spannin g 4 order s o f

magnitud e Exposure s continue d throug h metamorphosi s N FStag e 6 6 o r

day 7 5 (8 3 dpf ) Larva l densit y wa s 2 5 tadpole s pe r

7 L o f tan k exposur e solution s Stud y wa s conducte d wit h aligh t

rang e o f 1 0 0 t o 50 0 lu x Iodid e wa s no t

measure d Spawn s fro m1 0 breedin g pair s were use d

Th e highes t concentratio n fo r

thi s stud y wa s lOOug/L . Thes e intermediat e measurement s wer e no t collecte d Thes e intermediat e measurement s wer e no t collecte d

No t performe d Justificatio n fo r OSRI Same specie s a s EDS P Tie r 1 scree n Earlie r stagin g at

initiatio n woul dno t

impac t th e stud y sinc e exposur e at

Stag e 5 1 an dbeyon dwa s included . N o negativ e impact . Tes t

concentration s wer e selecte d t o includ e an dexcee denvironmentall y relevan t doses . N o negativ e impact . Stud y duratio n exceede dth e 2 1 da y requiremen t

an dcontinue dthroug h complet e metamorphosis . Larval densit y withi n th e prescribe drang e an dwithi n th e AST M guidanc e o f

<lg/L/da y A s evidence db y excellen t

biologica l performanc e o f th e frogs , ligh t

intensit y wa s suitable . Developmenta l rate s i n contro l frog s wer e a s expecte d base do n literatur e reports ; iodid e level s assume dt o b e

withi n idea l ranges . Multipl e spawn s were utilize dt o avoi dtestin g ales s geneticall y divers e grou p o f animals . Mor e tha n on e spawnin g wa s require dt o provid e th e larg e numbe r o f animal s neede dfo r

tw o testin g laboratorie s Atrazin e i s no t

acutel y toxi c t o amphibians , wit h reporte dLC5 0 rangin g fro mapproximatel y 41 0 ug/ L t o >48,00 0 ug/

L (Solomo n e t

al. , 2008) . Thes e concentration s excee denvironmentall y realisti c concentrations . A concentratio n o f 10 0 ug/ L (approximatel y 1/ 3 o f

th e lowes t

acut e valu e o f

41 0 ug/L ) i s ados e whic h coul db e delivere dvi aflow

throug h withou t

th e ai do f co-solvents . Al l measurement s wer e obtaine dupo n complet e metamorphosi s at

Stag e 6 6 (mean numbe r

o f day s show n i n fig . 1) . Tim e t o metamorphosi s di dno t indicat e effect s a t

earlie r tim e points . Al l measurement s wer e obtaine dupo n complet e metamorphosi s at

Stag e 66 . Tim e t o metamorphosi s di dno t indicat e effect s a t

earlie r tim e points . Th e primar y focu s o f

th e stud y wa s o n functiona l endpoint s o f

successfu l survival , growth , metamorphosi s an dsexua l differentiation . N o evidenc e o f

thyroi deffect s base do n metamorphi c success , rat e o f

metamorphosi s o r

bod y lengt h an d weigh t wa s observe dove r

arang e o f

atrazin e concentration s o f

4 order s o f

magnitude . Th e studie s conducte dwit h atrazin e greatl y excee dth e requiremen t o f th e OPPT S 890.110 0 protocol , wit h afe w mino r exceptions . Additionally , th e recen t publicatio n b y Solomo n e t al. , 200 8 include s acomprehensiv e revie w o f th e literatur e publishe do n potentia l effect s o f atrazin e o n amphibia n metamorphosis . Th e studie s wer e conducte dbetwee n 200 1 an d2008 , an dwer e generall y conducte dat exposur e concentration s whic h include dan dexceede d environmentall y relevan t

exposures . Thes e author s conclude dthat "studie s examinin g Repor t Number : TK002342 2 Pag e 1 3 o f 5 8

### effect s o f atrazin e o n metamorphosi s sugges t n o consisten t

concentration-relate deffec t o f atrazin e o n thyroi dfunction" . Further , Law s e t

al. , 200 0 examine dth e effect s o f atrazin e (12.5 , 25 , 50 , 100 , o r 20 0 mg/kg/day fro mpostnata l day 2 2 t o 41 ) o n thyroi dhormones , triiodothyronin e (Ts) , thyroxin e (T 4 

### ) , an dthyroi dstimulatin g hormon e (TSH ) evaluate di n femal e Wistar rats . Seru mTS , T4 , an dTS H wer e unaltere dat an y leve l o f

atrazin e treatmen t

no r wer e an y histopathologi c /morphologi c change s i n th e thyroi dobserved . Also , Stoke r e t al. , 200 0 looke dfo r effect s o f atrazin e (12.5 , 25 , 50 , 100 , o r 20 0 mg/kg/day fro mpostnata l day 2 2 t o 53 ) o n th e thyroi dan dTS , T4 , an dTS H i n mal e Wista r rats . N o difference s wer e observe di n (TSH ) an d(T4 ) betwee n atrazin e treate drat s an dth e contro l animals ; however , T S wa s elevate di n th e hig h dos e atrazin e group . Not e tha t th e 20 0 mg/k g hig h dos e i s at leas t fou r order s o f magnitud e greater

tha n th e chroni c referenc e dos e fo r atrazin e (USEPA , 2006) . Conclusions : Atrazin e registrant s reques t awaive r o f th e OPPT S 890.110 0 Tier

1 testin g requirement . Whil e deviation s fro mth e OPPT S Amphibian Metamorphosi s Assay ar e describe dabove , thes e ar e mino r an do f n o consequence . I n fac t

som e deviation s resul t

i n mor e robus t

testin g conditions . Th e submitte dOSRI represent s an unprecedente damphibia n assa y conducte dwit h th e input

an ddirectio n o f

th e USEP A an dth e FEFRA Scienc e Advisory Panel . Th e stud y wa s conducte dunde r GL P an dreplicate di n tw o laboratories . Ove r 300 0 frog s were utilize di n thes e studie s a s compare dto 300-40 0 i n th e Tie r 1 assay . N o evidenc e o f thyroi deffect s wa s observe dafte r lon g ter mexposure , throughou t complet e metamorphosis , t o concentration s o f atrazin e spannin g fou r

order s o f magnitude . 3. 2 Androge n Recepto r Bindin g Assa y (OPPT S 890.1150 ) Purpos e o f

Assa y Th e androge n recepto r

(AR ) bindin g assa

y i s an i n vitr o tes t

designe dt o scree n fo r substance s that

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 with a[ 3 

### H]-ligan dfo r bindin g i n a cytoso l homogenat e o f

rat ventra l prostat e (VP ) tissue , whic h i s replet e with androge n receptors . Th e A Rbindin g assay doe s no t asses s functiona l o r

transcriptiona l activity o n th e par t o f abinder . I n additio n t o detectin g androge n agonist s an dantagonists , th e A Rbindin g assa y i s complementary i n supportin g an agonisti c o r antagonisti c resul t

i n th e Hershberge r assay . Assa y Methodolog y Summar y Th e A Rbindin g assay i s conducte dwith aserie s o f 12x7 5 cultur e tube s tha t

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

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

aknow n hig h affinit y androge n 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- l 881 , th e hig h affinit y ligand , i s the n dispense dt o al l tubes , whic h ar e the n incubate dovernigh t at 4°C . O n th e followin g day aseparatio n ste p isolate s radioactivit y boun dt o A Ri n eac h tube , whic h ar e counte db y liqui dscintillation . Th e principl e o f th e assay i s tha t

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

### H]-labele dligan dan dproduc e anegativ e sigmoi ddose

Report Number : TK002342 2 Pag e 1 4 o f 5 8respons 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 know n binders . Th e assay principl e furthe r hold s tha t

acompoun dwhic h displace s n o radiolabele dligan di s unabl e t o bin dt o AR ; it s ligan ddose-respons e plo t wil l res t at 100 % maximu mbindin g an dhav e n o downwar dslope . Othe r Scientificall y Relevan t

Informatio n I n preparatio n fo r

adoptio n o f

th e Tie r 1 scree n battery , th e USEP A conducte dvalidatio n studie s fo r

th e A Rbindin g assay . Th e A Rbindin g assay validatio n Integrate dSummary Report (ISR ) (USEPA , 2008a ) als o describe dresult s fro madditiona l chemical s tha t wer e examine da s putativ e A Rbinder s b y th e laboratorie s testin g th e approve dmethodology . Atrazin e wa s on e o f

th e 1 6 compound s use dfo r inter-laborator y compariso n (USEPA , 2008a , Table s 6- 1 an d6.2 , pp . 31-33) . Result s o f testin g ar e describe do n p . 7 9 o f

th e ISR . Tie r 1 scree n Recombinan t

hA R Recombinan t

rA R AR-linke dreporte r

i n CH O cell s A R(cytoso l o f

rat ventra l prostate ) OSR I Reference s Yamasaki e t

al. , 200 4 Fan g e t al. , 200 3 Kojim ae t al. , 200 4 USEPA , 2008 a Comment s Recombinan t

huma n A R Recombinan t

rat A R hA Rexpresse di n cell s Teste di n T l scree n validatio n proces s Yamasak i e t

al. , 200 4 Thi s stud y conducte da n analysi s o f A Rbindin g usin g th e bindin g domai n fragmen t o f huma n A Rexpresse d(amplified ) i n Escherichi acoli . Th e radiolabele dtrace r wa s adifferen t high-affinit y ligan dfro mth e R-188 1 use di n th e EDS P Tie r Ivalidate dmethod . Th e separatio n step wa s als o different . Th e bindin g assa y desig n an dcalculatio n o f competitio n result s appeare dsimila

r t o th e validate dmethod . Th e studie s wer e conducte do n 1 2 compounds , includin g atrazine , a t aprivat e researc h institut e i n Japan . Fan g e t al. , 200 3 Thi s stud y assesse dbindin g o f 20 2 chemical s t o asolubilise drat A Rproduce db y recombinan t

technology . I t

wa s state dtha t

thi s molecul e ha s th e identica l bindin g domai n a s huma n AR . [ 3 H]-R188 1 wa s use dfo r competitio n studies . Hydroxyapetit e wa s use dfo r separation . Th e metho dappear s t o b e clos e t o th e validate dEDS 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 rat prostate) . Kojim ae t

al. , 200 4 Thi s stud y examine dth e ability o f 20 0 chemical s t o induc e arespons e i n an AR-linke d reporte r system . Huma n A Rwa s transfecte dint o Chines e hamste r ovary (CHO ) cell s with a vecto r an daluciferas e reporte r

gen e wa s linke ds o tha t

bindin g o f

aligan dt o A Rwoul d produc e aluminescen t

signal . Althoug h a n AR-linke dreporte r syste mha s no t

bee n validate d for inclusio n in the EDS P Tie r 1 scree n battery , the principl e is presentl y bein g evaluate dand wil l likel y b e adopte di n th e future . I t i s highl y specifi c fo r

A Rbinders . Atrazin e wa s on e o f th e 20 0 teste dchemicals . Report Number : TK002342 2 Pag e 1 5 o f 5 8USEPA , 2008 a

Thi s i s th e IS R(Integrate dSummary Report ) furnishe db y th e USEP A coverin g th e validatio n o f

th e A Rbindin g assa

y adopte dfo r

th e EDS P Tie r

1 screen . A s state dabove , atrazin e wa s on e o f

anumbe r o f compound s teste di n th e validatio n procedure , usin g GL P methods . Deviation s to OPPT S 890.115 0 Protoco l OPPT S 890.115 0 Requirement s Criteri a A Ri n Rat V P Cytoso l 3 H-competito r

i s R-188 1 Separatio n o f boun dan dfre e trace r usin g hydroxyapetit e ge l Yamasak i e t

al. , 200 4 Deviation s t o OPPT S 890.115 0 Huma n A Ri n E col i

3 H-labele dcompetito r i s miboleron e Separatio n o f

boun d an dfre e usin g dextran-charcoa l Fan g e t al. , 200 3 Deviation s t o DPPTS890.i l 5 0 Recombinan t RatA R Non e

Non e Kojim ae t

al. , 200 4 Deviation s t o OPPT S 890.115 0 Huma n A Ri n CH O cell s N o radiolabel ; bindin g respons e measure dwit h a linke dreporte r

gen e No separatio n step; A Rbindin g i s linke dt o areporte r USEPA , 2008 a

Deviation s t o OPPT S 890 . 1 1 5 0
non e

non e

non e Justificatio n fo r

OSRI Binder s reac t

same with both huma n an dra t A R Know n androge n ligand s respon di n a n identica l manne r Method s ar e equall y vali d Conclusions : Th e atrazin e consortiu mrequest s awaiver fo r th e A Rbindin g assa

y becaus e atrazin e ha s bee n already teste db y th e EP A validatio n progra man db y othe r researcher s usin g substantiall y similar methodology . Atrazin e wa s include di n th e EP A validatio n studie s wit h th e A RBindin g assay , an dthre e additiona l studie s ar e availabl e i n th e publishe d literatur e whic h al l use dcommo n method s tha t ar e widel y accepte da s reliabl e measure s o f A Rbinding , includin g on e that wa s substantiall y similar

t o th e validate dprotocol . Eac h stud y evaluate dman y varie dcompounds , whic h promote s confidenc e i n th e result s b y allowin g cros s compariso n betwee n studies . Methodologica l difference s i n thes e thre e studie s shoul dhav e n o impac t o n justificatio n fo r

usin g OSRI i n consideratio n o f atrazin e interactio n with androge n receptors . 3. 3 Aromatas e Assay-Huma n Recombinan t (OPPT S 890.1200 ) Purpos e of

Assa y Th e Aromatas e (Human Recombinant ) Assa y i s intende dt o scree n fo r substance s tha t

may affec t th e endocrin e syste m(e.g. , steroidogenesis ) b y inhibitin g catalyti c activity o f aromatase , th e enzym e responsibl e fo r th e conversio n o f

androgen s t o estrogens . Aromatas e (ofte n calle dCYP-19 ) i s acytochrom e P45 0 enzym e comple x that produce s estrogen s fro m an androge n substrate . Alteration s i n th e amoun t o f aromatase , o r

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

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

aromatas e can als o resul t

i n diminishe dbloo dlevel s o f estrogens .

Report Number : TK002342 2 Pag e 1 6 o f 5 8

### Assay Methodolog y Summar y

Th e assay selecte db y th e EP A fo r validatio n use s ahuma n placenta l microsoma l preparatio n that contain s aromatase . I n thi s assay , microsome s are isolate dfro mhuman ter mplacenta , consistin g o f

th e endoplasmi c reticulu mmembran e that contain s membrane-boun d cytochrom e P450aro man dth e NADP H (p-Nicotinamid e Adenin e Dinucleotid e Phosphate ) cytochrom e P45 0 reductas e complex . Complet e enzym e activity require s th e presenc e o f eithe r NADP H o r

an NADPH-generatin g system . A n alternativ e t o placenta l extract s i s a recombinan t

microsom e preparatio n manufacture db y Gentes t (Woburn , MA ) tha t

include s P45 0 reductas e SUPERSOMES® . Enzym e activity i s measure db y quantifyin g eithe r an estrogen produc t

o r [ 3 

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

an dplacenta

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

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

### H2 0 i s release ddurin g th e conversio n o f androstenedion e t o estrone , an dresult s ar e reporte da s adirec t measuremen t

o f aromatas e activity pe r

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

o f interest . Othe r Scientificall y Relevan t

Informatio n I n preparatio n fo r

adoptio n o f

th e Tie r 1 scree n battery , th e USEP A conducte dvalidatio n studie s fo r th e aromatas e assay . Th e aromatas e assa y validatio n report

(USEPA , 2008b ) als o describe dresult s fro madditional chemical s that wer e examine dfo r

aromatas e inhibitio n b y th e laboratorie s testin g th e approve dmethodology . Atrazin e wa s on e o f th e 1 0 compound s use dfo r inter-laboratory compariso n (USEPA , 2008b , ISR , Tabl e 9.0.1 , p . 88) . Result s o f atrazin e testin g with both recombinan t an dplacenta l extrac t

microsom e preparation s ar e describe do n pp . 97-10 1 o f th e report , an dth e overal l classificatio n o f finding s i s presente di n Table s 10.5. 1 an d10.5. 2 o f th e IS R(p . 171) . Tw o publishe dstudie s als o reporte dresult s o f atrazin e activit y o n aromatas e wit h tissu e preparation s containin g th e CYP-1 9 complex .

Tie r I scree n Microsome s extracte dfro mtrou t brai n & ovary Recombinan t

huma n microsome s Recombinan t

an dhuma n placenta ] microsome s OSR I Reference(s ) Hinfra y e t al. , 200 6 Hollowayetal.,200 8 USEPA , 2008 b Comment s Teleos t fis h aromatase , [ 3 H]-labele dsubstrat e Recombinan t

huma n microsomes , fluorescen t substrat e EDS P Validatio n ISR ; huma n placenta l and/o r recombinan t

microsom e aromatas e 

### Hinfra y e t

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

4 3 divers e chemical s t o affec t aromatas e activity . Th e methodolog y use d microsome s prepare dfro mrainbo w trou t ovary an dbrai n tissue . [ 3 

### H]-androstenedion e wa s incubate dfo r

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

an extensiv e validatio n o f th e Report Number : TK002342 2 Pag e 1 7 o f 5 8protocol . A wid e variet y o f environmentall y sensitiv e chemicals , includin g som e heav y metals , wer e evaluated . Atrazin e wa s incubate dat on e concentratio n (1 0 uMolar) , usin g brai n an dovarian microsom e preparations . Hollowa y e t

al. , 200 8 I n thi s reporte dstudy , fro mauniversit y laboratory i n Canada , th e aromatas e assay s wer e conducte dwit

h acell-fre e recombinan t

human microsome system(B D Gentes t Bioscience s CYP-1 9 Suprasomes) . Th e stud y protoco l use ddibenzyfluorescei n a s th e aromatas e substrate ; th e enzym e convert s substrat e t o aproduc t detecte db y aspecific fluorescen t emission . N o othe r

chemical s wer e teste di n thi s experiment , excep t fo r testosteron e a s a positiv e contro l fo r

enzym e inhibition . Atrazin e wa s incubate dat anumbe r o f concentrations , fro m1 pMolart o 10 0 uMolar . USEPA , 2008 b Thi s i s th e IS Rfurnishe db y th e USEP A coverin g th e validatio n o f th e aromatas e assay adopte dfo r

th e EDS P Tie r

1 screen . A s state dabove , atrazin e wa s on e o f 1 0 compound s teste di n th e validatio n procedure . Deviation s t o OPPT S 890.1200 Protoco l OPPT S 890.120 0 Requirement s Criteri a Eithe r huma n placenta ! extrac t or recombinant huma n microsome s a s CYP-1 9 sourc e Us e [3H]-androstenedion e a s enzym e substrat e Enzym e activit y measure d a s [ 3 H]-water , produce d fro msubstrat e b y enzym e actio n Hinfra y e t al. , 200 6 (eviation s t o OPPT S 890.120 0 Aromatas e i n microsome s extracte dfro mfis h brai n an dovary non e Enzym e activit y measure da s [ 3 H]

- estrone , th e steroi d produc t o f th e enzym e actio n Hollowa y e t al. , 200 8 Deviation s t o OPPT S 890.120 0 Recombinan t

huma n microsome s commerciall y prepare d(i.e . n o deviation ) Substrat e wa s a fluorescein compoun d Enzym e activit y measure da s fluorescen t

produc t create db y enzym e actio n USEPA , 2008 b

Deviation s t o OPPT S 890.120 0

non e

non e

non e Justificatio n fo r OSRI Aromatas e i s bioidentica l i n numerou s anima l specie s Substrate s use dhav e bee n validate da s tru e substrate s fo r CYP-1 9 Al l marker s hav e bee n validate da s specifi c product s o f

enzym e i n th e method s use d Conclusions : Th e atrazin e consortiu mrequest s awaive r fo r th e aromatas e assa

y becaus e atrazin e ha s previousl y bee n teste dvi ath e EP A validatio n progra man db y othe r researcher s usin g substantiall y similar methodology . Atrazin e wa s include di n th e EP A validatio n studie s wit h th e aromatas e assay . On e metho d(USEPA , 2008b ) wa s i n fac t th e validate d protoco l recommende dfo r th e EDS P Tier

1 scree n battery , an datrazin e wa s include damon g several compound s use dfo r validation . Anothe r

stud y use don e o f

th e tw o validate d microsom e preparations . Methodologica l deviation s shoul dhav e n o impac t o n justificatio n fo r usin g OSR I i n consideratio n o f atrazin e interactio n wit h th e aromatas e complex .

Report Number : TK002342 2 Pag e 1 8 o f 5 8

### 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

Assa y Thi s assay wil l b e use dt o provid e informatio n o n th e potentia l fo r acompoun dt o interac t wit h th e estroge n receptor s (ERs ) isolate dfro mth e rat uterus . Th e assay i s no t

intende dt o b e use dt o sho w tha t

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

ahormone' s activity begin s whe n i t bind s t o it s specifi c recepto r an d initiate s acascad e o f

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

cells , i n o r

nea r th e nucleus , an dhormone-boun dE Rinteract 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 compare dt o receptor s fo r othe r steroi dclasses . I t

i s highl y conserve dacros s species . Chemical s that compet e wit h endogenou s estrogen s hav e th e potentia l t o eithe r induc e (agonist ) hormone-dependen t transcriptiona l activity , o r t o bloc k (antagonist ) norma l hormon e functio n b y preventin g th e endogenou s hormon e fro mbindin g t o th e receptor . Thu s ates t o f acompound' s ability t o bin dt o E Rconstitute s adirect , simpl e evaluatio n o f it s estrogeni c potentia l i n vertebrates . Assay Methodolog y Summar y Th e estroge n recepto r

bindin g assa

y measure s th e abilit y o f

aradiolabele dligan d(170

- estradiol ) t o interac t

wit h th e E Ri n th e presenc e o f increasin g concentration s o f ates t chemical . Ra

t uterin e cytoso l (RUC ) containin g E Ri s incubate di n tes t tube s with concentration s o f

ates t substanc e an dan aliquo t

o f radiolabele d[ 3 H]-estradiol . I f th e tes t substanc e interact s wit h th e receptor' s HBD , les s radioligan dca n bind , an da n activ e competito r

produce s adescendin g dose-respons e plot . Similarly , compound s tha t d o no t displac e radiolabele destradio l fro mE Rar e presume dt o b e devoi do f receptor-bindin g activity . Th e assay ha s alon g histor y o f us e withi n th e researc h communit y fo r rapi dan d relativel y inexpensiv e detectio n o f chemical s wit h E Rbindin g capability . Th e Tie r 1 scree n E Rbindin g 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 adequate independent

run s constitut

e a n experiment . Othe r Scientificall y Relevan t

Informatio n A n Integrate dSummary Report (ISR ) wa s prepare dfo r

th e EP A that

detail s th e validatio n proces s fo r

th e E Rbindin g assay usin g ra t uterin e cytoso l (USEPA , 2009) . Part o f th e validatio n proces s include danalysi s o f competitiv e bindin g b y 2 2 chemical s selecte dfo r interlaboratory validatio n studie s (ISR , Tabl e 21 , p . 52) . Atrazin e wa s on e o f th e 2 2 chemical s tha t

wer e tested . A summary o f thes e studie s i s presente di n Tabl e 2 7 (p.60 ) o f th e 

### ISR . Report Number : TK002342 2 Pag e 1 9 o f 5 8Tie r I scree n RUC , [ 3 H]-estradio l RUC , [ 3 H]-estradio l RUC , [ 3 H]-estradio l OSRI Reference(s ) Blai r e t al. , 200 0 Tennant , 1994b USEPA , 200 9 Comment s Sam e a s Tie r

I scree n Sam e a s Tie r

I scree n Validate dProtoco l use dfo r Tie r 1 Scree n Blai r e t al. , 200 0 Thi s study , conducte dat

th e Nationa l Cente r

fo r Toxicologica l Researc h (NCTR ) i n Arkansas , state 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 . Some 20 0 chemical s wer e teste dfo r ability t o displac e radiolabele d estradio l fro mER . Hydroxyapetit e ge l wa s use dfo r separatio n o f

boun dan dfre e tracer . Atrazin e wa s assesse dat

concentration s u p t o 10" 4 Molar (10 0 uMolar) . Tennan t e t al. , 1994 b Thi s stud y use da n RU C preparatio n t o determin e whethe r atrazin e coul ddisplac e bindin g o f [ 3 H]-estradio l t o ER . Concentration s o f u p t o 10" 3 Molar

( 1 mMolar ) wer e tested . Th e onl y variatio n noted , i n contras t t o th e validatio n studies , wa s th e us e o f dextran-charcoal fo r

th e separatio n o f

boun dan dfre e tracer . It

shoul db e noted , however , that th e IS Rdi drepor

t o n evaluation s o f

th e dextran-charcoa l procedure , an dconclude d"The RBA s obtaine di n th e differen t separatio n system s were observe dt o b e i n th e sam e range . There di dno t appea r t o b e an y greate

r variatio n i n 1C5

0 value s betwee n th e tw o separatio n system s tha n tha t observe dwithi n separatio n system s " (USEPA , 2009 , IS Rp . 22) . USEPA , 200 9 Thi s i s th e Integrate dSummar y Repor t (ISR) prepare dfo r

th e USEPA o n validatio n o f th e RU C E Rbindin g assay protocol . Atrazin e wa s on e o f 2 2 chemical s teste da s part o f th e validatio n process . E Rbindin g studie s wer e conducte di n fou r differen t laboratories . Deviation s t o OPPT S 890.125 0 Protoco l OPPT S 890.125 0 Requirement s Criteri a E Rfro 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 t o OPPT S 890.125 0 Non e Non e Tennan t

e t al. , 1994 b Deviation s t o OPPT S 890.125 0 Non e Dextran-charcoa l separatio n step USEPA , 200 9

Deviation s to OPPT S 890.125 0 non e

non e Justificatio n fo r OSRI Non e neede d Th e IS Rstate dtha t hydroxyapetit e an ddextran

charcoa l gav e simila r

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

bindin g assay becaus e atrazin e ha s previousl y bee n teste db y th e EP A validatio n programan db y other researcher s usin g substantiall y simila r methodology . Al l thre e OSR I report s use d methodolog y tha t

i s widel y accepte dfo r

evaluatio n o f

E Rbinding , an dwer e i n fac t ver y similar t o th e Tie r

1 validate dprotocol s that includ e RU C an d[ 3 H]-estradiol . Further , th e IS Rinclude s result s fro mtest s o f atrazin e competitio n fo r

bindin g a s par t

o f th e validatio n Report Number : TK002342 2 Pag e 2 0 o f 5 8process . Methodologica l deviation s shoul dhav e n o impac t o n justificatio n fo r

usin g OSRI i n consideratio n o f

atrazin e interactio n with estroge n recepto r binding . 

### 3. 5 Estroge n Recepto r Transcriptiona l Activatio n Assa y - Huma n Cel l Lin e HeLa-990 3 (OPPT S 890.1300 ) Purpos e o f

Assa y Th e purpose o f

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

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

i n away that

promote s subsequen t

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

th e detectio n o f

estrogen-mediate dtransactivation . Nuclear ER s exis t a s at leas t tw o subtypes , terme daan dp . The y ar e encode db y distinc t genes , hav e differen t

tissu e distribution , relativ e ligan dbindin g affinitie s an dbiologica l functions . Nuclear ER a mediate s th e classi c estrogeni c responses , an dtherefor e model s currentl y bein g develope dt o measur e E Ractivatio n mainl y us e ERa-linke dreporters . Thi s OPPT S Tes t

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

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

regulatio n o f

cellular processes . Furthermore , thi s Tes t

Guidelin e doe s no t

addres s antagonis t

interactio n with th e hER aan d subsequen t

effec t o n transcription . Assa y Methodolog y Summar y Th e transcriptiona l activatio n (TA ) assa y usin g areporte r

gen e techniqu e i s an i n vitr o too l tha t provide s mechanisti c data . Th e assay i s use dt o signa l bindin g o f th e estroge n recepto r wit h aligand . Th e receptor-ligan dcomple x the n translocate s t o th e nucleus , bind s specifi c DN A respons e elements , transactivate s a n associate dfirefl y luciferas e reporte r gene , an d thereb y increase s cellula

r expressio n o f

luciferas e enzyme. Luciferi n i s asubstrat e tha t i s transforme db y th e enzym e t o abioluminescen t produc t that ca n b e simpl y an dquantitativel y measure dwith aluminometer . Th e tes t syste mprovide di n thi s guidelin e utilize s th e hERa-HeLa-990 3 cel l line , whic h i s derive dfro mahuma n cervica l tumor . Th e cell s contai n tw o stabl y inserte dconstructs : Th e hER aexpressio n construc t (encodin g th e full-lengt h huma n receptor) . A firefl y luciferas e 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 TAT A element . Th e mous e M T TAT A gen e construc t ha s bee n show n t o hav e th e bes t performance , an ds o i s commonl y used . Consequentl y thi s hERa-HeLa-990 3 cel l lin e ca n measur e th e abilit y o f a tes t chemica l t o induc e hERa-mediate dtransactivatio n o f luciferas e gen e expression . Report Number : TK002342 2 Pag e 2 1 o f 5 8Dat ainterpretatio n fo r thi s assa y i s base dupo n whethe r

o r no t th e maximu mrespons e leve l induce db y ates t

substanc e equal s o r

exceed s an agonis t

respons e equa l t o 10 % o f tha t induce db y amaximall y inducin g ( 1 nM ) concentratio n o f th e positiv e contro l 17 0 estradio l (E2 ) (i.e. , th e P C 10) . Othe r Scientificall y Relevan t

Informatio n Atrazin e ha s bee n teste dusin g anumbe r o f estroge n recepto r

transcriptiona l activatio n assay s simila r t o th e EDS P Tie r

1 protocol . Th e followin g thre e cite dstudie s com e fro mreputabl e an dexperience dlaboratorie s tha t hav e a n extensiv e publishe dliteratur e i n us e o f transfecte d steroi dreceptor-linke dcel l reporte r systems . Al l use dwidel y accepte dtechnolog y i n cel l line s that hav e bee n use dfo r

man y years . Tw o o f

th e thre e studie s (Kojim ae t al. , 200 4 an d Balaguer et al. , 1999 ) specificall y teste dth e ER asubtype , an dth e third(Connor et al. , 1996) appear s t o hav e use dth e ER asubtyp e althoug h i t wa s no t identifie dspecificall y i n th e publication . Methodologica l deviation s shoul dhav e n o impac t o n justificatio n fo r

usin g OSR I i n consideratio n o f

atrazin e interactio n wit h estroge n recepto r transactivatio n systems . Tie r I scree n E Rtransfecte dwith reporte r E Rtransfecte dwit h reporte r E Rtransfecte dwit h reporte r Reference(s ) Conno r etal. , 199 6 Kojim aetal. , 200 4 Balague r e t al. , 199 9 Comment s

hE Rtransfecte dint o MCF- 7 cell s hER atransfecte dint o CH O hER atransfecte dint o HeL acell s Conno r et al. , 199 6 Thi s study use dMCF- 7 cells , ahuma n breas t cancer-derive dcel l line , transientl y transfecte d wit h a n E Rchimer aan daluciferas e reporte r gene . Cell s wer e plate dan dincubate dwith medi acontainin g atrazin e ( 1 nMola r to 1 0 uMolar ) or

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

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

th e chimeri c recepto r include d"the ligan dbindin g domai n o f th e ER" , whic h i s no w wel l understoo dt o b e th e sequenc e late r

identifie da s th e ligan dbindin g domai n o f ERa . Kojim ae t

al. , 200 4 Thi s study examine dth e ability o f 20 0 chemical s to induc e arespons e i n a n ER-linke d reporte r system . Human ER awa s transfecte dint o Chines e hamste r ovary (CHO ) cell s wit h a vector , an daluciferas e reporte r gen e wa s linke ds o tha t

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

signal . Atrazin e (1 0 nMolar t o 1 0 uMolar ) wa s on e o f th e 20 0 teste d chemical s i n compariso n t o estradiol-17| 3 (0.0 1 pMolar t o 10 0 nMolar) . Balague r et al. , 199 9 Thi s study wa s conducte di n amanner mos t simila r to th e EDS P validate dprotocol . HeL a cell s wer e stabl y transfecte dwith achimeri c recepto r expressio n syste mcontainin g ER aan d luciferase . Eigh t

putativ e xenoestrogen s wer e incubate dat concentratio n range s o f

0. 1 nMolar t o 1 0 uMolar , an dcompare dt o estradio l at 0. 1 pMola

r t o 1 0 nMolar . Report Number : TK002342 2 Pag e 2 2 o f 5 8Deviation s t o OPPT S 890.130 0 Protoco l OPPT S 890.1300 Requirement s Criteri a HeL acel l lin e Cell s stabl y transfecte d wit h ER dlinke dt o reporte r Conne r

et al. , 199 6 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 ) Kojim ae t al. , 200 4 Deviation s t o OPPT S 890.130 0 Chines e Hamste r Ovar y Cell s

non e Balague r

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

non e

non e Justificatio n fo r OSRI Cel l line s i n thes e report s ar e commonl y an dreliabl y use dfo r T A studies , particularl y fo r

steroi d receptor s Th e same E Rconstruc t

i n tw o studies , an dlikel y i n th e

y d Conclusions : Th e atrazin e consortiu mrequest s awaive r fo r th e estroge n recepto r transcriptiona l activatio n assay becaus e atrazin 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 similar methodology . Al l thre e cite dstudie s originat e fro mreputabl e an dexperience dlaboratorie s tha t hav e extensiv e publishe dliteratur e describin g th e us e o f transfecte dsteroi dreceptor linke dcel l reporte r

systems . Al l use dwidel y accepte dtechnolog y i n cel l line s that hav e bee n studie s fo r

man y years . Tw o o f

th e thre e studie s specificall y teste dth e ER asubtype , an dth e thir dstudy appear s t o hav e use dth e ER asubtype . Methodologica l deviation s shoul dhav e n o impac t o n justificatio n fo r

usin g OSR I i n consideratio n o f atrazin e interactio n wit h estroge n recepto r transactivatio n systems . 3. 6 Fis h Shor t

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

Assay Th e fis h short-termreproductio n assay wa s develope di n a n effor t t o scree n fo r

change s i n morpholog y (secondary sexual characteristics) , histopathology , spawning , an dspecifi c biochemica l endpoint s whic h migh t reflec t interferenc e wit h th e norma l functionin g o f th e hypothalamic-pituitary-gonada l (HPG ) axis . Assay Methodolog y Summar y Th e OPPT S 890.135 0 guidelin e utilize s matur e fathea dminnow s (Pimephalespromelas ) tha t ar e expose dt o aminimu mo f thre e concentration s o f

tes t substanc e an dappropriat e control(s ) fo r

2 1 days . Spawnin g i s establishe di n apre-exposur e perio do f at leas t 1 4 days . I n eac h o f fou r replicat e tank s pe r

concentratio n ther e ar e fou r female s an dtw o males . Th e endpoint s assesse dinclud e survival , behavior , fecundit y measure da s numbe r o f spawn s an d numbe r o f egg s pe r femal e reproductiv e day , fertilizatio n succes s measure da s numbe r o f fertil e egg s pe r

femal e reproductiv e day an dpercen t fertil e eggs , gonadal histopathology , gonadosomati c inde x (GSI) , 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 d biochemica l measure s o f

vitellogenin , estradiol , an dtestosterone .

Report Number : TK002342 2 Pag e 2 3 o f 5 8Othe r Scientificall y Relevan t

Informatio n Atrazin e ha s bee n previousl y teste di n th e fis h short termreproductio n study usin g th e EDS P Tier 1 scree n methodolog y (Battelle , 2005 ) an di n anothe r study (Bringol f

e t al. , 2004 ) usin g very similar

methodolog y t o th e OPPT S 890.135 0 an dOEC D 22 9 tes t guideline s (OECD.2009) . I n additio n astandar dfis h ful l lifecycl e study with fathea dminno w i s available , alon g with dat afro mlon g termlifecycl e exposure s o f bluegil l sunfish , broo k

trout , an dfatheadminnow . Thes e dat aar e adequat e t o satisf y th e EDS P Tie r

I dat arequiremen t

fo r an atrazin e fis h shor

t termreproductio n assay . Stud y Fis h Shor t

Term Reproductio n scree n Fis h ful l lifecycl e stud y i n fathea d minno w Lon g ter mlifecycl e studie s i n bluegil l sunfish , broo k

trou t an dfathea dminno w Reference(s )

Battelle , 200 5 Bringol f e t al. , 200 4 Dionne , 199 2

Mace k e t al. , 197 6 Comment s

Negativ e /EDS P Tie r

1 Scree n Negativ e Fathea dminno w ful l lifecycl e stud y (274-day exposure ) indicatin g n o reproductiv e effect s u p t o an dincludin g th e highes t tes t concentratio n (2. 0 m g ai/L mea n measure d concentration) . Bluegil l sunfis h lifecycl e stud y (450-da y exposure ) indicatin g n o reproductiv e effects . Broo k trou t lifecycl e stud y (306-day exposure ) indicatin g n o reproductiv e effects . Fathea dminno w lifecycl e stud y (280-day exposure ) indicatin g n o reproductiv e effects . Battelle , 200 5 Atrazin e wa s include da s ates t substanc e i n thi s multi-chemical evaluatio n exercis e fo r th e fis h short termreproductio n assay . Group s o f 4 femal e an d2 mal e fathea dminnow s wer e expose dt o atrazin e unde r

flow-throug h condition s fo r 21-days . Al l endpoint s i n th e curren t OPPT S 890.135 0 protoco l wer e evaluate d(i.e. , survival , adul t behavior , fecundity/fertilizatio n success , hatchability , adul t appearance , GSI , vitellogenin , se x steroid s an dgonadal histopathology ) with th e exceptio n o f secondary se x characteristics . Th e study report conclude dtha t

atrazin e exposure at

level s a s hig h a s 22 3 ng/L ha dn o significan t effect s o n diagnosti c marker s fo r potentia l endocrin e effects . Bringol f etal. , 200 4 Bringol f e t al. , 200 4 expose dgroup s o f 4 femal e an d2 mal e fathea dminnow s t o atrazin e unde r static-renewa l conditions . Th e endpoint s wer e survival , eg g production , numbe r o f spawns , eggs/spawn , relativ e gona dweight , gona dhistology , numbe r o f nuptia l tubercle s an d plasm avitellogeni n concentrations . N o statisticall y significan t effect s wer e note dfollowin g atrazin e exposur e at

5 an d5 0 ng/L . Dionne , 199 2 A n atrazin e standar dfis h ful l lifecycl e stud y wa s conducte dwit h fathea dminnow . Th e stud y wa s conducte dfollowin g th e FIFRA guidelin e #72-5 . Fathea dminnow s wer e expose dfo r a complet e generatio n (274-days ) an dth e progen y (Fl ) fo r 30-day s pos t

hatc h t o fiv e concentration s o f

atrazin e rangin g fro m0.1 5 t o 2. 0 mg/L . Th e endpoint s include dhatch , Report Number : TK002342 2 Pag e 2 4 o f 5 8survival , reproductio n an dgrowt h o f th e F O generatio n an dhatch, surviva l an dgrowth o f th e F l generation . Therefore , thi s stud y provide s aful l evaluatio n o f effect s o f atrazin e o n fis h ove r a n entir e lifecycle . A N o Observe dEffec t Concentratio n o f

0.2 5 m g atrazine/L wa s establishe dbase do n th e growth paramete r measure di n th e F O an dF l generations . Mace k et al. , 197 6 Th e USEP A conducte da n investigatio n int o th e chroni c toxicit y o f atrazin e t o thre e fis h species . Thes e exposure s ar e analogou s t o lifecycl e studie s an di n som e case s ar e considerabl y longe r

i n duratio n (e.g . bluegil l sunfis h 450-day s exposure) . Fis h wer e expose d unde r flow-throug h conditions . Exposure s wer e initiate dwith youn g fis h (varie damon g species ) whic h wer e followe dthroug h t o reproductio n i n th e firs t seaso n fo r

fathea dminno w an dbroo k trou t an dth e secon dseaso n fo r bluegil l sunfish . Fecundit y wa s assesse di n al l species . F l progen y wer e maintaine dfo r mortalit y an dgrowt

h assessment s (bluegil l an d broo k trou t for 90-day s andfathea dminno w for 60-days) . Ther e wer e no reproductiv e effect s note di n an y species . Fo r bluegil l sunfis h an dfathea dminno w n o significan t effect s wer e observe di n th e F O an dF l generation s u p t o an dincludin g th e highes t concentration s teste do f 0.09 5 an d0.21 3 mg/L , respectively . Fo r broo k trout , F O an dF l growt

h wa s reduce dat

0.1 2 mg/L . Du e t o th e inclusio n o f

atrazin e i n th e EP A validatio n program , deviation s o f th e reporte d screenin g studie s fro mth e finalize dguidelin e ar e negligible . Deviation s t o OPPT S 890.135 0 Protoco l OPPT S 890.135 0 Requirement s Criteri a Fis h rhre e chemica l treatmen t level s require d Flo w throug h exposure s Pre-exposur e evaluatio n

Secondary sexua l character s Battelle , 200 5 Deviation s t o OPPT S 890.135 0 N o deviatio n Tw o concentration s o f atrazin e teste d(nominall y 2 5 an d25 0 ug/L ) N o deviatio n 9 day s pre-exposure : minimu mo f

1 4 require d

No t measure d Bringol f e t al. , 200 4 Deviation s t o OPPT S 890.135 0 Fis h wer e 2 2 month s old : recommende dag e 4. 5 - 6 month s Tw o concentration s o f Atrazin e teste d(nominall y 5 an d5 0 ug/L ) Static-renewa l assay . N o deviatio n

N o deviatio n Justificatio n fo r OSR I Stud y wit h fis h a t

recommende d ag e available . Sufficien t informatio n fo r

lac k o f a n effec t ca n b e determine dfro mth e fou r differen t concentration s teste d i n tw o studies . Flow-throug h stud y available . Battell e 2005 : Meet s requiremen t

a s state di n OEC D 22 9 (7-14-day s recommended) . Validit y criterio n o f >15eggs/female/da y wa s me t demonstratin g tha t

th e fis h were i n asuitabl e reproductiv e stat e fo r th e purpos e o f

th e test . Stud y assessin g secondar y se x character s available . Weigh t of evidenc e fro mal l othe r

endpoint s show s tha t a n effec t i s unlikely . Conclusions : Th e atrazin e consortiu mrequest s awaiver fo r th e fis h shor

t termreproductio n assa y becaus e atrazin e ha s alread y bee n thoroughl y teste di n existin g studie s usin g Report Number : TK002342 2 Pag e 2 5 o f 5 8

### substantiall y similar methodology . Atrazin e wa s teste di n th e EP A validatio n programusin g methodolog y substantiall y simila r t o th e finalize dOPPT S 890.135 0 guideline , an datrazin e wa s furthe r

teste db y Bringol f

e t al. , 2004 . I n additio n astandar dfis hful l lifecycl e stud y an d lon g termlifecycl e test s i n thre e specie s ar e available . Thes e dat asatisf y th e EDS P Tie r

I datarequirement

fo r an atrazin e fis h shor

t ter mreproductio n assay . 3. 7 Hershberge r

Assa y - Rat

(OPPT S 890.1400 ) Purpos e o f

Assay Th e Hershberge r

Bioassay i s ashort-termi n viv o screenin g tes t usin g accessory tissue s o f

th e mal e rat reproductiv e tract . Th e bioassay serve s a s amechanisti c i n viv o screenin g assay fo r androgen agonists , androge n antagonist s an d5a-reductas e inhibitors . It i s intende dto b e include di n abattery o f

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

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

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

asubstanc e t o elici t biologica l activitie s consistent

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

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

group s o f experimenta l animal s at on e dos e leve l pe r

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

mor e targe t

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

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

an androgen . Whe n screenin g fo r

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

subcutaneou s injectio n fo r aperio dofte n consecutiv e days , i n concert with dail y s c (subcutaneous ) injection s o f T P (0. 2 o r

0. 4 mg/kg/d) . Th e validatio n program determine dthat

either dos e o f TP 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 tha t

th e substanc e i s positiv e fo r antiandrogeni c activity . Report Number : TK002342 2 Pag e 2 6 o f 5 8Othe r Scientificall y Relevan t

Informatio n Atrazin e ha s bee n previousl y teste di n th e Hershberge r assa y usin g substantiall y similar methodolog y a s th e EDS P Tier 1 scree n guidelines . N o methodological deviation s wer e identifie di n th e studie s tha t woul dimpac t

th e justificatio n fo r

usin g OSRI i n consideratio n o f atrazin e activity i n th e Hershberge r bioassay screen . Tie r I scree n Hershberge r OSRI Reference(s )

Ashby , 200 4 Yamasaki e t

al. , 200 4 Comment s

Hershberge r

protoco l Hershberge r

protoco l Ashby , 200 4 Thi s study wa s conducte dat th e Centra l Toxicolog y Laboratory i n England . Six-wee k ol d rat s wer e orchidectomize dan dafte r recovery wer e administere datrazin e b y gavag e at dose s o f 0 , 10 , 25 , 5 0 an d10 0 mg/kg/day alon g with 0. 4 mg/kg/da y o f testosteron e propionat e b y s c injection , fo r

1 0 days . SV , VP , LABC , COW , an dG P wer e al l dissecte dan dweighed . Yamasaki et

al. , 2004 I n thi s stud y th e Hershberge r assay wa s conducte do n 1 2 chemicals , includin g atrazine , usin g th e OEC D draf

t protocol . Th e stud y als o assesse dth e androge n recepto r bindin g assay fo r th e same substance s t o confir mth e usefulnes s o f th e Hershberge r

assay . Rat s wer e castrate d at 4 2 day s o f

ag e an dwer e administere datrazin e (0 , 3.2 , 1 6 an d8 0 mg/kg/day ) b y gavag e fo r 1 0 days . A n antagonis massa y wa s als o conducte dwit h th e sam e dose s plu s concomitan t s c dos e o f T P at 0. 2 mg/kg/day . SV , VP , LABC , COW , an dG P wer e al l dissecte dan d weighed . Deviation 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 t o castrat e rat s Tes t fo r antagosis musin g concomitan t T P administratio n Weig h 5 tissues : VP , SV , LABC ,

COW , GP Ashby , 200 4 Deviation s t

o OPPT S 890.140 0 Non e Non e No t don e Non e Non e Yamasak i e t al. , 200 4 Deviation s to OPPT S 890.140 0 non e

non e

non e
non e

non e 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 administration ; thes e studie s both use dgavag e EDS P protoco l include s bot h agonis t

an d antagonis t

evaluatio n Sam e evaluatio n desig n a s EDS P protoco l Al l 5 tissue s weighed , a s include di n EDS P protoco l Conclusions : Th e atrazin e consortiu mrequest s awaive r fo r th e EDS P Tie r

1 Hershberge r assay becaus e atrazin e ha s alread y bee n thoroughl y teste di n existin g studie s usin g substantiall y similar

methodology . I t

appear s that

th e Yamasaki e t

al. , 200 4 stud y wa s Report Number : TK002342 2 Pag e 2 7 o f 5 8conducte dspecificall y a s atria l o f th e EDS P validate dprotoco l o n 1 2 chemical s o f widesprea dinterest , includin g atrazine . N o methodologica l deviation s wer e identifie dtha t woul dimpac t

th e justificatio n fo r

usin g OSR I i n consideratio n o f atrazin e activity i n th e Hershberge r

bioassa y screen . 3. 8 Femal e Puberta l Assa y - Rat (OPPT S 890.1450 ) Purpos e o f

Assa y Th e purpos e o f

th e femal e pubertal assa

y i s t o scree n substance s fo r th e potentia l t o interac t wit h th e endocrin e syste mb y evaluatin g effect s o n puberta l developmen t an dthyroi d functio n i n th e intac t

juvenile/peripuberta l femal e rat . Thi s assa y i s capabl e o f

detectin g anti

thyroid , estrogeni c o r

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

steroidogenesis) , o r

agent s whic h can alte r

puberta l developmen t

vi a change 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 . Assay Methodolog y Summar y Th e Femal e Pubertal scree n use s fiftee n femal e rat s i n eac h o f thre e treatmen t

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

t 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) , an dth e lowe r dos e at one-hal f th e highe r

dose . Al l dam s mus t

b e pregnan t

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

th e en do f

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

- t o 5-day cycles) , irregular cyclin g (som e regular

cyclin g bu t wit h 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 do f

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

r t o obtai n asufficien t

volum e o f

bloo dfo r th e hormon e an dchemistrie s measurements . 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 dth e weight s o f

each , excep t

th e thyroid/trache aan duterus , ar e recorde d(kidneys , adrenals , an dovarie s ar e weighe da s pairs. ) Th e ovaries , uterus , kidney , an dthyroi dtissu e Report Number : TK002342 2 Pag e 2 8 o f 5 8ar e place di n 10 % buffere dformali n fo r at leas t 2 4 hour s an dthe n store di n 70 % ethano l unti l embedde di n paraffin . Section s ar e cu t an dstaine dwith hematoxyli n an deosi n (H&E ) fo r subsequen t

histologica l evaluations . Hormonal Assays : Immunoassay s o f thyroxin e (TV ) an dTS H can b e conducte dusin g radioimmunoassa y (RIA) , immunoradiometri c assay (IRMA) , enzyme-linke d immunosorben t

assay (ELISA) , o r

time-resolve dimmunofluorescen t procedures . Bloo dChemistry : An y standar dpane l o f bloo dchemistr y 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 ar e subjectivel y evaluate dfo r

follicular cel l heigh t

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

(includin g presence/absenc 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 both primar y an datreti c follicles ) i n additio n t o an y abnormalities/lesions , suc h a s ovaria n atrophy . Uterin e histolog y shoul ddocumen t case s o f uterin e hyper

- o r

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

endometria l glan ddevelopment . Othe r Scientificall y Relevan t

Informatio n Atrazin e ha s bee n previousl y teste di n th e femal e puberta l assa y usin g substantiall y simila r methodolog y a s th e EDS P Tie r 1 scree n guidelines . N o methodologica l deviation s wer e identifie dtha t

woul dimpac t

th e justificatio n fo r

usin g OSR I i n consideratio n o f atrazin e activity i n th e femal e puberta l assay . On e o f th e tw o reference dstudie s parallel s th e eventua l EDS P T1 S assa

y ver y closely an d include dnearl y every require dmeasur e o f th e recommende dprotocol . Th e othe r stud y wa s designe dver y closel y t o th e EDS P protoco l an dassesse dsom e o f th e targete dparameter s o f thi s apica l test . Tie r I scree n Femal e Puberta l OSRI Reference(s ) Law s e t al. , 200 0 Ashb y e t al. , 200 2 Comment s Prototyp e fo r

EDS P Tie r

1 assa y validatio n Substantiall y simila r

t o Tie r 1 assa y Law s e t al. , 200 0 Thi s study , conducte db y aUSEP A laboratory researc h team , wa s designe da s an earl y prototyp e fo r

th e protocol s that

eventuall y becam e th e EDS P Tie r 1 puberta l rat

screen , an di t include datrazin e a s ates t compound . Report Number : TK002342 2 Pag e 2 9 o f 5 8

### Femal e rat s wer e dose db y oral gavag e fro mPN D 2 2 throug h 4 1 at 5 dose s o f

atrazin e (12. 5

- 20 0 mg/kg/day ) plu s avehicle-onl y group . Dat e o f vagina l openin g (VO ) wa s recorded , a s wer e estrou s cyclin g pattern s followin g VO . A t sacrific e seru mwa s prepare dfro mbloo d an danalyze dfo r

T 4 an dTS H content , amon g othe r hormones . Uterus , ovaries , thyroid , liver , kidney , pituitary , an dadrenal s wer e al l dissected , processe dfo r histolog y an dexamined . Ashb y e t al. , 200 2 Prio r t o th e developmen t

o f th e EDS P Tie r

1 process , Ashb y an dco-worker s evaluate dth e effec t o f atrazin e treatmen t

i n aprotoco l substantiall y similar t o th e validate dmethod . Beginnin g o n PN D 22 , atrazin e wa s administere dt o immatur e femal e rat s b y oral gavag e at dose s o f 0 , 10 , 3 0 o r

10 0 mg/kg/day ) throug h PN D 4 3 (Wista r strain ) o r PN D 4 6 (Sprague

Dawle y strain) . Som e o f

th e targete dparameter s o f

th e Tier 1 tes t wer e assessed , suc h a s ag e at vagina l opening , dail y bod y weights , an duterin e weight . Assay s o f seru mhormone s an d othe r chemistrie s wer e no t

done , no r wa s histolog y o f

necropsie dorgan s conducted . Th e stud y di dhoweve r

includ e certai n othe r

test s that ar e no t part o f th e curren t

EDS P Tie r

1 puberta l rat

protocol . 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 nea

r 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 Law s e t al. , 200 0 Deviation s t o OPPT S 890.145 0 Non e PN D 22-4 1 5 doses ; highes t

dos e suppresse dB W Non e excep t

non-endocrin e seru mchemistr y measure s no t don e Non e Ashb y e t al. , 200 2 Deviation s t o OPPT S 890.145 0 non e PN D 22-4 3 o r

4 6 3 doses ; highes t

dos e suppresse dB W VO , E cycl e pattern s an d B W assesse d N o histologica l examinatio n Justificatio n fo r OSRI Non e neede d Age s o f coverag e ar e essentiall y identica l t o th e validate dEDS P metho d Essentiall y i n lin e wit h T l S protoco l Man y o f targete d parameter s o f

thi s apica l tes t wer e assesse d On e o f tw o cite dstudie s performe dhistologica l examination s 

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

1 Femal e Pubertal assay becaus e atrazin e ha s already bee n thoroughl y teste di n existin g studie s usin g substantiall y similar

methodology . N o methodologica l deviation s wer e identifie dthat woul d impac t th e justificatio n fo r

usin g OSRI i n consideratio n o f atrazin e activity i n th e femal e ra t prepuberta l screen . 3. 9 Mal e Puberta l Assa y - Rat (OPPT S 890.1500) Purpos e o f

Assa y Th e purpos e o f

th e mal e puberta l assay i s t o scree n fo r substance s tha t

hav e th e potentia l t o interac t wit h the endocrin e system , by identifyin g effect s on puberta l developmen t and 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 vi aalteration s i n recepto r

bindin g o r

steroidogenesis . Report Number : TK002342 2 Pag e 3 0 o f 5 8

### Assay Methodolog y Summar y

Thi s protoco l use s fiftee n mal e rat s i n eac h treatmen t grou p ( 1 vehicle-treate dan d2 treate d doses) . Th e Sprague-Dawle y o r Wistar strain s ar e preferred . Th e highes t dos e leve l shoul d b e at o r jus t belo w th e Maximu mTolerate dDos e (MTD) , an dth e lowe r dos e at one-hal f th e highe r dose . 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 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 bod y weigh t at 0700-090 0 daily , fro mPN D 2 3 throug h PN D 53 . Th e followin g observation s ar e made : Preputia l Separation : Beginning 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 dprepuc e ar e al l recorde do n th e day s the y ar e observed . Th e day o f

complet e preputia l separatio n i s th e designate dendpoin t fo r th e parameter . Necropsy : Subject s are sacrifice do n PND 53 . Trunk 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 colder fo r subsequen t

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

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

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

histologica l evaluations . Th e thyroid , with attache dtrachea , i s fixe di n 10 % buffere dformali n fo r at leas t 2 4 hours . The n th e thyroi d (with parathyroids ) i s dissecte dfro mth e trachea , blotte dan dweighed , place di n 70 % ethano l unti l embedde di n paraffin , sectioned , staine dwith H&E , an dhistologicall y evaluated . Kidne y i s als o fixe di n 10 % buffere dformali n fo r at leas t 2 4 hours , the n place di n 70 % ethano l unti l embedde di n paraffin , sectioned , staine dwith H&E , an dhistologicall y evaluated . Hormona l Assays : Hormona l measurement s 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 . Bloo dChemistry : An y standardpane l o f bloo dchemistr y 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 . Report Number : TK002342 2 Pag e 3 1 o f 5 8Histology : 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

follicular cel l heigh t

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

Informatio n Atrazin e ha s bee n previousl y teste di n th e mal e puberta l assa y usin g substantiall y simila r methodolog y a s th e EDS P Tie r 1 scree n guidelines . Th e cite dstud y (Stoke r e t al. , 2000 ) evaluate dessentiall y al l o f th e importan t

endocrine-relate dtarget s identifie di n th e OPPT S 890-150 0 protocol . Importantly , th e anima l strain , metho do f administration , an dduratio n o f dosing , use di n th e Stoke r e t al. , 200 0 study wer e identica l t o th e EDS P protocol . N o methodologica l deviation s wer e identifie dtha t woul dimpac t

th e justificatio n fo r

usin g OSRI i n consideratio n o f

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

Stoke r e t al. , 200 0 Comment s

EDS P Tie r

1 scree n Stoke r e t al. , 200 0 Thi s study o f

atrazine , conducte db y th e USEP A Reproductiv e Toxicolog y Divisio n o f th e Nationa l Healt

h an dEnvironmenta l Effect s Researc h Laboratory , wa s atria l o f th e propose d desig n fo r th e EDS P Tie r

1 mal e rat

puberta l scree n that

wa s eventuall y adopted . Group s o f intac t 2 3 day ol dweanlin g mal e Wistar rat s wer e administere datrazin e at dos e level s o f

0 , 6.2 5 (preputia

l separatio n study only ) 12.5 , 25 , 50 , 100 , 150 , an d20 0 mg/kg/day for 3 0 days . A n additiona l grou p o f

untreate dmale s wa s pair-fe d(PF ) t o matc h th e bod y weight s o f th e 20 0 mg/k g grou p i n orde r

t o identif y effect s fro mreduce dfoo dconsumptio n observe dat 20 0 mg/kg . Preputia l separatio n wa s monitore dfro mpost-nata l da y 3 3 onwards . Th e pituitary , testes , ventra l prostate , latera l prostate , epididymides , an dsemina l vesicle s wit h coagulatin g glan dwer e remove dan dweighed . Tissu e sample s wer e examine db y histology . Bloo dwa s analyze dfo r

TSH , T 4 , TS , luteinizin g hormon e (LH) , prolacti n (PRL ) an destrogens . Additionally , L H an dPR L wer e determine di n th e anterio r pituitaries . Body weight s o f

hig h dos e (200 mg/kg ) atrazin e treate drat s wa s significantl y decrease db y 17 % at day 5 3 an dreturne dt o norma l at day 120 . Bod y weights o f pai r fe danimal s wer e decrease dt o acomparabl e exten t (-14 % at PN D 53 ) a s th e 20 0 mg/kg/day atrazin e treate d group .

Report Number : TK002342 2 Pag e 3 2 o f 5 8

### Deviation s t o OPPT S 890.1500 Protoco l OPPT S 890.1500 Requirement s Criteri a S D o r Wistar rat 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 , dorsolatera l prostate , semina l vesicl e wit h coagulatin g gland s an dfluid , levato r an i plu s bulbocavernosu s muscles , liver , kidneys ,

pituitary , an dadrenal s Histologi c evaluatio n of

testis , epididymis, thyroid , an dkidne y fro mal l treate dsubject s Measur e thyroxine , TSH , testosterone , creatinine , BU N i n seru m Stoke r e t al. , 200 0 Deviation s t o OPPT S 890.1500

Non e Non e 7 doses , includin g 2 at

MT D Weighe dpituitary , testes , ventra l prostate , dorsolatera l prostate , semina l vesicl e wit h coagulatin g gland s an d

fluid, 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 OSRI Non e neede d Non e neede d Non e neede d Nearl y al l androge n target s weighe d Specifi c scree n target s were evaluate dhistologicall y a t highes t treatmen t

dos e Measures fo r

EDS P screen relate dtarget s wer e conducte d 

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

1 Mal e Pubertal assay becaus e atrazin e ha s alread y bee n thoroughl y teste di n existin g studie s usin g substantiall y simila

r methodology . N o methodologica l deviation s wer e identifie dthat woul d impac t th e justificatio n fo r

usin g OSRI i n consideratio n o f atrazin e activity i n th e mal e rat prepuberta l screen . 3.1 0 Steroidogenesi s Assay - 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 intende dt o scree n fo r substance s that

may affec t

th e steroidogeni c pathwa y beginnin g wit h reaction s occurrin g afte r th e gonadotropi n o r corticotrophi n receptor s ar e occupied . Thi s i n vitr o assay i s no t intende dt o identif

y substance s tha t

ma y affec t

Steroidogenesi s du e t o actio n o n th e hypothalamu s o r pituitar y gland . Assa y Methodolog y Summar y Th e purpos e o f

thi s i n vitr o assay i s t o scree n fo r substance s that

potentiall y may affec t

th e synthesi s o f

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

adrenocortica l tissu e tha t

normall y secrete s very littl e estroge n o r testosterone , an dwhic h require s ACT H t o initiat e Steroidogenesis . Thi s assa y i s conducte db y incubatin g H295 Rcell s wit h 7 concentration s o f eac h tes t substanc e 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 17p-estradio l an dtestosteron e conten t o f th e incubatio n medi aar e performe dusin g appropriat e steroi dhormon e assays . Report Number : TK002342 2 Pag e 3 3 o f 5 8Cel l viability , an importan t inde x o f potentia l toxicity o f

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

Informatio n Atrazin e ha s bee n teste di n anumbe r o f studie s usin g th e H295 Rcel l system , a s wel l a s othe r i n vitr o system s tha t

produc e steroi dhormone s (reviewe db y Y i e t al. , 2010b). Aromatas e i s th e on e steroidogeni c enzym e tha t convert s androgen s t o estrogens . Level s o f aromatas e ar e assesse db y measurin g level s o f 3 H-wate r produce dfro m3 H-androstenedion e substrate o r b y measurin g level s o f

mRN A fro mth e aromatas e enzym e itself . Th e EDS P Tie r

1 scree n tha t

use s H295 Rcell s i s calle daSteroidogenesi s Assay . Th e protoco l doe s no t

cal l fo r measure s o f

aromatas e expressio n pe r

s e (i.e. , 3 H-wate r

productio n o r enzym e mRNA) . Th e targete dmeasure s ar e level s o f testosteron e an destradio l i n th e incubatio n media , a s representative s o f th e entir e steroidogeni c pathway . B y thes e criteria , tw o report s presen t

result s appropriat e t o OSRI consideration . On e report (USEPA , 2008c ) wa s i n fac t

th e validatio n proces s o f

th e protoco l recommende dfo r th e EDS P Tie r

1 scree n battery , an datrazin e wa s include damon g several compound s evaluated . Th e othe r report include dmeasure s o f

estron e outpu t

amon g th e result s presented . 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 atrazin e activity i n th e H295 RSteroidogenesi s screen . Tie r I scree n Steroidogenesi s H295 R Steroidogenesi s H295 R Cell viabilit y OSRI Reference(s ) USEPA , 2008 c Sanderso n etal. , 200 1 Yietal. , 2010 a Comment s EDS P Validatio n Report H295 Rcells ; measure destron e outpu t Review s issue s o f

cytotoxicity , aromatas e expression , an d publication s o f

atrazin e o n bot h USEPA , 2008 c A validatio n wa s conducte dfo r th e USEP A EDS P progra munde r th e directio n o f

Entrix , Inc. , o f Canada . Th e proces s include dtestin g o f th e basi c incubatio n methodology o f H295 R cells , evaluatio n o f

contro l substance s t o stimulat e an dinhibi t Steroidogenesis , harves t

o f incubatio n medi afo r

testosteron e an destradio l assay , an dtest s o f exposur e t o aselectio n o f substances . A n inter-lab compariso n surve y o f result s wa s conducte dwit h 7 differen t laboratorie s i n 7 countries . Atrazin e wa s on e o f 1 2 substance s teste d(USEPA , 2008c , ISR , Tabl e 6.2 , page s 31-32) . Result s fro mincubation s wit h atrazin e at 7 concentration s (0. 1 nMola

r - 10 0 uMolar ) wer e returne dfro m5 laboratorie s fo r testosteron e outpu t

(Fig . 9.7 , p . 55 ) an dfo r

estradio l outpu t

(Fig . 9.10 , p . 58) . Cel l viabilit y wa s als o reporte dfo r al l 7 concentration s o f

atrazin e (Fig . 9.5 , p . 53) . Sanderso n et

al. , 200 1 Thes e experiment s conducte di n aDutc h universit y laboratory wer e focuse dprimaril y o n th e enzym e aromatas e i n H295 Rcells , whic h i s no t th e steroida l endpoin t

evaluate di n th e Tie r

1 screen . However , studie s i n thi s report di dmeasur e estron e outpu t int o th e incubatio n media . Whil e estron e i s no t

th e targete dproduc t

o f th e validate dEP A 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 Report Number : TK002342 2 Pag e 3 4 o f 5 8concentration s was muc h narrowe r tha n the recommende dscree n but was nea r the highes t level s calle dfo r

i n th e screen . Cel l viabilit y wa s evaluated . Y i e t al. , 2010 a Th e researc h reviewe di n thi s report i s importan t

regardin g th e potentia l fo r response s du e t o cytotoxi c actions . A n importan t componen t

o f thi s assa y i s t o certif

y that cel l viabilit y ha s no t bee n compromise db y th e tes t substance . A s reviewe db y Y i e t al. , 20 1 Oa , aromatas e expressio n an destroge n outpu t ca n b e enhance db y cytotoxi c processes . Studie s wit h atrazin e usin g H295 Rcells , an dothe r cel l types , sho w n o evidenc e o f cytotoxicity at

level s that alte r aromatas e expression . Deviation s t o OPPT S 890.155 0 Protoco l OPPT S 890.1SS O 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 d estradio l outpu t

i n medi a USEPA , 2008 c

Deviation s to OPPT S 890.155 0 non e non e non e

non e 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 dtha n EDS P T l scree n Estrone measured ; testosteron e an destradio l no t measure d Y i e t

al. , 2010 a Deviation s t o OPPT S 890.155 0 non e non e non e

non e Justificatio n fo r OSR I Non e neede d Non e neede d Method s ar e al l reliabl e Estron e i s als o aproduc t

o f steroidogenesi s Conclusions : Th e atrazin e consortiu mrequest s awaive r fo r th e EDS P Tier

1 H295 R Steroidogenesi s assa

y becaus e atrazin e ha s alread y bee n thoroughl y teste di n existin g studie s usin g substantiall y similar methodology . 3.1 1 Uterotrophi c Assay

- Ra t (OPPT S 890.1600 ) Purpos e o f

Assay Th e Uterotrophi c Assay i s intende da s a n 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 do n th e long-know n principl e tha t

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

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

i s acomplet e assessmen t

o f th e abilit y o f

ates t substanc e t o bin dt o E Ran dpromot e well-know n an deasil y measure dresponse s t o E Rbinding , i n a livin g animal . N o other

hormon e clas s accomplishe s any for mo f thi s "pur e action" . Assay Methodolog y Summar y Th e goa l o f

thi s assa y i s t o scree n substance s fo r estrogenicity throug h asimpl e i n viv o assay . Validatio n studie s wer e carrie dou t unde r OEC D auspices . Approve dmethod s permi t us e o f eithe r rat s (Sprague-Dawle y o r

Wistar strain s preferred) , o r

mice . Subject s can b e intac t immatur e (PND 21-2 4 at

th e beginning ) o r

ovariectomize d(OVX ) adults . Tes t material s can b e administere db y ora l gavag e o r injecte dsubcutaneously . Ther e shoul db e at leas t tw o dos e groups , with th e highe r dos e a t o r near th e animals ' maximu mtolerate ddose , plu s a Report Number : TK002342 2 Pag e 3 5 o f 5 8grou p administere dvehicl e only . There shoul db e at leas t 6 animals/dos e groups . Administratio n o f

areferenc e hormone , suc h a s 17a-ethiny l estradio l i s considere dhelpfu l bu t i s no t mandatory . Treatmen t shoul db e administere ddail y fo r 3 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 ope n t o releas e intralumina l fluid , blotte dbu 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 ma y b e importan t

i f th e administere dcompoun daffecte dbod y weights . Othe r Scientificall y Relevan t

Informatio n Atrazin e ha s bee n previousl y teste di n th e uterotrophi c assa y 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 dtarge t

identifie di n th e OPPT S 890-160 0 protocol . Th e animal strain , low-estroge n conditio n o f th e tes t animals , an dmetho do f

administration , wer e acceptabl e a s describe di n th e protocol . Ther e 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 atrazin e activity i n th e rat uterotrophi c assay . Tie r I scree n Uterotrophi c Reference(s )

Tenant etal. , 1994a Conno r etal. , 199 6 Ashb y e t al. , 200 2 Comment s Adul t ovariectomize drat s Intac t immatur e rat s Intac t immatur e rat s Tennan t e t al. , 1994 a Adul t S D rat s wer e purchase dafte r ovariectom y an dwer e administere datrazin e b y gavag e fo r 3 day s at dose s u p t o 30 0 mg/kg . Group mea n B W (body weight ) at th e highes t

dos e wa s 12 % belo w th e vehicl e grou p weight . Tes t group s wer e 5- 6 animal s pe r dose . A positiv e contro l grou p receive destradio l injecte dsubcutaneously . Uter i wer e dissected , cleane dan d weighed . Conno r et al. , 199 6 Thi s study , fro madifferen t universit y 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 (PN D 21 ) wer e administere dthre e dose s o f atrazin e a

t aslightl y lowe

r dosin g range . A positiv e contro l grou p receive destradio l b y intraperitonea l injection . Uter i wer e dissected , cleane dan d weighed . Ashb y e t al. , 200 2 Thi s stud y include damor e comprehensiv e assessmen t o f atrazin e effect s i n prepuberta l rats . Group s o f intac t Wistar female s wer e administere datrazin e b y gavag e beginnin g at PN D 21

- 22 . On e se t

o f animal s wa s sacrifice dat

PN D 29-30 , i.e . afte r

8 day s o f treatment . Grou p mea n B W at

th e highes t

dos e wa s 92 % o f

vehicl e mea n BW . Report Number : TK002342 2 Pag e 3 6 o f 5 8Deviation s t o OPPT S 890.160 0 Protoco l OPPT S 890.1600 Requirement s Criteri a S D o r Wista r rats , intac t OV X adul t

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

o r nearMT D Gavag e o r injecte ds c - 3 day s treatment , sa c o n da y 4 Dissect , clean , weig h uteru s Tennan t e t al. , 1994 a Deviation s t o OPPT S 890.1600 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 890.160 0 None : use dimmatur e S D rats , treatmen t starte da t PN D 2 1 None : 3 dose s plu s vehicle , MT D exceede d None : gavag e Non e Non e Ashb y e t al. , 200 2 Deviation s t o OPPT S 890.160 0 None : use dimmatur e Wista r rats , treatmen t starte dat PN D 2 1-2 2 None : 3 dose s plu s vehicle , nea r

t o MT D None : gavag e 8 day s treatment , sa c o n day 9 Non e ratificatio 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 atrazin e consortiu mrequest s awaive r fo r th e EDS P Tie r

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

methodology . 

### 4. 0 REPRODUCTIV E AN D DEVELOPMENTA L TOXICIT Y CHARACTERISTIC S O FATRAZIN E Atrazin 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 al l eleve n o f

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

tier studie s availabl e fo r

atrazin e whic h evaluate s apical endpoint s i n intac t animals . Change s i n thes e endpoint s upo n exposur e t o ates t substanc e woul dindicat e th e potentia l fo r

th e substanc e t o interferenc e wit h endocrin e systems . Sinc e thes e dat aar e al l supplementary i n th e cas e o f atrazine , whic h ha s arobus t se t o f actual Tie r

1 assay s available , onl y abrie f

summary o f

th e finding s fro mth e mos t relevan t studie s i s provide dbelow . Base do n result s fro mstudie s i n th e rat an drabbi t

summarize dan dreviewe di n an FA O Worl dHealt

h Organizatio n report

(Pfei l e t al. , 2007 ) an dals o i n th e USEPA Interi m Registratio n Eligibilit y Decisio n (USEPA , 2006) , i t wa s conclude dtha t

atrazin e doe s no t caus e an y specifi c developmenta l effect s whe n administere dt o pregnan t dam s durin g th e perio do f organogenesis . Atrazin e di dno t caus e embry o toxicit y o r

feta l malformatio n i n guidelin e developmenta l toxicit y studie s conducte di n both th e rat an drabbit . Th e developmental an dmaterna l NOEL s wer e both 2 5 mg/kg/da y i n th e ra t an d5 mg/kg/da y i n th e rabbi t a s indicate di n Tabl e 2 . Base dupo n aweigh t o f th e evidenc e fro mal l relevan t studies , EP A (USEPA , 2006 ) conclude dthat th e NOAE L fo r

developmenta l toxicit y i n rat an drabbi t wa s 1 0 mg/kg/day . Atrazin e ha dn o effect s o n embry o toxicity , feta l malformation , o r feta l developmen t

i n a two-generatio n reproductio n stud y conducte di n rat s at feedin g level s o f

0 , 10 , 5 0 an d50 0 ppm . Atrazin e doe s no t

caus e an y impairmen t

i n reproductiv e performanc e i n rat s fe dat dietary level s u p t o 50 0 pp mfo r tw o consecutiv e generations . Th e parenta l an dfeta l n o observe deffec t

level s i n thi s stud y wer e 5 0 pp m(3. 5 an d3. 8 mg/kg/day fo r male s an d females , respectively ) base dupo n reduce dfoo dconsumptio n an dbod y weigh t gai n at 50 0 

### ppm . Repor t Number : TK002342 2 Pag e 3 7 o f 5 8TABL E 2 EP A Guidelin e Reproductiv e an dDevelopmenta l Toxicit y Studie s o n Atrazin e Technica l Stud y Typ e Ra t Reproductio n (Mainier o e t al. , 1987 ) Rat Teratolog y (Giknis , 1989 ) Ra t Teratolog y (Infurna , 1984 ) Rabbi t Teratolog y (Arthur , 1984 ) MRI D Numbe r 4043130 3 4106520 1 0014300 8 0014300 6 Targe t

Organ(s ) Non e indicate d Non e indicate d Non e indicate d Non e indicate d MT D

(rag/kg ) 2 5 10 0 7 0 7 5 Develop . NOE L

(mg/kg ) 3.7 8 2 5 1 0 5 Materna l

NOE L (mg/kg ) 3.7 8 2 5 1 0 5 

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

r 1 assay s s o th e protoco l followe di n thes e case s wa s essentiall y identical t o th e fina l OPPT S 89 0 Serie s guidelin e study . I n thre e o f th e assays , atrazin e wa s teste db y eithe r substantiall y simila

r method s (E Ran duterotrophic ) o r i n th e cas e o f

th e castrat e mal e rat followe dth e standar dHershberge r protocol . Th e amphibia n stud y conducte do n atrazin e goe s beyon dth e requirement s o f ascreenin g study an di s mor e aptl y viewe da s amode l fo r Tie r 2 testin g i n amphibians . I n addition , atrazin e ha s bee n teste dvi ath e standardhighe r tie r roden t studie s require dfo r

registratio n whic h measur e anumbe r o f additional endpoints , whic h ar e functionall y equivalen t t o th e Tie r

1 assay s wit h numerou s overlappin g endpoint s evaluated . I n conclusion , atrazin e ha s bee n thoroughl y teste dfo r al l o f th e endpoint s covere db y Tie r 1 assay s o f th e EDSP , an dth e atrazin e consortiu mreques t waiver s fo r

al l o f th e Tie r 1 assay s i n th e EDS P tes t

order .

TK0023422-AtrazineEDSPAssessment.do c sbh : 4/6/1 0 ve r 1. 1

Report Number : TK002342 2 Pag e 3 8 o f 5 8

### 6. 0 REFERENCE S

Arthu r A . 1984 . Segmen t

I I Teratolog y Stud y i n Rabbits : Toxicology/Patholog y Report No . 68-84 . Unpublishe dstud y prepare db y Ciba-Geig y Corp . 22 0 p . MRI D 00143006 . Ashb y J , Tinwel l H . 2002 . Th e Effect s o f Atrazin e o n th e Sexua

l Maturatio n o f

Femal e Alderle y Park-Wistar

an dSprague-Dawle y Rats : Fina l Report : La b Projec t Number : 1775

- 02 . Unpublishe dstud y prepare db y Centra l Toxicolog y Laboratory . 3 2 p . MRI D 45722401 . Ashb y J . 2004 . Atrazine : Studie s o n Atrazin e Usin g th e Hershberge r Castrate dRat

Assa y fo r Antiandrogens . Centra l Toxicolog y Laboratory , Manchester , UK . Report No : CTL/026222/ GEN/REPT . 05.11.2004 . Balague r P , Francoi s F , Comunal e F , Fene t H , Boussiou x A-M , Pon s M , Nicola s J-C , Casella s C . 1999 . Reporter cell line s to study the estrogeni c effect s o f xenoestrogens . Sci . Total Environ . 233:47-56 . Battelle . 2005 . Draf

t Fina l Report

W A 2-2 9 o n Multi-chemica l Evaluatio n o f th e Short-term Reproductio n Assa y wit h th e Fathea dMinnow . Report t o U.S . Environmenta l Protectio n Agency , Endocrin e Disrupte r Screenin g Program . http://www.epa.gov/oscpmont/oscpendo/pubs/edmvac/draf t fis h repr o multichemical.pd f Blai r RM , Fan g H , Branha mWS , Has s BS , Dia l SL , Molan dCL , Ton g W , Sh i L , Perkin s R , Sheehan DM . 2000 . Th e Estroge n Recepto r Relativ e Bindin g Affinitie s o f 18 8 Natura l an d Xenochemicals : Structura l Diversity o f Ligands . Toxicol . Sci . 54 : 138-153 . Bringol f RB , Belde n JB , Summerfel t RC . 2004 . Effect s o f

Atrazin e o n Fathea dMinno w i n a Short-TermReproductio n Assay . Environmenta l Toxicolog y an dChemistry 23(4) : 1019

- 

### 1025 . Conno r K , Howel l J , Che n I , Li u H , Berhan e K , Sciarrett aC , Saf e S , Zacharewski T . 1996 . Failur e o f Chloro-s-Triazine-Derive dCompound s t o Induc e Estroge n Receptor-Mediate d Response s i n viv o an di n vitro . Fundam . Appl . Toxicol . 30:93-101 . Dionn e E . 1992 . Chroni c Toxicity t o th e Fathea dMinno w (Pimephale s promelas ) Durin g a Ful l Lifecycl e Exposure . Atrazin e Technical . Springborn Laboratorie s Inc . Report 92-7

- 4324 . MRI D 42547103 . Fan g H , Ton g W , Branha mWS , Molan dCL , Dia l SL , Hon g H , Xi e Q , Perkin s R , Owen s W , Sheeha n DM . 2003 . Stud y o f 20 2 Natural , Synthetic , an dEnvironmenta l Chemical s fo r Bindin g to th e Androge n Receptor . Chem . Res . Toxicol . 16 . 1338-1358 .

Report Number : TK002342 2 Pag e 3 9 o f 5 8

### FIFR A SAP . 2007 . Report N o 2007-08 ; FIFR A Scientifi c Advisor y Pane l Meeting , Octobe r 9-11 , 200 7 Arlingto n VA ; A Se t o f Scientifi c Issue s Bein g Considere db y th e Environmenta l Protectio n Agenc y Regarding : Th e Potentia l fo r Atrazin e t o Affec t

Amphibia n Gonadal Development ; 2007 . Gikni s M . 1989 . A Teratolog y (Segmen t II ) Stud y i n Rats : Atrazin e Technical : Laboratory Stud y No . 882049 . Unpublishe dstud y prepare db y Ciba-Geig y Corp . 35 7 p . MRI D 41065201 . Hinfra y N , Porche r

JM , Brio n F . 2006 . Inhibitio n o f Rainbo w Trou t

(Oncorhynchu s mykiss ) P45 0 Aromatas e Activitie s i n Bai n an dOvarian Microsome s b y Variou s Environmental Substances . Comp . Biochem . Physiol . C . Toxicol . Pharmacol . 144(3):252-62 . Holloway , AC , Anger , DA , Crankshaw , DJ , Wu , M , Foster , WG . 2008 . Atrazine-Induce d Change s i n Aromatas e Activit y i n Estroge n Sensitiv e Targe t Tissues . J . Appl . Toxicol . 28(3):260-70 . Hosme r A , Springer

T , Kloa s W , Lut z I , Wol f

J , Holde n L . 2007 . Respons e o f Larva l Xenopu s laevi s t o Atrazin e Exposure : Assessmen t o f Metamorphosi s an dGonada l Morphology : Overvie w an dCompariso n o f Studies . Syngent aReport submitte dt o USEPA , MRI D 4715 3 501 . Infurn aR . 1984 . A Teratolog y Stud y o f Atrazin e Technica l i n Charle s Rive r Rats : Toxicology/

Patholog y Report

No . 60-84 . Unpublishe dstud y prepare db y Ciba-Geig y Corp . 26 9 p . MRI D 00143008 . Kloa s W , Lut z I , Springe r

T , Kruege r

H , Wol f J , Holde n L , Hosme r

A . 2009 . Doe s Atrazin e Influenc e Larva l Developmen t an dSexual Differentiatio n i n Xenopu s laevis ! Toxicol . Sci . 107(2) , 376-384 . Kojim aH , Katsur aE , Takeuch i S , Niiyam aK , Kobayash i K . 2004 . Screenin g fo r Estroge n an dAndroge n Recepto r

Activitie s i n 20 0 Pesticide s b y i n vitr o Reporte r Gen e Assay s usin g Chines e Hamster

Ovar y Cells . Environ . Healt

h Perspect . 112(5):524-31 . Law s SC , Ferrel l JM , Stoker TE , Schmi dJ , Coope r

RL . 2000 . Th e Effect s o f

Atrazin e o n Femal e Wistar

Rats : A n Evaluatio n o f

th e Protoco l fo r

Assessin g Puberta l Developmen t an d Thyroi dFunction . Toxicol . Sci . 58 : 366-376 . Lut z I , Kloa s W , Springe r

T , Holde n L , Wol f

J , Kruege r

O , Hosme r

A . 2008 . Development , Standardizatio n an dRefinemen t o f Procedure s fo r

Evaluatin g Effect s o f

Endocrin e Activ e Compound s o n Developmen t an dSexual Differentiatio n o f Xenopu s laevis . Anal . Bioanal . Chem . 390(8):2031-48 .

Report Number : TK002342 2 Pag e 4 0 o f 5 8

### Mace k KJ , Buxto n KS , Saute r

S , Gnilk aS , Dean JW . 1976 . Chroni c Toxicity o f Atrazin e t o Selecte dAquati c Invertebrate s an dFishes . USEPA , Off . Res . Dev. , Environ . Res . Lab . Duluth , MN . EPA-600/3-76-047 . 4 9 p . MRI D 000243-77 . Mainier o J , Yourenef

f M , Gikni s M . 1987 . Two-generatio n Reproductio n Stud y i n Rats : Atrazin e Technical : Laboratory Stud y No . 852063 . Unpublishe dstud y prepare db y Ciba

Geig y Corp . 1395p . MRI D 40431303 . OEC D 2009 . OEC D 229 : Fis h Shor t TermReproductio n Assay . Paris , Organisatio n fo r Economi c Co-operatio n an dDevelopment : 1-40 . Pfei l R , Dellarc o V , Davie s L . 2007 . Atrazine . In : Pesticide Residue s i n Food—2007 : Toxicologica

l Evaluations . FAO/WHO , 37-138 . Sanderso n JT , Letche r

R , Henewee r

M , Gies y JP , van de n Ber g M . 2001 . Effect s o f Chloro

s-Triazin e Herbicide s an dMetabolite s o n Aromatas e Activit y i n Variou s Huma n Cel l Line s an do n Vitellogeni n Productio n i n Mal e Car p Hepatocytes . Environ . Healt h Perspect . 109(10) : 1027-1031 . Solomo n KR , Car

r JA , D u Pree z LH , Gies y JP , Kendal l RJ , Smith EE , Van De r Kraa k GJ . 2008 . 'Effect s o f

Atrazin e o n Fish , Amphibians , an dAquati c Reptiles : A Critica l Review' , Critical Review s i n Toxicology , 38:9 , 721-772 . DOI : 10.1080/10408440802116496 . Stoke r TE , Law s SC , Guidic i DL , Coope r RL . 2000 . Th e Effec t

o f Atrazin e o n Puberty i n Mal e Wista

r Rats : A n Evaluatio n i n th e Protoco l fo r th e Assessmen t

o f Puberta l Developmen t

an dThyroi dFunction . Toxicol . Sci . 58 : 50-59 . Tennan t MK , Hil l DS , Eldridg e JC , Wetze l LT , Breckenridg e CB , Steven s JT . 1994a . Possibl e Antiestrogeni c Propertie s o f Chloro-s-Triazine s i n Rat

Uterus . Toxicol . Environ . Healt h 43(2):183-96 . MRI D 43598617 . Tennan t MK , Hil l DS , Eldridg e JC , Wetze l LT , Breckenridg e CB , Steven s JT . 1994b . Chloro-s-Triazin e Antagonis mo f Estroge n Action : Limite dInteractio n wit h Estroge n Recepto r Binding . J . Toxicol . Environ . Healt h 43:197-21 2 . MRI D 43598618 . USEPA . 2006 . Finalizatio n o f Atrazin e IRED , an dCompletio n o f Toleranc e Reassessmen t an dReregistratio n Eligibilit y Proces s (Apri l 6 , 2006) . http://www.epa.gov/oppsrr d1 /REDs/atrazin e combine ddocs.pd f USEPA . 2007 . Whit e Paper o n th e Potentia l fo r

Atrazin e t o Affec t

Amphibia n Gonadal Development ; Submitte dt o th e FIFR A Scientifi c Advisory Pane l fo r Revie w an dComment ; Octobe r 9-12 , 2007 . http://www.epa.gov/scipolv/sap/meetings/2007/october/200 7 amphibia n whit e paper.pd f

Report Number : TK002342 2 Pag e 4 1 o f 5 8

### USEPA . 2008a . Integrate dSummar y Report fo r th e Validatio n o f

an Androge n Recepto r Bindin g Assa y a s aPotentia l Scree n i n th e Endocrin e Disrupte r Screenin g Program . Revisio n o f

February 18 , 2008 . USEPA . 2008b . Integrate dSummary Report o n Aromatase . Revisio n o f Februar y 18 , 2008 . USEPA . 2008 c Multi-Laboratory Validatio n o f th e H295 RSteroidogenesi s Assay t o Identif y Modulator s o f

Testosteron e an dEstradio l Production , Entri x Report Prepare db y M . Hecker , J.P . Giesy , an dG . Timm . February , 2008 . USEPA . 2009 . Integrate dSummary Report fo r Validatio n o f

an Estroge n Recepto r

Assay 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 ProgramTier

1 Battery . March , 2009 . Yamasak i K , Sawak

i M , Nod aS , Muro i T , Takakur aS , Mitom aH , Sakamot o M , Naka i M , Yakab e Y . 2004 . Compariso n o f th e Hershberge r

Assay an dAndroge n Recepto r Bindin g Assa y o f Twelv e Chemicals . Toxicology . 195(2-3) : 177-186 . Y i KD , Breckenridg e CB , Simpkin s JW . 2010a . Cytotoxicity o f Atrazin e an dit s Metabolite s i n vitro . J Toxicol . Environ . Health.?ar t A . (I n press) . MRI D 47972810 . Y i KD , Simpkin s JW , Breckenridg e CB . 2010b . Evaluatio n o f th e Potentia l Mechanism s o f Action s Underlyin g th e Effec t o f Triazine s o n Aromatas e Expressio n i n vitro : Relevanc e t o Ma n an dth e Environment . Revie w submitte dt o th e U S Environmenta l Protectio n Agenc y (MRI D 47972809 ) an dals o to Rev . Environ . Contam . Toxicol . (I n press) . MRI D 47972809 .

Report Number : TK002342 2 Pag e 4 2 o f 5 8

### APPENDICE S SECTIO N

Repor t Number : TK002342 2 Pag e 4 3 o f 5 8

### APPENDI X 1 Titl e Page s o f Cite dStudie s

Report Number : TK002342 2 Pag e 4 4 o f 5 8Ct§A-GEIG Y 

### Te 

# " in 9 Facility : 55 6 Morri s Avenu e

Summit , Ne w Jerse y0790 1 C.A.NO. : 66 9

SPONSOR : Agricultura l Divisio n

CIBA-GEIG Y Corporatio n P.O . Bo x 18300 Greensboro , Nort h Carolin a2741 9 TOXICOLOGY/PATHOLOG Y REPOR T NO. : 68-8 4 WIN . NO. : 83211 0 REPOR T DATE : Septembe r 18 , 198 4 STUD Y TITLE : Segmen t I I Teratolog y Stud y i n Rabbit s TES T ARTICLE : Atrazin e STUD Y DIRECTOR : A la n T . Arthur , Ph.D . MRI D 0014300 6 Classified: . Acceptabl e Guidelin e (4/1992 )

Th e subjec t nonclinica l laboratorystud ywa s conducte d an d reporte d i n complianc e wit h th e require ment s o f th e Goo d Laborator yPractic e regulation s (2 1 CF R 58 , 4 0 CF R 160,4 0 CF R 792 ) an d th e OEC D Guideline s fo rTestin g o f Chemicals . 

## AJU . Ala n T . Arthur , Ph.D . • StudyDirecto r Associat e Director , Reproductiv e and Geneti c Toxicolog

y Dat e

Rober t Katz , Ph.D . Directo r Chemica l Toxicolog y Dat e



### ^ Spono r / V D^fc e

i o



### Report Number : TK002342 2 Pag e 4 5 o f 5 8MRID : 4572240 1 VOLUM E O F O F SUBMISSIO N ATRAZINE : FINA L REPOR T

TITL E Th e Effect s o

f Atrazin e o n th e Sexua l Maturatio n o f Femal e Alderley Park-Wista r an d Sprague-Dawle y Rat s DATA REQUIREMEN T

. No t Applicabl e AUTHOR S Joh n Ashby , Ph.D . Hele n Tinwell , Ph.D . COMPLETIO N DAT E Jul y 3 , 200 2 PERFORMIN G LABORATOR Y Centra l Toxicolog y Laborator y Alderle y Park , Macclesfiel d Cheshir e (UK ) LABORATOR Y STUDY IDENTIFICATIO N Syngenta Numbe r1775-02 SUBMITTER/SPONSO R Syngent a Cro p Protection , Inc . 41 0 Swin g Roa d Post Offic e Bo

x18300 Greensboro , N C 2741 9 VOLUM E J

_O F J

_O F STUDY PAG E 1 O F 3 2



### Repor t Number : TK002342 2 Pag e 4 6 o f 5 8MRI D A254710 3 VOLUM E O F SUBMISSIO N ATRAZZM

E TECHNICA

L CHRONI C TOXICIT

Y T O TH E FATHEA

D MINNO

W (PIMEPHALE S FROMELAS ) DURIN G AFULL LIFE-CYCL

E EXPOSUR

E EP AGUIDELINE S NO . 72- 5
AUTHO R - EMIL

Y DIONN

E STUD Y COMPLETE D O

N- SEPTEMBE R 25 , 199 2 CONDUCTE D BY : SPRINGBOR

NLABORATORIES , INC . ENVIRONMENTA

L SCIENCE S DIVISIO N 79 0 MAI NSTREE T WAREHAM , MASSACHUSETT S 0257 1 LABORATOR

Y STUD Y NUMBE R - 92-7-432 4 VOLUME 1 O F 2 O F STUD Y PAG E 1 O F 43 9 INCLUDE S PAGE180

a AGRICULTURA

L DIVISIO

N CIBA-GEIG

Y CORPORATIO

N POS T OFFICE BO

X 1833 0 GREENSBORO , N

C 2741 9

R301CRM1007RS. 2 (MONBOEC . REPORT ] Report Number : TK002342 2 Pag e 4 7 o f 5 8

## -1
- MRI D 4106520 1 Classified : Acceptabl e Guidelin e (6/1998 ) VOLUM E O F O F SUBMISSIO N ATRAZIN E TECHNICA

L ATERATOLOG

Y (SEGMEN

T II ) STUD

Y I NRAT S EP AGUIDELINE S NO . 83- 3
AUTHO R - M.L.A . Gikni s STUD Y COMPLETE D O

N Februar y23 , 198 9 RESEARC H DEPARTMENT , PHARMACEUTICAL S DIVISIO N CIBA-GEIG

Y CORPORATIO

N 55 6 MORRI S AVENUE SUMMIT , NE W JERSE Y 0790 1

LABORATOR

Y STUD Y NUMBE R 88204 9
VOLUM E 1 O F 1 O F STUD Y PAGE 1 O F 35 7
AGRICULTURA

L DIVISIO

N CIBA-GEIG

Y CORPORATIO

N POST OFFIC E BO

X 1830 0 GREENSBORO , N

C 2741 9

K8/32/ 1 (MI

N8820A9 ) 

### Report Number : TK002342 2 Pag e 4 8 o f 5 8MRI D 4715350 1



# syngenta
Atrazin e Respons e o f

Larva l Xenopu s laevi s t o Atrazin e Exposure : Assessmen t o f Metamorphosi s an dGonada l Morphology : Overvie w an dCompariso n o f Studie s Conducte db y Wildlif e Internationa l Ltd . Easto n M D

an d Th e Leibniz-Institut e o f Freshwate r

Ecolog y an dInlan dFisherie s (IGB ) Berli n German y Final Report DATA REQUIREMENT : AUTHOR(S) : STUD Y COMPLETIO N DATE : PERFORMIN G LABORATORIES : Wildlif e International , Ltd . 859 8 Commerc e Driv e Easton , M D 2160 1 US A Experimental Patholog y Laboratories , Inc . 4560 0 Terminal Driv e Sterling , V A 2016 6 US A LABORATOR Y PROJEC T ID : SPONSOR :

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# CIBA-GEIG Y C . A . No. : 71 2 Testin g Facility : Pharmaceutical s Divisio n

CIBA-GEIG Y Corporatio n 55 6 Morri s Avenu e Summit . Ne w Jerse y0790 1 SPONSOR : Agricultura l Division

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Y Corporatio n P.O . Bo x 1830 0 Greensboro , Nort h Carolin a 2741 9 Ref . No. : 2-001-2 4 MRI D 0014300 8 Classified : Supplementary(9/1984 )

TOXICOLOGY/PATHOLOG Y REPOR T NO. : 60-8 4 MIN . NO. : 83210 9 REPOR T DATE : Septembe r 18 , 198 4 STUD Y TITLE : A Teratolog y Stud y o f Atrazin e Technica l i n Charle s Rive r Rat s TES T ARTICLE : Atrazin e Technica l STUD Y DIRECTOR : Rober t N . Infuma , Ph.D . Th e subjec t nonclinica l laborator ystud ywa s conducte d an d reporte d i n complianc e wit h th e require ment s o f th e Goo d Laborator y Practic e regulation s (2 1 CF R 58 , 4 0 CF R 160 , 4 0 CF R 792 ) an d th e OEC D Guideline s fo rTestin g o f Chemicals .
Rober t N . Infurna , Ph.D . Stud y Directo r Manager , Reproductiv e Toxicolog y Dat e

Ala n T . Arthur , Ph.D . Associat e Directo r Reproductiv e an dGeneti c Toxicolog y Dat e

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VOLUM E O F O F SUBMISSIO N ATRAZIN E TECHNICA

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L NAM E ATRAZINE AND SIMAZIN E POSSIBL E ANTIESTROGENI C PROPERTIE S O FCHLORO-s-TRIAZINE S I N RAT UTERU S

DAT A REQUIREMENT S NO T APPLICABL

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A CONFIDENTIALITY CLAI M 

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L NAM E CHLOROTRIAZINE S CHLORO-s-TRIAZIN

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Report Number : TK002342 2 Pag e 5 5 o f 5 8STATEMENT O F N O DATA COMFTDEyqiALITY CLAI M 

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# syngent a MRI D 4797281 0

Atrazin e Cytotoxicity o f

Atrazin e an dit s Metabolite s i n Vitr o Fina l Repor t

DAT A REQUIREMENT(S) : No t Applicabl e AUTHOR(S) : Charle s B . Breckenridg e Ku n Do n Y i Jame s W . Simpkin s STUD Y COMPLETIO N DATE : Februar y 2 , 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 : Repor t Number : TK002554 4 Tas k Number : TK0025544 SUBMITTER/SPONSOR : Syngent aCro p Protection , Inc . 41 0 Swin g Road Pos t Offic e Bo x 18300 Greensboro , N C 27419-830 0 US A VOLUM E J

_O F J

_O F STUD Y PAGE 1 O F 3 1

Report Number : TK002342 2 Pag e 5 7 o f 5 8VOLUM E O FO FSUBMISSIO N



# syngenta MRI D 4797280 9

Atrazin e Evaluatio n o f

th e Potential Mechanism s o f Action s Underlyin g th e Effec t

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Environmen t Summar y
DATA REQUIREMENT(S) : No t Applicabl e AUTHOR(S) : Charle s B . Breckenridg e Ku n Do n Y i Jame s W . Simpkin s COMPLETIO N DATE : February 2 , 201 0 PERFORMIN G LABORATORY : Syngent aCro p Protection , Inc . 41 0 Swin g Road Greensboro , N C 2740 9 US A LABORATORY PROJEC T ID : Repor t Number : TK002552 9 Tas k Number : TK002552 9 SUBMITTER/SPONSOR : Syngent aCro p Protection , Inc . 41 0 Swin g Road Pos t Offic e Bo x 18300 Greensboro , N C 27419-830 0 US A NOTE : Thi s repor t

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_O FSTUD Y PAG E 1 O F 6 3 Report Number : TK002342 2 Pag e 5 8 o f 5 8