### Makhteshi m Ago n of Nort h America , Inc .

• •

- • • • L • C » . . 

# M AN A Vi aCourie r Deliver y Marc h11,201 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 (Sout hBldg ) On e Potoma c Yard , Roo mS-490 0 277 7 Sout hCrysta l Driv e Arlington , V A 22202-450 1 ••••• • "••'• J Attention : Ms . Melani e Bisco e

Chemica l Revie w Manage r - Endosulfa n Subject . EDS PTes t

Orde r (EDS P-079401-102 ) Submissio no f

90-da y respons e fo r

Endosulfan , Thione x Technica l (EP A Reg . No . 11678-5 ) Dea r Ms . Biscoe : Herewith , Makhteshim-Aga n o f NorthAmerica , Inc . (MANA ) o n behal f o f Makhteshi m Chemica l Work s Ltd . (MCW ) i s submittin g th e Initia l o r 90-day EDS PTes t

Orde r respons e fo r endosulfa n(Thione x Technical , EP A Reg . No . H678-5 ; Order/DC I # : EDSP

- 079401-102) . Ou r respons e include s th e followin g documents :

• Transmitta l Documen t • Execute dInitia l Respons e Formfo r Individua l Orde r

Recipient s • Volum e 1 o f

2 ( 3 copies) : Respons e t o th e U.S . Environmenta l Protectio n Agenc y Testin g Orde r unde r th e Endocrin e Disrupto r

Screenin g Progra m(EDSP) : Discussio n o f Othe r Scientificall y Relevan t

Informatio n (OSRI ) fo r

Endosulfa n • Volum e 2 o f

2 ( 3 copies) : Supplementa l Dat afo r Makhteshi mChemica l Works , Ltd . - Endosulfa nEndocrine Disruptio n Screenin g Progra m I n th e attache drespons e (Vol . 1 o f 2) , w e identifie dtw o source s regardin g th e Othe r Scientificall y Relevan t Informatio n(OSRI ) fo r

endosulfan : 1 ) th e peer-reviewed , publishe dscientifi c literature ; an d2 ) th e endosulfa npesticid e registratio n studies . I n orde r t o ensur e tha t

onl y reliable , scientificall y vali dOSR I wer e include di n thi s assessment , eac hstud y wa s analyze dusin g th e scientifi c metho dse t

fort hb y Klimisc he t

al . (1997) , whic hi s consisten t wit hth e proces s outline d b y EP A i n 200 3 i n it s documen t entitle dEP A Assessment Factor s Guidanc e (2003) . Thus , th e proces s o f

selectin g studie s fo r

endosulfa n OSR I include didentifyin g areliabilit y indicato r fo r eac hstudy . A s outline db y Klimisc he t al . (1997) , reliabilit y ca n b e differentiate dint o fou r categories : Categor y 1 : "reliabl e withou t restriction" ; categor y 2 : "reliabl e wit hrestrictions" ; 451 5 Fall s o

f Neus e Road , Suit e 300 , Raleigh , N C 2760 9 Tel : 919-256-930 5 Fax:919-256-930 8

### categor

y 3 : "not reliable" ; an dcategor

y 4 : "no t assignable" . I n our

evaluatio n o f

endosulfa n OSRI , onl y studie s tha t

coul db e give n acategory ratin g o f eithe r 1 o r 2 wer e used . Thi s means , that onl y studie s that

wer e GLP-compliant

o r comparabl e to aGLP-compliant study protoco l wer

e give n a ratin g o f category 1 an dstudie s give n acategor y 2 rating were one s that , althoug hno t performe d accordin g t o GLP , wer e considere dt o b e well-documente dan dscientificall y acceptabl e studies . Th e studie s an dpublication s meetin g thos e criteri aar e enclose di n Volum e 2 o f 2 o f thi s submission . Th e enclose drespons e i s adetaile ddescriptio n o f availabl e endosulfa n OSR I that i s use dt o suppor t waiver s fo r

al l eleve n o f

th e EDS PTie

r 1 screenin g assays . Th e endosulfa n OSR I i s very extensive , includin g informatio n specifi c t o assessin g th e potentia l fo r estrogen , androge n an d thyroi dhormon e syste meffect s a s wel l a s th e effect s o f endosulfa n o n specifi c endpoint s (i.e. , potentia l mode s of

action) . MCWis seekin g waiver s for performanc e of

all eleve n of

the EDS P Tie r 1 studies : 890.110 0 (Amphibia n Metamorphosis) ; 890.115 0 (Androge n Recepto r Binding) ; 890.120 0 (Aromatase) ; 890.125 0 (Estroge n Recepto r Binding) ; 890.130 0 (Estroge n Recepto r Transcriptiona l Activation) ; 890.135 0 (Fis hShort-TermReproductio n Assay) ; 890.140 0 (Hershberger

assay) ; 890.145 0 (Puberta l Femal e Ra

t Development) ; an d890.150 0 (Puberta l Mal e Rat Development) ; 890.155 0 (Steroidogenesis) ; an d890.160 0 (Uterotrophi c assay) . I f yo u hav e an y question s o r nee dfurthe r

information , pleas e contac t m e at 610-793-322 2 o r

sen d m e an e-mai l at

bertvolger@comcast.net . Sincerely ,

Ber t Volger , Ph.D . Consultan t

fo r MAN A

cc : L . Pruett

- MAN A

## TRANSMIT!AL DOCUMEN T EDS P TES T ORDE R ENDOSULFA N INITIA LRESPONS E

SUBMITTE R Makhteshi mChemica

l Work s Ltd . (EP A Compan y No . 11678 ) c/ o Makhteshi mAga n o f

Nort hAmerica , Inc . 451 5 Fall s o f

Neus e Rd. , Suit e 30 0 Raleig hN C 2760 9 

### DAT E Marc h11 , 201 0 

### TES T ORDE RNUMBE R EDSP-079401-10 2


### DOCUMENT S

• Transmitta l Documen t

• Execute dInitial Respons e Formfo r Individual Order

Recipient s

• Volum e 1 o f

2 ( 3 copies) : Respons e t o th e U.S . Environmenta l Protectio n Agenc y Testin g Orde r unde r th e Endocrin e Disrupte r

Screenin g Progra m(EDSP) : Discussio n o f Othe r Scientificall y Relevan t

Informatio n (OSRI ) fo r

Endosulfa n

• Volum e 2 o f

2 ( 3 copies) : Supplementa l Dat afo r Makhteshi mChemica l Works , Ltd . - Endosulfan Endocrin e Disruptio n Screenin g ProgramTITL E Respons e t o th e U.S . Environmenta l Protectio n Agenc y Testin g Orde r unde r th e Endocrin e Disrupte r Screenin g Progra m(EDSP) : Discussio n o f

Othe r Scientificall y Relevan t

Informatio n (OSRI ) fo r Endosulfa n DAT A REQUIREMENT S

N/ A STUD Y AUTHOR S Laur aM . Plunkett , Ph.D. , DAB T Integrativ e Biostrategies , LL C Houston , Texa s Pramil aSingh , Ph.D , DAB T Corporat e Toxicologist , MAN A Raleigh , N C STUD Y COMPLETIO N DAT E Marc h11,201 0 STUD Y NO . MA-END-EDSP-2010031 3 SUBMITTER Makhteshi mChemica l Works , Ltd . Beer Shev a84100 , Israe l c/o Makhteshi mAga n o f

NorthAmerica , Inc . 451 5 Fall s ofNeus e Road , Suit e 30 0 Raleigh , N C 2760 9 VOLUM E1 O F2 Pag e 1 o f 6 4 • - • C ( , • J L . o o •Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3

STATEMEN T O FN O DAT A CONFIDENTIALIT Y CLAIM S N o clai mo f

confidentialit y i s mad e fo r an y informatio n containe di n thi s 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 1 0 (d ) (1 ) (A) , (B ) o r (C) . Thi s statemen t

override s an y confidentialit y marking s withi n thi s study . Company : Makhteshi mChemica l Works , Ltd . Beer Shev a84100 , Israe l Compan y Representative :

Bert Volger , Authorize dRepresentativ e Makhteshi mChemica l Works , Ltd .

Date : Marc h11,201 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3

GOO D LABORATOR Y PRACTIC E COMPLIANC E STATEMEN T Th e materia l presente di n thi s report i s descriptiv e i n natur e and , a s such , i s no t subjec t t o th e Goo dlaboratory Practic e regulation s a s se t fort hi n 4 0 CF R160 .

Author :

Laur aM . Plunkett , Ph . D. , DABT . Date : Marc h11,201 0

Submitter/Sponsor :

Bert Volge r Authorize dRepresentativ e Makhteshi mChemica l Works , Ltd . Date : Marc h11,201 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3

Tabl e o f Content s I . Introductio n 5 II . Backgroun d5 III. . Discussio n o f

Endosulfa n OSR I 1 1 A . Directl y o r

Functionall y Equivalen t

Endosulfa n OSR I Identifie di n th e Publishe d Literatur e 2 0 B . Directl y o r

Functionall y Equivalen t

Endosulfa n OSR I Identifie di n Endosulfa n Registratio n Studies.... . 3 6 IV . MAN A Seek s aWaive r fo r Al l o f th e Tie r 1 EDS PAssay s tha t

Scree n fo r

Endocrin e Disruptio n Potentia l o f

Endosulfa n 4 8 A . MAN A Seek s aWaive r fo r Performanc e o f

Bot hTie r

1 I n Vitr o Assay s fo r Estroge n Activit y (Estroge n Recepto r Bindin g 890.125 0 an dEstroge n Recepto r Transcriptiona l Activatio n 890.1300 ) 5 0 B . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 I n Vitr o Assay s fo r

Androge n Activit y (Androge n Recepto r Bindin g 890.115 0 an dAromatas e Activit y 890.1200)...5 1 C . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Steroidogenesi s Assa y (890.1550 ) 5 2 D . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Amphibia n Metamorphosi s Assa y (890.1100 ) 5 2 E . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Femal e Puberta l Assa y (890.1450 ) 5 4 F . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Mal e Puberta l Assa y (890.1500 ) 5 5 G . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Uterotrophi c Assa y (890.1600 ) 5 6 H . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Hershberge r

Assa y (890.1400 ) 5 7 I . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Fis hShort-Ter mReproductio n Assa y (890.1350 ) 5 8 V . Overal l Summar y 5 8 VI . Reference s Cite d6 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3

I . Introductio n O n Decembe r

3 , 2009 , th e U.S . Environmenta l Protectio n Agenc y (EPA ) issue da Testin g Orde r

(TO ) fo r endosulfa n (CA S #115-29-7 ) relate dt o testin g unde r th e EP A Endocrin e Disrupte r

Screenin g Progra m(EDSP) . I n th e TO , EP A ha s require d submissio n o f

EDS PTie r

1 testin g fo r

endosulfan . Th e Tie r

1 test s liste dfo r

submissio n include : 1 ) Amphibia n Metamorphosi s assay (890.1100) ; 2 ) Androge n Recepto r Bindin g assay (rat prostate ) (890.1150) ; 3 ) Aromatas e assay (huma n recombinant ) (890.1200) ; 4 ) Estroge n Recepto r

Bindin g assa y (890.1250) ; 5 ) Estroge n Recepto r Transcriptiona l Activatio nassay (huma n cel l lin e HeLa-9903)(890.1300) ; 6 ) Fis hShort-ter m Reproductio n assay (890.1350) ; 7 ) Hershberge r assa y (890.1400) ; 8 ) Femal e Puberta l assay (rat ) (890.1450) ; 9 ) Mal e Puberta l assay (rat ) (890.1500) ; 10 ) Steroidogenesi s assa y (huma n cel l lin e H295R ) (890.1550) ; an d11 ) Uterotrophi c assay (890.1600) . Thi s documen t i s arespons e t o th e TO , an dha s bee n prepare db y Makhteshim-Aga n o f North Americ a(MANA ) o n behal f o f Makhteshi mChemical s Work s (MCW) . Thi s respons e 1 ) outline s th e extensiv e dat aalread y availabl e o n th e endocrin e disruptio n potentia l o f endosulfan , referre dt o b y EP A a s "othe r scientificall y relevan t

information " o r

OSRI , 2 ) discusse s ho w availabl e OSR I provide s fo r screenin g fo r

endpoint s include di n EDS P Tie r 1 assays , an d3 ) provide s MANA' s respons e regardin g th e performanc e o f specifi c EDS PTie r

1 testing , includin g rational e fo r waiver s fo r

al l o f th e EDS PTie r

1 assays . II . Backgroun d EDS PTie r

1 assay s wer e designe dt o provid e dat arelevan t t o screenin g fo r endocrin e disruptio n potentia l o f chemical s b y identifyin g potentia l mode s o f actio n o f chemicals , specificall y th e effect s o f chemical s o n estrogen , androge n an dthyroi d hormon e linke dsystems . Th e potentia l mode s o f actio n screene dfo r

i n Tie r 1 assay s include , i n vitr o estroge n an dandroge n hormon e recepto r binding , i n vitr o effect s o n enzyme s involve di n hormon e synthesi s (e.g. , assay s directe dtoward s Steroidogenesi s an daromatas e activity) , a s wel l a s i n viv o test s o f mammalia n an dfis hreproductio n involvin g bot hmal e an dfemal e organisms . Befor e discussin g detail s o n th e EDS PTie r 1 studies , i t

i s usefu l t o revie w wha t ha s alread y bee n establishe dabou t chemically-induce dendocrin e toxicity . Ther e ar eMakhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3

well-know n biologica l effect s tha t ca n b e monitore di n viv o i n anima l testin g an duse dt o identif y compound s tha t

ar e anti-androgens , compound s tha t ar e estrogenic , an d compound s tha t

affec t thyroi dhormon e activity . Th e biologica l endpoint s ar e liste d belo w i n Tabl e 1 . I t

i s importan t

t o not e tha t

th e variou s endpoint s liste di n Tabl e 1 ar e apica l endpoint s tha t

ar e alread y routinel y monitore di n standar dtoxicolog y studie s tha t ar e part o f th e pesticid e registratio n process . Therefore , i n th e cas e o f chemical s suc ha s endosulfan , whic har e currentl y registere dfo r us e i n th e Unite dStates , ther e i s awealt h o f toxicolog y dat aavailabl e o n endpoint s tha t addres s endocrin e toxicity . Tabl e 1 Know n Chemically-Induce dEffect s o n Hormone-Relate dApica l Biologica l Endpoint s Anti-androge n Agent s Altere dtesti s weigh t

an d histopatholog y Altere depididymi s weigh t an d histopatholog y Altere dprostat e weigh t

an d histopatholog y Altere dprostat e weigh t

an d histopatholog y Altere dsemina l vesicl e weigh t an dhistopatholog y Decrease dfertilit y an dfecundit y Increase dtim e t o matin g Altere duterin e histopatholog y Altere dovaria n histopatholog y Estrogeni c Agent s Increase duterin e weigh t

an d altere dhistopatholog y Decrease dteste s weigh t

an d altere dhistopatholog y Decrease depididymi s weigh t an d altere dhistopatholog y Decrease dovary weigh t Altere dhistopatholog y o f vagina , testis , an dmammar y glan d Decrease dlitte r

siz e Decrease dfertilit y an dmatin g inde x Increase dimplantatio n los s Increase dgestatio n tim e an dtim e t o matin g Decrease dfecundit y Thyroi dHormon e Activ e Agent s Enlarge dthyroid , increase d thyroi dweigh t Altere dhormon e level s (T3/T 4 an dTSH ) Altere dthyroi dhistopatholog y Increase dlive r

weigh t Increase dbod y weigh t Altere dbrai n morpholog y (developmenta l study )

Wit hth e informatio n i n Tabl e 1 i n mind , i t i s clea r tha t an y discussio n o f endosulfa n OSR I wil l includ e adiscussio n o f th e guidelin e toxicolog y studie s tha t wer e 6Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3



### performe dto suppor

t registratio n o f

endosulfan a s wel l a s scientifi c studie s o n endosulfan publishe di n th e ope n literature . Befor e discussio n o f eac hendosulfa n OSRI stud y i n som e detail , i t

i s importan t

t o understan dwha t

specifi c endpoint s o f

endocrin e toxicit y ar e bein g monitore di n EDS PTie r 1 studies . Wit hthi s approach , th e OSR I discussio n ca n b e clearl y focuse do n th e issu e o f th e adequac y o f

existin g endosulfa n OSR I t o support a waive r o f certai n EDS PTie r

1 studies . A s aresult , Tabl e 2 belo w ha s bee n constructe dt o provid e a n organizationa l ai dtha t list s th e Tie r

1 EDS Passay s an deac htyp e o f endpoin t measure di n th e combine dassays . Th e endpoint s liste di n Tabl e 2 wer e foun db y revie w o f eac ho f th e EP A tes t guideline s (890.110 0 throug h890.1600) . I n som e cases , th e endpoin t liste di n Tabl e 2 ha s bee n generalize dt o includ e mor e tha n on e specifi c endpoint . Fo r example , th e endpoin t liste da s "developmental measures " include s avariety o f specifi c finding s i n i n viv o studie s includin g objectiv e measure s suc ha s tim e t o preputia l separation , tim e t o vagina l patenc y o r vaginal opening , tim e to ey e opening , an dmore subjectiv e measure s suc ha s behaviora l change s i n lactatio n o r nursin g behavior , o r

neurobehaviora l tes t

observations . Th e endpoin t

liste da s "reproductiv e organ " weigh t o r histopatholog y include s avariet y o f organs , whic hma y b e measure di n an y specie s an di s mean t t o includ e measure s relate dt o th e mammar y gland , uterus , ovaries , cervi x an dvagin ai n femal e animal s an d th e testes , epididymides , prostate , semina l vesicle , glan s penis , levato r ani

bulbocavernou s muscle , an dCowper' s gland s i n mal e animals . Th e endpoin t liste da s "hormon e synthesis " i s mean t t o encompas s endpoint s suc ha s enzym e activit y measures . Th e endpoin t

liste da s "sex hormones " include s measuremen t o f actua l level s o f vitellogenin , estradiol , testosterone , seru mluteinizin g hormone , an dfollicula r stimulatin g hormone . Finally , th e endpoin t liste da s "thyroi dhormones ' include s measuremen t o f level s o f thyroxin e (T4 ) an dthyroid-stimulatin g hormone . Finally , th e appearanc e o f "yes " throughou t

th e tabl e i n an y colum n o r ro w indicate s tha t

th e particula r

endpoin t i s measure di n th e particula r

EDS PTie r

1 assay , wherea s n o entr y o r a"blank box " i n th e tabl e indicate s tha t

th e endpoin t

i s no t measure di n tha t

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1 assay .O O o ( N i a . C/ 5 Q U Q

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### Inspectio n o f

th e endpoint s measure di n variou s EDS PTie r 1 assay s (Tabl e 2 ) reveal s ther e i s awid e variet y o f dat acollecte di n rat s (mammals) , an don e study eac hi n fis han damphibians . Th e amphibia n metamorphosi s assay , however , i s designe dt o b e a screenin g assay fo r

potentia l effect s o nthyroi dhormon e system s i nmammals , includin g human s (se e OPPT S 890.1100) . Th e variou s assay s cove r differen t stage s o f developmen t (e.g. , earl y development , peripuberta l period , adult) . Th e assay s scree n fo r effect s o n estrogen , androge n an dthyroi dhormon e systems . Th e endpoint s liste di n Table 2 wil l b e specificall y identifie di nOSR I availabl e fo r endosulfan . Befor e discussin g th e OSR I fo r endosulfa n specifically , i t i s importan t

t o differentiat e betwee n "direct " equivalenc e dat aversu s "functional " equivalenc e data . I n it s EDS PT O documents , EP A ha s indicate dthat companie s ca n submi t

OSR I dat atha t

i s either "directly equivalent " to dat agathere di n EDS P Tier 1 test s o r dat athat i s "functionall y equivalent " t o EDS PTie r 1 data . Therefore , directl y equivalen t dat awoul d b e dat acollecte do n th e exac t sam e endpoint s i n th e sam e assay s a s specifie dunde r EDS PTie r

1 , althoug hth e assay involve dmigh t diffe r slightl y i n term s o f

th e protoco l use do r th e condition s o f

th e assay , i.e. , dat agathere di n a n estroge n recepto r bindin g assay i n atissu e other

tha n rat uterin e cytoso l (OPPT S 890.1250) . Als o i t follow s that , functionall y equivalen t

refer s t o quality , reliabl e dat atha t provide s th e sam e essentia l endpoin t informatio n o n achemical , eve n i f method s an dprocedure s othe r tha n EDS P Tie r 1 assay s wer e use dt o obtai n th e dat a(i.e. , fertilit y dat agathere di n amulti

generatio n ra t

reproductiv e toxicit y study) . Us e o f functionall y equivalen t

endpoint s an d studie s i s allowe dunde r

th e concep t

o f OSRI an davoid s unnecessar y additiona l us e o f resource s an dsacrific e o f

animals , whil e ensurin g tha t sufficien t dat aar e availabl e t o evaluat e th e potentia l endocrin e toxicit y o f chemicals . I n orde r t o identif y OSR I fo r

endosulfan , tw o source s wer e examined : 1 ) th e peer reviewed , publishe dscientifi c literature ; an d2 ) th e endosulfa n pesticid e registratio n studies , h i orde r

t o ensur e tha t

onl y reliable , scientificall y vali dOSR I wer e include di n thi s evaluatio n fo r

endosulfan , eac hstud y wa s analyze dusin g th e scientifi c metho dse t fort hb y Klimisc he t

al . (1997) , whic hi s consistent withth e proces s outline db y EP A i n 200 3 i n it s documen t

entitle dEP A Assessmen t

Factor s Guidanc e (2003) . Thus , th e proces s o f selectin g studie s fo r

endosulfa n OSR I include didentifyin g areliabilit y

1 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3

indicato r fo r eac hstudy . A s outline db y Klimisc he t al . (1997) , reliabilit y ca n b e differentiate dint o fou r

categories a s follows , "reliabl e withou t restriction " o r

categor y 1 ; "reliabl e wit hrestrictions " o r categor y 2 ; "not

reliable " o r

categor y 3 ; an d"no t assignable " o r

category 4 . I n ou r

evaluatio n o f

endosulfa n OSRI , onl y studie s tha t coul d b e give n acategor y ratin g o f eithe r 1 o r 2 wer e use di n th e OSR I evaluatio n presente d here . Thi s means , tha t

onl y studie s tha t

wer e GLP-compliant ' o r comparabl e t o aGLP

complian t stud y protoco l wer e give n aratin g o f category 1 an dstudie s give n acategory 2 ratin g wer e one s that , althoug hno t performe daccordin g t o GLP , wer e considere dt o b e well-documente dan dscientificall y acceptabl e studies . Th e endosulfa n OSR I wa s evaluate db y scientist s wit htrainin g i n toxicolog y an dfamiliarit y wit hpesticid e registratio n an dEDS P testin g protocols . I t i s th e objectiv e o f

th e OSR I identificatio n proces s t o determin e whethe r equivalen t

OSR I exis t

tha t support th e waive r

o f a n EDS PTie r

1 study . Interpretatio n o f th e result s gathere di n specifi c OSR I studie s i s beyon dth e scop e o f thi s process . Th e followin g (Sectio n II I o f

thi s document ) i s adiscussio n o f th e OSR I fo r

endosulfa n tha t

i s eithe r directl y equivalen t

o r functionall y equivalen t

t o th e EDS PTie r

1 assay endpoint s liste di n Tabl e 2 . Th e discussio n i s focuse do n describin g EDS PTie r 1 endpoint s tha t

ar e adequatel y evaluate dthroug hus e o f OSRI . Followin g th e discussio n o f th e OSR I data , MAN A ha s provide daspecifi c rational e fo r wh y certai n EDS PTie r

1 assay s shoul db e waive dfo r

endosulfa n base do n th e availabl e OSR I (Sectio n I V o f thi s document) . III . Discussio n o f

Endosulfa n OSRI Ther e ar e OSRI fo r

endosulfa n tha t

ca n b e identifie da s bein g eithe r directl y o r functionall y equivalen t

t o dat acollecte di n EDS PTie r 1 testing . Table s 3 an d4 belo w provid e alistin g o f

eac hsourc e o f

OSR I fo r endosulfa n tha t

ha s bee n identified , an d organize s th e OSR I fro mtha t sourc e base do n th e EDS PTie r 1 endpoints , simila r

t o th e presentatio n of

the EDS P Tie r

1 endpoint s in Tabl e 2 above . Tabl e 3 provide s alistin g of th e mammalia n endosulfa n OSR I organize db y endpoint s fro mth e mammalia n Tie r I EDS P assays . Table 4 provide s alistin g o f non-mammalia n endosulfa n OSR I (i.e. , 1 "GLP-compliant " mean s tha t astud y wa s performe dunde r Goo dlaborator y Practic e (GLP ) a s outline db y EPA . 1 1Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3

studie s i n birds , amphibians , an dfish ) organize db y endpoint s fro mnon-mammalia n Tie r 1 EDS Passays . Som e endosulfa n publishe dOSRI appear multipl e time s becaus e the y contai n multipl e OSR I endpoints . Th e appearanc e o f "yes " throughou t

th e table s i n an y colum n o r ro w indicate s tha t

th e particula r

endpoin t i s measure di n th e particula r endosulfa n OSR I referenced , wherea s n o entr y o r a"blan k box " i n th e tabl e indicate s tha t th e endpoin t

i s no t measure di n th e particula r endosulfa n OSR I studie s referenced . Followin g Table s 3 an d4 , th e variou s OSR I ar e discusse di n mor e detail , includin g a discussio n o f

whethe r th e OSR I ar e eithe r

directl y equivalent , functionall y equivalent , o r both .



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### A . Directl y o r

Functionall y Equivalen t

Endosulfa n OSRI Identifie di n th e Publishe dLiteratur e A searc ho f

th e publishe dscientifi c literatur e identifie d2 1 differen t source s o f endosulfa nOSR I tha t

ar e directl y equivalen t

t o dat acollecte di n EDS P Tie r 1 assays . Thes e studie s include : Sot o e t al . (1994) ; Sot o e t

al . (1995) ; Wad e e t al . (1997) ; Ramamoorth y e t al . (1997) ; Arcar o e t

al . (1998);Wilso n an d LeBlan c (1998) ; Hunte r

e t al . (1999) ; Legle r

e t al . (1999) ; Won g an dMatsumur a (2000) ; Newbol de t

al . (2001) ; Anderso n e t

al . (2002) ; Fan g e t

al . (2003) ; Gormle y an dTeathe r

(2003) ; Grunfel dan dBonefeld-Jorgense n (2004) ; Bpnefeld

Jorgense n e t

al . (2005) ; Lemair e e t

al . (2006) ; Lavill e e t

al . (2006) ; Varayou de t al . (2008) ; Vinggaar de t

al . (2008) ; Judso n e t

al . (2009) ; an dBrunell i e t al . (2009) . Th e followin g i s adiscussio n o f th e relevan t

OSR I fro meac hstud y an da statemen t a s t o whethe r

th e dat aar e directl y o r

functionall y equivalen t

OSRI . 1 . Sot o e

t al , (1994 ) Revie w o f thi s stud y indicate s tha t

i t provide s reliable , non guidelin e OSR I dat afo r

screenin g fo r

th e estroge n recepto r

bindin g activit y o f endosulfan . I n thi s i n vitr o study , si x pesticide s compounds , includin g endosulfan , wer e individuall y teste dfo r estroge n recepto r

bindin g activit y usin g th e E-SCREE N assay . I n additio n t o testin g th e racemi c mixtur e o f endosulfan , bothendosulfan- aan dendosulfan- p wer e als o tested . Th e E

SCREE N i s astandar dassay employe di n man y laboratorie s an dmeasure s th e comparativ e potenc y o f individua l tes t

compound s t o induc e proliferatio no f

MCF- 7 cell s (huma n breas t cance r cells ) i n culture . Thi s cel l lin e expresse s estroge n receptors , i.e. , estroge n receptor- a(ERa) , an d stimulatio no f

estroge n receptor s b y compound s suc ha s estradio l lead s t o increase dproliferatio no f

MCF- 7 cells . Th e author s repor t us e o f 1?P

estradio l t o validat e th e responsivenes s o f thei r assay . 17p-estradio l i s th e positiv e contro l agen t

use di n EDS PTie r

1 assays . I n Tabl e 1 fro mth e paper , th e author s report

finding s fo r

th e teste dcompounds . Endosulfa n i s 2 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### reporte dt o sho w estroge n recepto r bindin g activit y at

dose s o f 1 0 an d2 5 u M an darelativ e potenc y a s compare dt o 17p-estradio l o f 0.0001 % o r activit y tha t

i s fou r order s o f magnitud e les s tha n th e positiv e contro l compound , indicatin g onl y wea k activit y i n thi s assay . Th e dat aprovide d ar e directl y equivalen t

t o th e typ e o f

dat acollecte di n th e EDS PTie r 1 890.125 0 assay an dar e reliabl e endosulfa n OSRI . 2 . Sot o et

a l (1995 ) Revie w o f thi s stud y indicate s tha t

i t provide s reliable , non guidelin e OSR I dat afo r

screenin g fo r

th e estroge n recepto r

bindin g activit y o f endosulfan . I n thi s i n vitr o stud y o f

avariety o f

compounds , includin g endosulfan , th e author s repor t result s o f testin g usin g th e E-SCREE N assay . Again , thi s i s astandar dassay employe di n man y laboratorie s an d measure s th e comparativ e potenc y o f individua l tes t

compound s t o induc e proliferatio n o f

MCF- 7 cell s i n culture . Th e author s report us e o f 170

- estradio l as wel l as avariet y of othe r know n estroge n recepto r

agonist s to validat e th e responsivenes s o f thei r assay . l?p-estradio l i s th e positiv e contro l agen t

use di n EDS PTie r

1 assays . I n Tabl e 6 fro mth e paper , th e author s report

finding s fro mth e E-SCREE N fo r endosulfa n an deac ho f

it s isomers , separately . Endosulfa n an dbothisomers ar e reporte dt o sho w estroge n recepto r

bindin g activit y at

dose s o f 1 0 u M tha t

represent s a relativ e potenc y a s compare dt o 17(3-estradio l o f 0.0001% , o r

activit y tha t i s fou r order s o f magnitud e les s tha n th e positiv e contro l compound , indicatin g onl y wea k

activit y i n thi s assay . Th e dat aprovide dar e directl y equivalen t to the typ e of

dat acollecte din the EDS PTie r 1 890.125 0 assay an dar e reliabl e endosulfa n OSRI . 3 . Wad e et

al . (1997 ) Revie w o f thi s stud y indicate s tha t

i t provide s reliable , non guidelin e OSR I dat afo r

screenin g fo r

th e estroge n recepto r

bindin g 2 1Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 potenc y o f

endosulfa n i n vitr o a s wel l a s providin g reliabl e i n viv o dat ao n uterin e weigh t

change s fro ma n Uterotrophi c assay i n endosulfan-expose d immatur e rats . I n th e i n vitr o portio n o f

th e study , ara t

uterin e estroge n recepto r bindin g displacemen t

assay wa s performed . Th e assay employe dtw o concentration s o f

ra t uterin e cytoso l t o provid e fo r determinatio n o f bindin g i n th e presenc e o f ahig hconcentratio n o f

estroge n receptor s (0.4 8 nM ) an dalo w concentratio n o f estroge n receptor s (0.06 5 nM) . Dat awer e collecte dusin g standar dcompetitiv e displacemen t bindin g technique s wit hdisplacemen t

o f [ 3 H]-labele d17(3-estradio l bein g quantified . Scatchar danalysi s wa s performe dan dth e ICs o value s fo r unlabele d17(3

- estradio l wer e 0.3 7 n M (hig hrecepto r concentration ) an d0.1 9 n M (lo w recepto r concentration) . I n contrast , endosulfa n exhibite donl y wea k estroge n recepto r

bindin g abilit y wit hbindin g inhibite donl y a t ados e o f 1 0 u M i n th e presenc e o f

hig hconcentration s o f

estroge n receptor s an dat 2 a s wel l a s 1 0 u M whe n teste dwit hlo w concentration s o f estroge n receptors. Next , endosulfa n wa s teste di n aMCF- 7 cel l proliferatio n assay . A s alread y discusse dabove , thi s i s astandar dassay fo r screenin g fo r estroge n recepto r

bindin g abilit y o f

chemicals . Endosulfa n increase dcel l proliferatio n i n thi s assay bu t onl y a t th e highes t

tes t dos e o f 5 0 uM . Th e author s characteriz e endosulfa n a s "weak " i n thi s assa y o f i n vitr o estrogeni c activity . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f

dat acollecte di n th e EDS PTie r 1 890.125 0 assay , an dar e reliabl e endosulfa n OSRI . Th e i n viv o portio n o f

th e stud y i s consisten t

i n desig n withth e Tie r 1 EDS P Uterotrophi c assay (890.1600) . Immatur e (1 8 day s old ) Sprague-Dawle y rat s wer e injecte dfo r thre e day s subcutaneousl y wit h3 mg/k g endosulfan . A s acontrol , anothe r grou p o f immatur e rat s wa s injecte dwit h0. 1 m g diethylstilbestero l (DBS) , aknow n estrogeni c agent . Th e positiv e contro l agent , DBS , produce da n 80 % increas e i n uterin e weigh t i n treate drat s whil e treatmen t wit hendosulfa n wa s no t

associate d 

### 2 2Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### wit han y increase duterin e weigh t i n th e immatur e rats . Th e dat aprovide d i nthi s stud y ar e directl y equivalen t t o th e typ e o f

dat acollecte di n th e EDS PTie r

1 890.160 0 assay , an dar e functionall y equivalen t t o th e dat a collecte di n th e 890.125 0 assay , an dar e reliabl e endosulfa n OSRI . 4 . Ramamoorth y e t

al . (1997 ) Revie w o f thi s stud y indicate s tha t

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e estroge n recepto r

transactivatio n potentia l o f

endosulfan . I n thi s study , ayeast-base destroge n recepto r assay wa s use dt o asses s th e transactivatio n potentia l o f endosulfan . Th e syste memploye da mous e estroge n recepto r

an dareporte r

plasmi dcontainin g asingl e ER E linke dt o th e Lac Zgene . Th e assay measure dp-galactosidas e activit y a s a n endpoin t o f estroge n recepto r

transactivation . Endosulfa n wa s on e o f fou r pesticide s tha t

wa s teste dindividually . Th e activit y o f endosulfa n wa s reporte drelativ e t o th e activit y o f th e positiv e contro l agen t

DES . Th e author s reporte dtha t

endosulfa n increase dgen e expressio n onl y a t ados e o f 2. 5 x 10- 5 M (2 5 uM ) an dtha t thi s leve l o f

activit y wa s fiv e order s o f magnitud e les s tha n DES . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f

dat acollecte di n th e EDS PTie r 1 890.130 0 assay , an dar e functionall y equivalen t t o th e dat acollecte di n th e 890.125 0 assay . Thus , thes e dat aals o provid e reliabl e endosulfa n OSRI . 5 . Arcar o et

al . (1998 ) Revie w o f thi s stud y indicate s tha t

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e estroge n recepto r

bindin g potentia l o f

endosulfan . Th e author s report

result s o f testin g i n vitr o usin g aMCF- 7 foc i assay an dacompetitiv e estroge n recepto r bindin g assay . Severa l compound s were teste dindividually , includin g endosulfan . Compound s wer e als o teste dfo r

potentia l synerg y i n thes e sam e assays . Th e MCF- 7 2 3Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 foc i assay i s simila r

t o th e basi c cel l proliferatio n assay use dt o scree n fo r estroge n recepto r

binding . I n th e assay , endosulfa n wa s teste dfo r it s abilit y t o induc e foc i (estrogeni c activity ) an dt o inhibi t foc i induce db y 1 n M 17(3-estradio l (anti-estrogeni c activity) . I n th e competitiv e bindin g assay , huma n recombinan t estroge n receptor s (1. 2 n M concentration ) wer e teste dusin g [ 3 H]-labele d17p-estradio l wit hstandar drecepto r bindin g methods . Endosulfa n increase dfoc i formatio n i n MCF- 7 cell s onl y a t concentration s greate r

tha n 1 0 u M an dth e author s reporte dn o detectabl e bindin g t o estroge n receptor s i n th e competitiv e bindin g assay . Therefore , consisten t wit hresult s o f

othe r studies , endosulfa n exhibite donl y wea k i n vitr o activity . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f dat acollecte di n th e EDS PTie r 1 890.125 0 assay , an dar e reliabl e endosulfa n OSRI . 6 . Wilso n an dLeBlan c (1998 ) Revie w o f

thi s stud y indicate s tha t

i t provide s reliable , non guideline , i n viv o dat afo r screenin g fo r

th e potentia l fo r

endosulfa n t o affec t steroi dhormon e homeostasi s i n vivo . I n thi s i n viv o study , mal e an dfemal e CD- I mic e tha t wer e sexuall y matur e wer e administere dendosulfa n i n th e die t fo r seve n day s (dose s = 0 , 3.8 , 7. 5 an d1 5 mg/kg/day) . Th e goa l o f th e stud y wa s t o determin e whethe r

endosulfa n affect s testosteron e metabolis man d eliminatio n i n mal e an dfemal e mic e an di f an y effect s resulte di n alteration s i n testosteron e homeostasi s (i.e. , wer e biologicall y relevant) . Th e author s examine dformatio n o f testosteron e metabolite s i n live r preparation s fro mmic e a t sacrifice . I n on e grou p o f femal e mic e administere d7. 5 mg/k g endosulfa n fo r seve n days , th e author s als o monitore deliminatio n o f

[ 14 C]-radiolabele dtestosteron e that wa s injecte d intraperitoneally . Thes e animal s wer e the n house di n metabolis mcage s fo r collectio n o f

fece s an durin e i n orde r

t o monito r

eliminatio n o f testosterone . 

### 2 4Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### Th e author s reporte dthat

ther e wer e several death s i n th e tw o highes t dos e group s i n mal e animal s only . The y report als o tha t i n femal e mic e onl y ther e wa s adose-dependen t increas e i n th e rat e o f

testosteron e hydroxylate dmetabolit e formation , a s wel l a s a n increase drat e o f urinar y eliminatio n o f

testosteron e i n female s only . N o change s i n testosteron e o r estradio l level s i n seru mwer e reporte di n thes e animal s eve n thoug hther e wer e effect s o n enzym e activit y an drat e o f elimination . Th e author s conclude dtha t

althoug hther e wer e endosulfan-induce deffect s o n testosteron e metabolis man deliminatio n kinetics , thes e change s wer e no t sufficien t t o affec t hormon e homeostatsis . Th e dat aprovide di n thi s stud y ar e functionall y equivalen t t o th e dat acollecte di n th e 890.145 0 an d890.150 0 assay s (i.e. , se x hormon e levels) . Thus , thes e dat aals o provid e reliabl e endosulfa n OSRI . 7 . Hunte r

e t al (1999 ) Revie w o f thi s stud y indicate s tha t

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e estroge n recepto r

bindin g potentia l an dth e estroge n recepto r transactivatio n potentia l o f endosulfan . I n thi s study , estroge n recepto r bindin g wa s directl y measure da s wa s estroge n recepto r

transactivatio n potential . Inhibitio n o f [ 3 

### H]-labele d estradio l bindin g t o recombinan t huma n ER awa s measure di n aSf 9 baculovira l system . Th e author s report tha t endosulfa n exhibite dver y weak activit y i n thi s assay a s the y wer e unabl e t o calculat e a n ICso . Competitiv e bindin g activit y wa s onl y observe da t a1 0 u M endosulfa n concentration , ados e tha t

wa s associate dwit hles s tha n 20 % bindin g inhibition . Then , i na ni nvitr o estroge n receptor-controlle dcel l proliferatio nassa y usin g ELT 3 cells , th e abilit y o f endosulfa n t o affec t

cel l proliferatio n wa s measured . These cell s exhibi t proliferativ e response s a t a n estradio l concentratio n o f 1 0 nM . Whe n endosulfan- p wa s tested , i t exhibite dn o activit y i n thi s assay . Endosulfan- ashowe dlimite dactivit y a t \ 

### a1 0 u M exposur e leve l (25 % o f th e 1 0 n M estradio l response) . Finally , 2 5Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 th e author s report

result s fro mareporte r

gen e transactivatio n assay (ELT 3 cell s transfecte dwit hfirefl y luciferas e linke dt o vitellogeni n ER Ean da promoter) . I n thi s assay , endosulfan- aan dendosulfan- p bot hshowe d activit y a t a1 0 u M dos e level , a s compare dt o activit y wit hestradio l i n thi s assay tha t

exhibite da n ECs o o f

10 0 pM . Dat afro mal l thre e o f th e assay s reporte di n thi s pape r confir mth e wea k

activit y o f

endosulfa n t o produc e estrogeni c effect s i n vitro . Th e dat aprovide di n thi s stud y ar e directl y equivalen t

t o th e typ e o f

dat acollecte di n th e EDS PTie r 1 890.125 0 an d890.130 0 assays . Thus , thes e dat aals o provid e reliabl e endosulfa n OSRI . 8 . Legle r

et a l (1999 ) Revie w o f

thi s stud y indicate s tha t

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e estroge n recepto r

transactivatio n potentia l o f

endosulfan . I n thi s study , a n ER-CALU X assay wa s use dtha t employe dT47 D (huma n breas t

cance r cells ) Lu c cells . Thi s luciferas e gen e expressio n assa y wa s use dt o scree n fo r estrogeni c activit y o f

endosulfan , wher e luciferas e expressio n i s unde r estrogen-mediate dcontrol . Endosulfa n wa s weakl y estrogenic , wit han ECs o o f 5. 9 |j M an dmaxima l effect s see n at 1 0 u M endosulfan . I t

shoul db e note dtha t

th e potenc y o f

endosulfa n i n thi s assa y wa s simila r

t o th e potenc y reporte db y othe r paper s alread y discusse dabove . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f

dat acollecte di n th e EDS PTie r 1 890.130 0 assay , an dar e functionall y equivalen t

t o th e dat acollecte di n th e 890.125 0 assay . Thus , thes e dat aals o provid e reliabl e endosulfa n OSRI . 9 . Won g an dMatsumur a(2000 ) Revie w o f

thi s stud y indicate s tha t

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e estroge n recepto r

bindin g potentia l o f

endosulfan . 

### 2 6Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 Th e author s o f

thi s stud y wer e focuse do n examinin g th e ligand

independen t

activit y o f

chemical s t o activat e estroge n receptors . Th e assay use di s state dto detec t

estrogeni c activity even i n chemical s that exhibi t onl y wea k o r partia l agonis t

activit y a t

estroge n receptors . Th e assay i s a serum-fre e assay performe di n a n ovaria n cel l line , BG- 1 cells ; cel l proliferatio n response s ar e measure dfollowin g exposur e t o chemicals . The activit y of

tes t chemical s is compare dto 17(3-estradiol . Endosulfa n wa s on e o f

nin e organochlorin e agent s teste dindividuall y an dth e author s report i t induce dcel l proliferation . Endosulfan' s effect s wer e partiall y antagonize db y tamoxifen , a n estroge n recepto r antagonist . Th e dat a provide di n thi s study ar e directl y equivalen t t o th e typ e o f

dat acollecte d i n th e EDS PTie r

1 890.125 0 assay . Thus , thes e dat aals o provid e reliabl e endosulfa n OSRI . 10 . Newbol det

a l (2001 ) Revie w o f

thi s stud y indicate s tha t

i t provide s reliable , non guidelin e OSR I dat ascreenin g fo r uterin e weigh t

change s fro ma n Uterotrophi c assa y i n endosulfan-expose dimmatur e mice . Thi s i n viv o study i s consisten t i n desig n wit hth e Tie r

1 EDS P Uterotrophi c assa y (890.1600) , however , mic e ar e use dinstea do f rats . I n on e part o f th e study , immatur e (PND17 ) CD- I mic e wer e injecte dfo r thre e day s subcutaneousl y (PND17-PND19 ) withvariou s individua l compounds . Th e tes t

compound s include dth e positiv e contro l agent s 17(3

- estradio l an dDES , th e negativ e contro l tamoxifen , an dendosulfa n a t dose s o f fro m0. 1 t o 100 0 mg/kg/day . I n asecon dpart o f th e study , matur e ovarectomize dCD- I mic e wer e administere deithe r apositiv e contro l agent (DES ; 1 0 ng/kg) , anegativ e contro l agen t (cor n oi l vehicle) , o r endosulfa n at

ados e o f 1 0 mg/kg , subcutaneousl y fo r 1 4 days . I n eac h experiment , th e endpoin t

monitore dwa s increase duterin e weight . Th e author s reporte dthat

endosulfa n wa s no t

Uterotrophi c i n either

experiment . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f

dat aMakhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 collecte di n th e EDS PTier 1 890.160 0 assay , an dare functionally equivalen t

t o th e dat acollecte di n th e 890.125 0 assay , an dar e reliabl e endosulfa n OSRI . 11 . Anderse n e t

al (2002 ) Revie w o f

th e dat aindicate s tha t

i t provide s reliable , non-guidelin e dat ascreenin g fo r

estroge n recepto r

bindin g an dtransactivation , androge n recepto r transactivation , an daromatas e activit y tha t ma y b e associate d wit hexposur e t o endosulfan . I n thi s study , th e abilit y o f individua l compound s t o affec t estroge n recepto r binding , androge n recepto r bindin g an daromatas e (CYP19 ) activit y wa s examined . Th e stud y report s us e o f standar dmethod s fo r

eac h typ e o f assay . Estroge n recepto r

bindin g wa s examine di n MCF- 7 cell s usin g method s simila r

t o thos e alread y discusse dabove . I n th e assay , endosulfa n exhibite dalowest-observe deffec t concentratio n (LOEC ) o f 1 u M an damaximum-observe deffec t concentratio n (MOEC ) o f 2 5 uM , bu t wa s reporte dt o b e si x order s o f magnitud e les s poten t

that th e positiv e contro l compoun d17(3-estradiol . I n an estroge n recepto r transactivatio n assay , MCF-7BU S cell s wer e use d(measurin g (3-galactosidas e activity) . Th e author s reporte dtha t endosulfa n exhibite daLOE C o f 1 u M an da MOE C o f 2 5 u M an dwa s fiv e order s o f magnitud e les s poten t

i n th e assay a s compare dt o th e positiv e contro l 17p-estradiol . Androge n recepto r transactivatio n wa s measure di n CHOK 1 cell s (Chines e hamste r ovar y cells ) transfecte dwit hth e huma n androge n recepto r expressio n vecto r pSVAR O an dth e MMTV-LU C reporte r plasmid . Th e author s reporte d tha t endosulfa n exhibite daLOE C o f 2 0 u M an daMOEC o f

2 0 u M an d wa s seven order s o f

magnitud e les s poten t

i n th e assa

y a s compare dt o th e positiv e control . Finally , th e activit y o f endosulfa n wa s examine di n a n aromatas e assa

y i n huma n placental microsomes . Th e author s reporte dthat a t a5 0 u M dos e endosulfa n inhibite daromatas e activit y b y 13 % wherea s th e positiv e control agen t inhibite daromatas e activit y b y 100 % a t a1 u M 

### 2 8Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### dos e level . Considere dtogether , thes e dat ademonstrate dth e lac k o f significan t estrogeni c an dandrogeni c activit y i n vitr o withendosulfa n exposure , an dar e consisten t wit hothe r

endosulfa n OSRI alread y discussed . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f dat acollecte di n th e EDS PTie r 1 890.120 0 assay , th e 890.125 0 assay , an dth e 890 . 1300 assay , ar e functionall y equivalen t t o th e dat acollecte d i nth e 890.115 0 assay , an dar e reliabl e endosulfa n OSRI . 12 . Fan g et

al (2003 ) Revie w o f th e dat aindicate s tha t

i t provide s reliable , non-guidelin e dat afo r screenin g fo r

th e androge n recepto r

bindin g potentia l o f endosulfan . I nthi s i n vitr o study , 20 2 chemical s wer e individuall y screene dfo r androge n recepto r

bindin g activit y usin g asyste memployin g ra t recombinan t

androge n receptor s expresse di n E . coli . Standar dtechnique s fo r recepto r bindin g wer e employe da s describe di n th e paper . Competitiv e bindin g wa s assesse dusin g th e androge n recepto r agonis t 17R-methyl

- [ 3 

### H]methyltrienolon e (als o know n a s R1881) . R188 1 wa s employe da t a dos e o f 1 nM . Th e IC 5 

### o fo r

endosulfa n reporte db y th e author s wa s 2. 3 x 10" 5 

### M . Th e author s describ e endosulfa n a s aweak androge n recepto r agent . Thes e dat aar e als o consisten t withdat areporte db y other s a s discusse dabove . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f

dat acollecte di n th e EDS PTie r 1 890.115 0 assay , an dar e reliabl e endosulfa n OSRI . 13 . Gormle y an dleathe r (2003 ) Revie w o f th e dat aindicate s tha t

i t provide s reliable , non-guidelin e dat afo r screenin g fo r

th e potentia l o f

endosulfa n t o affec t

developmen t an dreproductio n i n fish . 2 9Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 I n thi s i n viv o study , Japanes e medak awer e use dan dth e effect s o f endosulfa n o n behavior , developmen t an dreproductio n wer e assessed . Th e fis hwer e expose dfo r

2 4 hour s an dexposur e began eithe r 4- 6 hour s afte r fertilizatio n o r

4- 6 hour s afte r

hatching . Endosulfa n wate r concentration s teste dinclude d0.01 , 0. 1 an d1 ji g endosulfan/L , level s state db y th e author s t o b e relevan t

t o level s encountere di n th e environment . Th e author s reporte dn o effect s o n endpoint s directl y relevan t t o endpoint s monitore di n Tie r

1 EDS Passays , suc ha s tim e t o hatchin g an dhatchin g success . Ther e wer e als o n o effect s o n surviva l an dn o consisten t effect s o n eg g production . Th e author s reporte ddecrease dfr y siz e a t th e tw o highes t exposur e level s an ddecrease dswimmin g behavio r whe n th e fis h wer e teste d2 week s afte r

hatching , bu t

onl y whe n th e exposur e t o endosulfa n wa s post-fertilization . Th e dat aprovide di n thi s stud y ar e directl y equivalen t

t o th e typ e o f

dat acollecte di n th e EDS P Tie r 1 890.135 0 assay , an dar e reliabl e endosulfa n OSRI . 14 . Grunfel dan dBonefeld-Jorgense n (2004 ) Revie w o f

th e dat aindicate s tha t

i t provide s reliable , non-guidelin e dat afo r screenin g fo r

estroge n recepto r

transactivatio n potentia l o f endosulfan . I n thi s i n vitr o study , estroge n recepto r mRN A level s wer e monitore di n MCF-7BU S cells . A s alread y discussed , thi s i s astandar d assa y fo r examinin g estrogeni c activit y o f chemical s a s i t

relate s t o transactivatio n o f

th e estroge n receptor . Bot hER aan dER| 3 mRN A level s wer e measure dfollowin g exposur e t o endosulfan . Th e activit y o f nin e differen t chemicals , includin g endosulfan , wa s teste dindividually . Th e activit y wa s compare dt o th e positiv e contro l agen t 17(3-estradiol , presen t at tw o differen t

dose s (lo w dose : 0.0 6 nM ; hig hdose : 1 0 nM) . Endosulfa n exhibite donl y weak

activit y i n thi s assay , decreasin g level s o f ER aa t a dos e o f fro m1 t o 2 5 uM , bu t

havin g n o effec t

o f level s o f ER 0 mRNA . Thes e dat aar e als o consisten t wit hpreviou s finding s (discusse dabove ) 3 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### regardin g estroge n recepto r transactivatio n potentia l o f endosulfan . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f

dat a collecte di n th e EDS PTie r 1 890.130 0 assay , an dar e functionall y equivalen t t o th e dat acollecte di n th e 890.125 0 assay . Thus , thes e dat a als o provid e reliabl e endosulfan OSRI . 15 . Bonefeld-Jorgense n et a l (2005 ) Revie w o f th e dat aindicate s tha t

i t provide s reliable , non-guidelin e dat afo r screenin g fo r

estroge n recepto r

transactivatio n potentia l o f endosulfan . I n thi s i n vitr o study , estroge n recepto r transactivatio n wa s examine di n MCF-7BU S cells . A s already discussed , thi s i s astandard assa y fo r assessin g estrogeni c activit y o f chemical s a s i t

relate s t o transactivatio n o f

th e estroge n receptor . Th e activit y o f fou r differen t chemicals , includin g endosulfan , wa s teste dindividually . Th e activit y wa s compare dt o th e positiv e contro l agen t 17|3-estradiol . Endosulfa n exhibite donl y wea

k activit y i n thi s assay , wit hth e author s reportin g a LOE C o f 1 u M an daMOE C o f

2 5 uM . Thes e dat aar e consisten t with previou s finding s (discusse dabove ) regardin g estroge n recepto r transactivatio n potentia l o f endosulfan . Th e dat aprovide di n thi s stud y ar e directl y equivalen t

t o th e typ e o f

dat acollecte di n th e EDS PTie r 1 890.130 0 assay , andare functionall y equivalen t to the dat acollecte din the 890.125 0 assay . Thus , thes e dat aals o provid e reliabl e endosulfa n OSRI . 16 . Lemair e et

al . (2006 ) Revie w o f th e dat aindicate s tha t

i t provide s reliable , non-guidelin e dat afo r screenin g fo r

estroge n recepto r

bindin g an dtransactivatio n potentia l o f

endosulfan . I n thi s i n vitro study , 4 9 differen t pesticides , teste dindividually , wer e screene dfo r

estroge n recepto r

transactivatio n an dbindin g (competitiv e bindin g displacement ) i n HEL N cells . I n th e recepto r 3 1Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 transactivatio n assays , HEL N ER aan dER| 3 responsiv e reporte r cel l line s wer e used . Thes e cell s wer e use di n astandar dassa y fo r assessin g estrogeni c activit y o f

chemical s a s i t

relate dt o transactivatio n o f th e estrogen receptor . Endosulfan wa s on e o f th e 4 9 pesticide s teste da t a concentratio n o f

1 0 uM . Th e activity o f

al l tes t agent s wa s compare dt o th e positiv e contro l agen t

17p-estradio l at

ados e o f 1 0 nM . Fo r competitiv e bindin g detereminations , ER aan dER 0 cell s i n cultur e wer e incubate dfirs t

wit h[ 3 H]-labele destradiol . Then , th e cell s wer e expose dt o pesticides , includin g endosulfan , at aconcentratio n o f

1 0 uM . Endosulfa n exhibite dn o appreciabl e activatio n activit y i n thi s assay , an dinhibite d ER( 3 bindin g b y onl y 20-30 % a t aconcentratio n o f

1 0 uM . Thes e dat aare consisten t wit hpreviou s finding s (discusse dabove ) regardin g estroge n recepto r bindin g an dtransactivatio n potentia l o f endosulfan . Th e dat a provide di n thi s stud y ar e directl y equivalen t t o th e typ e o f

dat acollecte d i n th e EDS PTie r

1 890.125 0 assay an dth e 890.130 0 assay , an dals o provid e reliabl e endosulfa n OSRI . 17 . Lavill e e

t a l (2006 ) Revie w o f

th e dat aindicate s tha t

i t provide s reliable , non-guidelin e dat afo r screenin g fo r

th e potentia l o f

endosulfa n t o affec t

aromatas e activity . I n thi s study , th e i n vitr o activit y o f aromatas e i n JEG- 3 huma n choriocarcinom acell s wa s examine dfollowin g exposure to 3 0 different pesticides , teste dindividually . Endosulfa n wa s on e o f th e compound s tested . Additionally , th e author s repor t measuremen t

o f CYP1 9 mRN A level s i n thes e cells . Thi s assay ha s bee n develope dt o asses s effect s o f chemical s o n steroidogenesi s i n embryoni c an dfeta l tissue . Th e assa y wa s performe dusin g bothshort ( 2 hours ) an dlon g (2 4 hours ) exposur e time s fo r eac hcompound . Th e author s reporte dtha t endosulfa n increase d aromatas e activity , bu t

onl y afte r th e 2 4 hou r

exposure perio dan donl y at th e highes t

tes t dos e leve l o f

1 0 u M (n o effect s at

1 an d3 uM) . Ther e wa s 

### 3 2Makhteshi mChemical Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### n o effec t o n CYP1 9 mRN A level s afte r endosulfa n treatment . I n comparison , th e positiv e contro l compound s produce deffect s t o increas e aromatas e activit y a t

lowe r dose s an dafte r

bot hshort

an dlon g ter m

. exposures . Thes e dat aar e consisten t wit hpreviou s finding s (discusse d above ) regardin g th e potentia l o f endosulfa n t o affec t

aromatas e activity . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f

dat a collecte di n th e EDS PTie r 1 890.120 0 assay , an dals o provid e reliabl e endosulfa n OSRI . 18 . Varayou det

a l (2008 ) Revie w o f thi s stud y indicate s tha t

i t provide s reliable , non guidelin e OSRI dat afo r

screenin g fo r

uterin e weigh t

change s fro ma n Uterotrophi c assa y i n endosulfan-expose dovarectomize drats . Thi s i n viv o stud y i s consisten t i n desig n wit hth e Tier

1 EDS P Uterotrophi c assa y (890.1600) . I n th e study , sexuall y matur e Wista r rat s (9 0 day s old ) wer e ovarectomized . Followin g recover y fro msurger y (1 4 days) , rat s tha t

exhibite da t

leas t 7 days o f atrophi c vagina l smear s wer e injecte dfo r

thre e day s (subcutaneously ) wit hendosulfa n (0 , 0.006 , 0.06 , 0. 6 an d6 mg/kg) . Th e contro l agent s teste dinclude daknow n Uterotrophi c dos e o f estradio l (0.0 2 mg/kg ) an danon-uterotrophi c dos e o f estradio l (0.000 2 mg/kg) . Th e primar y endpoin t monitore dwa s increase duterin e weight . Th e author s reporte dtha t endosulfa n wa s no t

Uterotrophi c a t

an y tes t dose . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f dat acollecte di n th e EDS PTie r 1 890.160 0 assay , an dar e functionall y equivalen t

t o th e dat acollecte di n th e 890.125 0 assay , an dar e reliabl e endosulfa n OSRI . 19 . Vinggaar det

al . (2008 ) Revie w o f th e dat aindicate s tha t

i t provide s reliable , non-guidelin e dat afo r screenin g fo r

androge n recepto r

bindin g potentia l o f

endosulfan .
3 3Makhteshi mChemica l Works , Ltd . Study No . MA-END-EDSP-2010031 3 

### I n thi s i n vitr o study , androge n recepto r transactivatio n wa s assesse dfo r

39 7 chemicals , individually , i n aluciferas e reporte r assay usin g CHOK 1 cell s transfecte dwit hhuma n androge n receptor . Standar d technique s fo r

wer e employe da s describe di n th e paper . R188 1 wa s employe da s apositiv e contro l agent a t ados e o f 0. 1 nM . Chemicals , includin g endosulfan , wer e teste dat dose s o f 1 , 3 , 10 , an d3 0 uM . Th e IC2 5 fo r endosulfa n reporte db y th e author s wa s betwee n 3 an d1 0 uM , indicatin g onl y very wea k

activit y base do n th e author s ow n rankin g system . Thes e dat aar e consisten t wit hdat areporte db y other s a s discusse d above . Th e dat aprovide di n thi s study are directl y equivalen t t o th e typ e o f dat acollecte di n th e EDS PTie r 1 890.115 0 assay , an dar e reliabl e endosulfa nOSRI . 20 . Judso n e t

al . (2009 ) Th e citatio n reference dher e refer s t o th e publishe dpaper an dth e accompanyin g file s foun do nth e EP A websit e fo r th e ToxCas t

program 2



### . Revie w o f th e dat aindicate s tha t

i t provide s reliabl e dat afo r

screenin g fo r estroge n recepto r

binding , androge n recepto r binding , estroge n recepto r transactivation , aromatas e activity , an dthyroi dhormon e bindin g potentia l o f endosulfan . I n apape r publishe donlin e i n Decembe r 200 9 (Judso n e t

al . 2009) , th e author s report

o n th e result s o f

extensiv e i n vitr o testin g o f ove r 30 0 individua l chemicals , includin g endosulfan . Endosulfan , a s wel l a s th e al l o f th e othe r compounds , wa s teste dfo r estroge n recepto r

binding , androge n recepto r

binding , estroge n recepto r transactivation , aromatas e activity , an dthyroi dhormon e bindin g potential , amon g avariet y o f othe r endpoints . Th e specifi c test s performe dinclude dth e following : bindin g t o bovin e estroge n receptor s fro muterin e membran e (bER) ; bindin g t o
2 Th e ToxCas t

progra mi s aprogra mwithi n EP A tha t i s currentl y validatin g method s fo r testin g chemical s usin g avariet y o f

i n vitr o assay s a s awa y t o develo p .testin g paradigm s fo r replacin g i n viv o toxicit y testin g o r t o predic t result s o f

i n viv o toxicit y testing . Include di n th e progra mar e i n vitr o assay s examinin g th e abilit y o f chemical s t o affec t

estrogen , androgen , an dthyroi dhormon e systems . Detail s o f th e progra man d th e dat aca n b e foun da t

www.epa.gov/ncct/toxcast . 3 4Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 huma n estroge n receptor s fro mbreas t cancer cell s (hER) ; bindin g t o bovin e progesteron e receptor s fro muterin e membran e (bPR) ; bindin g t o huma n progesteron e receptor s fro mMCF- 7 cell s (hPR) ; bindin g t o ra t androge n receptor s (rAR ; recombinant) ; bindin g t o huma n androge n receptor s fro mLnCA Pcell s (hAR) ; bindin g t o ra t thyrotropin-releasin g hormon e recepto r

fro mforebrai n membrane s (rTRH) ; bindin g t o huma n thyroi dhormon e receptor-alph a(hTRa ; recombinant) ; activit y t o inhibi t th e aromatas e enzyme ; estroge n recepto r transactivation ; androge n recepto r transactivation ; an dthyroi dhormon e recepto r transactivation . Th e dat afro mthes e specifi c tests wa s examine dsinc e th e endpoint s ar e directl y o r

functionall y equivalen t

t o endpoint s o f

EDS P Tie r 1 assays . Th e result s wer e a s follows . I n th e cell-fre e screenin g assays , endosulfa n teste da t asingl e initia l concentratio n o f 2 5 \i M wa s inactiv e fo r

bindin g t o al l endocrin e recepto r proteins . Becaus e i t

wa s inactiv e at

thi s dose , n o othe r

dose s wer e tested . Mor e specifically , endosulfa n di dno t

sho w evidenc e o f

bindin g t o bovin e estroge n receptors , bovin e o r huma n progesteron e receptors , ra t o r huma n androge n receptors , ra t thyrotropin-releasin g hormon e receptor , o r huma n thyroi dhormon e receptor-a . Endosulfa n di dexhibi t aweak leve l o f bindin g t o huma n estroge n receptor s (11.4 1 ^ M dose ) i n on e assa y only . I n cell-base d assays , endosulfa n di dno t activat e huma n androge n receptors , huma n ERa , o r huma n thyroi dhormon e receptor-(3 , an dals o di dno t bloc k activit y o f establishe dligand s at

thes e recepto r

site s (i.e. , n o antagonis t

activity demonstrated) . I n th e hormon e recepto r transactivatio n assays , endosulfa n di dno t activat e th e huma n androge n receptor , huma n estrogen-relate d receptor-a , huma n estrogen-relate dreceptor-y , o r thyroi dhormon e receptor-a . Endosulfa n showedwea k activit y i n ahuma n estroge n receptor- atransactivatio n assay , wit hactivit y see n at aconcentratio n o f 2. 1 uM . I t

i s note dtha t

th e positiv e contro l compound , 17p-estradiol , ha d activit y i n thi s sam e assay i n th e lo w nanomola r range . 

### 3 5Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f dat acollecte di n th e followin g EDS PTie r 1 assays : 890.1150 , 890.1200 , 890.1250 , an d890.1300 . Th e dat ao n thyroi dhormon e bindin g ar e functionall y equivalen t t o a n endpoin t

o f EDS PTie r

1 assay s 890.1100 , 890.1450 , an d890.1500 . Thes e dat aar e reliabl e endosulfa n OSRI . 21 . Brunell i e t

al (2009 ) Revie w o f th e dat aindicate s tha t

i t provide s reliable , non-guidelin e dat afo r screenin g fo r

i nviv o reproductiv e effect s o f endosulfa n i n tadpoles , anon-mammalia n specie s tha t i s use di n aTie r

1 EDS Passay (890.1100) . I nthi s study , tadpole s wer e expose dt o endosulfa n i nwate r a t level s o f 0.01 , 0.0 5 an d0. 1 ug/Lfo r 43-5 2 days (fro mhatchin g throughou t

metamorphosis) . Th e larva e wer e observe dfo r mortality , larva l growth , development , metamorphosi s stage , an dbehavio r (swimmin g , activity) . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f dat acollecte di n th e followin g EDS PTie r 1 assay 890.1100 , an dar e functionall y equivalen t

t o dat acollecte di n EDS PTie r 1 assay 890.1350 . Thes e dat aar e reliabl e endosulfa n OSRI . 8 . Directl y o r

Functionall y Equivalen t Endosulfa n OSRI Identifie di n Endosulfa n Registratio n Studie s A searc ho f

th e guidelin e toxicolog y studie s performe dt o support th e registratio n o f

endosulfa n identifie dsevera l source s o f endosulfa n OSR I tha t

ar e directl y o r functionall y equivalen t

t o endpoints/dat acollecte di n EDS PTie r 1 assays . Thes e studie s includ e th e followin g (se e Table s 3 an d4 above) : achroni c toxicit y stud y i n rat s (Ruckma n 1989) , achroni c toxicit y stud y i n dog s (Brunk 1989) , amulti-generatio n reproductiv e toxicit y stud y i n rat s (Edward s 1983) , a prenata l developmenta l toxicit y stud y i n rat s (MacKensi e 1980) , adevelopmenta l neurotoxicit y stud y i n rat s (Gilmor e 2006) , aone-generatio n reproductio n stud y i n 3 6Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 Bobwhit e quai l (Beaver s 1987) , aone-generatio n reproductio n stud y i n Mallar d duck s (Beaver s 1987) , an daful l lif e cycl e stud y i n fis h(Dionn e 2002) . Th e followin g i s adiscussio n o f th e relevan t

OSR I fro meac hstud y an dastatemen t a s t o whethe r

th e dat aar e directl y o r

functionall y equivalen t

OSRI . 1 . Ruckma n (1989 ) Revie w o f

thi s GLP-complian t

guidelin e chroni c toxicit y stud y i n rat s indicate s tha t

i t provide s reliabl e dat arelatin g t o avariet y o f EDS P Tie r 1 stud y endpoint s a s show n i n Tabl e 3 above, endpoint s relatin g t o estrogen , androge n an dthyroi dhormon e syste mactivity . h i thi s guidelin e stud y i n rats , youn g adul t mal e an dfemal e Sprague-Dawle y rat s wer e administere d97.1 % pur e endosulfa n i n die t a t dose s o f 0 , 3 , 7.5 , 1 5 an d7 5 pp mendosulfa n fo r 10 4 weeks . I t

shoul db e note dtha t adietar y rout e o f

administratio n i s mor e relevan t t o th e expecte dhuma n exposur e t o endosulfa ntha n dat atha t migh t b e collecte d usin g othe r

route s o f administratio n i n animal s (e.g. , ora l gavag e dosing , intraperitonea l dosing , subcutaneou s dosing) . A s i n an y standar dguidelin e roden t chroni c toxicit y study , endpoint s relevan t t o endocrin e activit y wer e assesse d(se e Table 3) . Th e endpoint s measure di n th e chroni c stud y tha t ar e directl y equivalen t

t o endpoint s monitore di n EDS PTie r 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival , bod y weight , clinica l chemistry , orga n weight s (adrenal , thyroid , pituitary , kidney , liver) ; reproductiv e orga n weight s (ovaries , testes) ; orga n histopatholog y (adrenal , thyroid , pituitary , kidney , liver) ; an d reproductiv e organ histopatholog y (femal e mammar y gland , ovaries , uterus , vagina , testes , epididymides , prostate , semina l vesicle) . I t shoul d b e note da s wel l tha t

th e chroni c ra t

stud y involve s repeate ddosin g fo r 2 years , atim e perio dmuc hlonge r tha n an y tim e perio do f

exposur e i n EDS PTie r

1 assays , an dcoverin g adevelopmenta l perio dno t covere db y EDS PTie r

1 studies , i.e. , senescence .

3 7Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 Th e developmenta l period s assesse di n th e chroni c stud y includ e youn g adult , adul t

an dsenescence , i n bothmal e an dfemal e animals . Th e mos t sensitiv e NOAE Li n th e stud y wa s 1 5 pp man dwa s base do n kidne y toxicit y a t th e 7 5 pp mexposur e level . A s show n i n Tabl e 3 , estrogen responsiv e (e.g. , ovaries , uterus) , androgen-responsiv e (e.g. , testes , prostate ) an dthyroid-responsiv e tissue s (e.g. , thyroid , brain) , wer e examine dhistopathologicall y i n thi s study . Ther e wer e n o effect s o n orga n weight s o f

an y orga n relevan t

t o endocrin e activity , an dn o effect s o n orga n histopatholog y wer e reported , includin g endocrin e organ s (i.e. , thyroid , pituitary , an dvariou s reproductiv e organ s i n male s an dfemales) . Thes e dat ademonstrat e tha t endosulfa n wa s no t

toxi c t o endocrin e orga n system s i n thi s study i n rats . Th e dat aprovide di n thi s chroni c stud y ar e directl y and/o r functionall y equivalen t

t o specifi c dat acollecte di n th e followin g EDS P Tie r 1 assays : 890.1350 , 890.1400 , 890.1450 , 890.1500 , an d890.1600 . Th e dat aprovid e informatio n o n estrogen , androgen , an dthyroi dhormon e syste mtoxicit y o f

endosulfan . Thes e dat aar e reliabl e endosulfa n OSRI . 2 . Brun k

(1989 ) Revie w o f

thi s GLP-complian t

guidelin e chroni c ( 1 year ) toxicit y stud y i n dog s indicate s tha t i t provide s reliabl e dat arelatin g t o avariet y o f EDS PTie r

1 stud y endpoint s as show n in Tabl e 3 above , endpoint s relatin g t o estrogen , androge n an dthyroi dhormon e syste mactivity . I n thi s guidelin e stud y i n dogs , youn g adul t mal e an dfemal e Beagl e dog s were administere d96.5 % pure endosulfan i n diet a t dose s o f 0 , 3 , 10 , 3 0 an d6 0 pp mendosulfa n fo r 1 year . I n th e hig hdos e group , th e animal s were firs t

administere d3 0 pp mi n die t an dth e dos e wa s increase d t o 6 0 pp mdurin g th e study . I t shoul db e note dtha t

adietary rout e o f administratio n i s mor e relevan t t o th e expecte dhuma n exposur e t o endosulfa n tha n dat atha t

migh t b e collecte dusin g othe r

route s o f administratio n i n animal s (e.g. , ora l gavag e dosing , intraperitonea l dosing , 3 8Makhteshi mChemical Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 subcutaneou s dosing) . A s i n an y standar dguidelin e do g chroni c toxicit y study , endpoint s relevan t

t o endocrin e activit y wer e assesse d(se e Table 3) . Th e endpoint s measure di n th e chroni c study tha t ar e directl y equivalen t

t o endpoint s monitore di n EDS PTie r 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival , bod y weight , clinica l chemistry , orga n weight s (adrenal , thyroid , pituitary , kidney , liver) ; reproductiv e orga n weight s (ovaries , uterus , vagina , testes , epididymides , prostate) ; organ histopatholog y (adrenal , thyroid , pituitary , kidney , liver) ; an dreproductiv e orga n histopatholog y (femal e mammar y gland , ovaries , uterus , vagina , testes , epididymides , prostate) . I t shoul db e note da s wel l tha t

th e chroni c do g stud y involve s repeate ddosin g fo r 1 year , atim e perio dmuc hlonge r tha n an y tim e perio do f

exposur e i n EDS P Tie r 1 assays . Th e developmenta l period s assesse di n th e chroni c stud y includ e youn g adul t

an dadult , i n bothmal e an dfemal e animals . Th e mos t sensitiv e NOAE Li n th e study wa s 1 0 pp man dwa s base do n decrease d bod y weigh t

an dtoni c spasm s observe dat th e 3 0 pp mexposur e level . Ther e wer e n o effect s o n orga n weight s o f an y orga n relevan t

t o endocrin e activity , an dn o effect s o n organ histopatholog y wer e reported , includin g th e endocrin e organ s studie d(e.g. , thyroid , pituitary , an dvariou s reproductiv e organ s i n male s an dfemales) . Thes e dat ademonstrat e tha t endosulfa n wa s no t

toxi c t o endocrin e organ system s i n thi s chroni c stud y i n dogs . Th e dat aprovide di n thi s chroni c stud y ar e directl y and/o r functionall y equivalen t

t o specifi c dat acollecte di n th e followin g EDS P Tie r 1 assays : 890.1350 , 890.1400 , 890.1450 , 890.1500 , an d890.1600 . Th e dat aprovid e informatio n o n estrogen , androgen , an dthyroi dhormon e syste mtoxicit y o f

endosulfan . These dat aar e reliabl e endosulfa n OSRI .



### 3 9Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 3 . Edward s (1983 ) Revie w o f

thi s GLP-complian t

guidelin e multi-generatio n (two ) reproductiv e toxicit y stud y i n rat s indicate s tha t i t provide s reliabl e dat a relatin g t o avariety o f

EDS P Tier

1 study endpoint s a s show n i n Tabl e 3 above , endpoint s relatin g t o estrogen , androge n an dthyroi dhormon e syste mactivity . Thi s stud y ha s th e mos t extensiv e coverag e o f

EDS P Tie r 1 endpoint s o f

an y singl e endosulfa n stud y an dcover s al l developmenta l period s covere db y EDS P Tie r 1 assay s (i n utero , lactation , post-natal , peri-pubertal , youn g adult , an dadult) . I n thi s guidelin e stud y i n rats , mal e an dfemal e Sprague-Dawle y rat s wer e administere dendosulfa n i n die t at dose s o f 0 , 3 , 1 5 an d7 5 pp m endosulfa n fro m6 week s o f ag e (pre-matin g i n PI) , durin g matin g (PI) , an dthroug hPND2 1 i n th e F2 b generation . Pup s o f th e F l generatio n were , therefore , expose di n utero , durin g lactation , an dthe n place do n endosulfan i n die t

immediatel y at

weanin g throug hth e post-natal perio d an dthroug hmatin g an dsubsequen t parturition , delivery o f

th e F 2 generation . Thus , thi s stud y include dexposur e o f F l animal s o f

bothsexe s durin g peri-puberty , a n importan t consideratio n fo r

late r discussio n o f

th e puberta l ra t

EDS P Tie r

1 assays . A s wit hth e othe r guidelin e studie s alread y discussed , th e dietar y rout e o f administratio n i s mor e relevan t t o th e expecte dhuma n exposur e t o endosulfa n tha n dat atha t migh t b e collecte dusin g othe r

route s o f administratio n i n animal s (e.g. , ora l gavag e dosing , intraperitonea l dosing , subcutaneou s dosing) . Th e multi-generatio n reproductiv e toxicity i s expecte dt o b e a critica l EDS P Tie r

2 study . Althoug hth e endosulfa n study wa s no t performe daccordin g t o th e mos t recent EP A guidelin e fo r

multi

generatio n reproductiv e toxicit y studie s (OPPT S 870.3300) , i t wa s performe daccordin g th e guideline s i n plac e at th e tim e th e stud y wa s performed . Lik e th e mos t curren t guidelin e stud y design , th e stud y wa s designe dt o examin e potentia l effect s o f endosulfa n o n endocrin e organs , in particular

the estroge n andandroge n system s that are critica l to 4 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 reproductiv e function . A s aresul t o f bein g areproductiv e toxicit y study , man y endpoint s relevan t

t o endocrin e activity wer e assesse d(se e Tabl e 3) . Th e endpoint s measure di n th e multi-generatio n reproductiv e toxicit y study that are directl y equivalen t

t o endpoint s monitore di n EDS PTie r 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival ; bod y weigh t

(bot hadult s an dpups) ; developmenta l measure s (matin g inde x i n bot hsexes , gestatio n index , gestatio n length , se x rati o o f tw o successiv e generations , malformation s i n bothgenerations , postnata l developmen t

measure s i n F l pups) ; reproductiv e orga n weight s (uterus , prostate , an depididymides) ; reproductiv e orga n histopatholog y (femal e mammar y gland , ovaries , uterus , vagina , cervix , testes , epididymides , prostate , semina l vesicle) ; fertilit y indices ; fecundit y indices ; an destrou s cyclicity . Th e mos t sensitiv e NOAE Li n th e stud y wa s 3 pp man dwa s base d o n adecrease dmaterna l an dpu p bod y weigh t an dincrease dlive r

an d kidne y weight s a t

th e highe r

exposur e level s (1 5 an d7 5 pp mendosulfan) . Ther e wer e n o effect s o n endocrin e organ weights . Ther e wer e n o effect s o n endocrin e orga

n histopatholog y at

an y dos e leve l teste d(e.g. , thyroid , pituitary , an dvariou s reproductiv e organ s i n male s an dfemale s expose d eve n durin g sensitiv e developmenta l periods) . Ther e wer e n o effect s o n reproductiv e indice s i n eithe r generatio n (al l monitore dendpoints) . Ther e wer e n o reproductiv e syste mabnormalitie s note di n pup s o f eithe r generation . Thes e dat ademonstrat e tha t endosulfa n wa s no t

toxi c t o endocrin e orga n system s i n thi s sensitive , i n viv o tes t fo r reproductiv e toxicit y durin g critica l period s o f development . Th e dat aprovide di n thi s multi-generatio n reproductiv e toxicity stud y ar e directl y and/o r

functionall y equivalen t

t o dat acollecte di n al l o f th e EDS P Tie r

1 assay s tha t

examin e aspect s o f

mammalia n endocrin e functio n(i.e. , 890.1100 , 890.1150 , 890.1200 , 890.1250 , 890.1300 , 890.1400 , 890.1450 , 890.1500 , 890.1550 , an d890.1600) . Th e dat a provid e informatio n o n estrogen , androgen , an dthyroi dhormon e syste m 

### 4 1Makhteshi mChemica l Works , Ltd . Study No . MA-END-EDSP-2010031 3 toxicit y o f

endosulfa n i n viv o an dar e mor e tha n adequat e t o asses s th e endocrin e toxicit y potentia l o f endosulfan . Moreover , i n conjunctio n wit h th e othe r availabl e publishe dtoxicit y data , th e multi-generatio n reproductiv e stud y dat aar e als o directl y applicabl e t o identifyin g actua l mode s o f actio n fo r

endosulfa n (se e Tabl e 1) . Fo r example , th e lac k

o f effect s o n fertilit y an dfemal e an dmal e endocrin e orga n histopatholog y i n th e multi-generatio n stud y i s consisten t wit hfinding s i n vitr o tha t endosulfa n i s no t

apoten t estrogeni c agent , i s no t

apoten t androge n recepto r bindin g agent , an dha s n o effect s o n aromatas e activity . Further , th e lac k o f effect s o n thyroi dorga n histopatholog y an dultimat e effect s o f alterin g thyroi dhormon e activit y i n th e multi-generatio n stud y ar e consisten t withth e lac k

o f thyroi dhormon e recepto r bindin g b y endosulfa n (Judso n e t

al . 2009) . 4 . MacKenzi e (1980 ) Revie w o f

thi s GLP-complian t

guidelin e prenata l (teratology ) toxicity study i n rat s indicate s that i t provide s reliabl e dat arelatin g t o a variet y o f EDS PTie r

1 stud y endpoint s a s show n i n Tabl e 3 above . Thi s stud y cover s aspecifi c developmenta l perio dno t examine di n mammal s i n th e EDS PTie r

1 assays , i n uter o exposure . I n uter o exposur e i n EDS P Tie r 1 studie s i s limite dt o assay s conducte dwit hamphibian s an dfish . I n thi s guidelin e stud y i n rats , femal e Sprague-Dawle y rat s wer e administere dendosulfa n (97.3 % pure ) b y gavag e at dose s o f 0 , 0.66 , 2 , an d6 m g endosulfan/

k g bod y weigh t

fro mgestatio n day si x (GD6 ) throug hGD I 9 . I t

shoul db e note dtha t

th e gavag e rout e o f

administratio n i s no t a s relevan t

fo r huma n healt hris k

assessmen t

bu t i s th e mos t

routin e rout e o f exposur e fo r

teratolog y studies . Lik e al l guidelin e prenata l toxicit y studies , thi s stud y wa s designe d t o examin e th e teratogeni c potentia l o f endosulfan . A s aresult , certai n endpoint s relevan t

t o endocrin e activit y wer e assesse d(se e Tabl e 3) . Th e endpoint s measure di n th e prenata l toxicit y study tha t ar e directl y 

### 4 2Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### equivalen t t o endpoint s monitore di n EDS PTie r 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival ; bod y weigh t (bot hadult s an dpups) ; developmental measure s (fetotoxicity ; skeletal an d sof t tissu e malformations) ; reproductiv e organ weight s (gravi duterus) ; an dfertilit y indice s (survival , pre - an dpost-implantatio n loss , dystocia , etc.) . Th e mos t

sensitiv e NOAE Li n th e stud y wa s 0.6 6 mg/kg/da y an d wa s base do n decrease dmaterna l an dfeta l bod y weight , clinica l sign s i n dams , an dmisaligne dsteraabra e i n fetuse s at th e 2 mg/k g exposur e level . Ther e wer e som e developmenta l abnormalitie s reporte di n th e hig hdos e group wher e materna l toxicity wa s als o evident . There were n o effect s o n endocrin e orga n histopatholog y at an y dos e leve l teste d(e.g. , variou s reproductiv e organ s i n male s an dfemale s expose deve n durin g development) . Ther e wer e n o statisticall y significan t effect s o n fertilit y indices , excep t

whe n materna l toxicit y wa s evident . These dat a demonstrate dtha t

endosulfa nwa s no t

toxi c t o endocrin e orga n system s i n thi s sensitive , i n viv o tes t

fo r toxicit y durin g critica l period s o f developmen t

i n utero . Th e dat aprovide di n thi s prenata l toxicit y study ar e functionall y equivalen t t o dat acollecte di n EDS PTie r 1 assay s tha t

examin e aspect s o f mammalia n endocrin e functio n(i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , an d890.1600) . Th e dat aprovid e informatio n o n estrogen , androgen , an dthyroi dhormon e syste mtoxicit y o f endosulfa n i n vivo , an d ar e reliabl e endosulfa n OSRI . 5 . Gilmor e e t

al (2006 ) Revie w o f thi s GLP-complian t

guidelin e developmenta l neurotoxicit y stud y i n rat s indicate s tha t i t provide s reliabl e dat arelatin g t o avariet y o f

EDS PTie r

1 stud y endpoint s a s show n i n Tabl e 3 above . Thi s study cover s aspecifi c developmenta l perio dno t examine di n mammal s i n th e EDS PTie r 1 assays , i n uter o exposure , a s wel l a s 4 3Makhteshi mChemica l Works , Ltd . Stud y No. MA-END-EDSP-2010031 3 

### postnata l developmen t

i n rats . Thi s stud y include s tw o sensitiv e measure s o f developmen t

i n animals , tim e t o preputita l separatio n i n male s an dtim e t o vagina l openin g i n females . In thi s guidelin e stud y in rats , femal e Wista r rat s wer e administere d99.1 % pur e endosulfa n i n die t at dose s o f 0 , 50 , 150 , an d40 0 pp mendosulfa n fro mGD 6 throug hPND2 1 (weaning) , wit hF l pup s evaluate dat

abou t PND60 , an dsacrifice db y PND75 . I t shoul db e note d that adietar y rout e o f

administratio n i s mor e relevan t t o th e expecte d huma n exposur e t o endosulfa n tha n dat atha t migh t b e collecte dusin g othe r route s o f administratio ni n animal s (e.g. , ora l gavag e dosing , intraperitonea l dosing , subcutaneou s dosing) . A s aresul t o f bein g adevelopmental neurotoxicir y study , certai n endpoint s relevan t

t o endocrin e activit y wer e assesse d(se e Tabl e 3) . Th e endpoint s measure di n th e developmenta l neurotoxicit y stud y tha t ar e directl y equivalen t

t o endpoint s monitore di n EDS PTie r 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival ; bod y weigh t

(bot hadult s an dpups) ; an ddevelopmenta l measure s (fetotoxicity , preputia l separation , vagina l opening) . Th e mos t sensitiv e NOAE Li n th e study wa s 5 0 pp mendosulfa n an dwa s base do n decrease dmaterna l bod y weigh t an ddecrease dfeta l bod y weigh t

a t endosulfa ndose s o f

15 0 pp man dgreater , an dadela y i n preputita l separatio n bu t

onl y a t th e mi ddos e leve l (15 0 pp mendosulfan) . Ther e wer e n o accompanyin g change s i n spermparameter s measure dat terminatio n i n thi s group . N o gros s lesion s o f any kin dwere reported , includin g lesion s i n endocrin e organs . N o effect s o n an y reproductiv e indice s wer e reported . Th e onl y findin g note dthat coul db e relate dt o endocrin e effect s wa s th e smal l dela y i n preputia l separatio n i n male s bu t onl y at th e mi dtes t

dos e leve l (10. 8 mg/kg/day) . Th e lac k o f adose

respons e i n th e findin g note dargue s agains t endosulfan bein g an endocrin e toxican t

i n thi s study . These dat ademonstrate dtha t endosulfa n wa s no t directl y toxi c t o endocrin e organ s system s i n thi s sensitive , i n viv o

4 4Makhteshi mChemical Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### tes t fo r toxicit y durin g critica l period s o f development , i n uter o an ddurin g lactation . Th e dat aprovide di n thi s developmenta l neurotoxicit y stud y ar e functionall y equivalen t

t o dat acollecte di n EDS PTie r 1 assay s tha t examin e aspect s o f

mammalia n endocrin e functio n(i.e. , 890.1400 , 890.1450 , 890.1500 , an d890.1600) . Thi s stud y provide s reliabl e endosulfa nOSRI . 6 . Beaver s et

a l (1987 ) Revie w o f thi s GLP-complian t

guidelin e one-generatio n reproductio n stud y i n Bobwhit e quai l indicate s tha t i t provide s reliabl e dat arelatin g t o EDS PTie r 1 stud y endpoint s a s show n i n Tabl e 4 above . I n thi s study , youn g adul t

Bobwhit e quai l wer e expose dt o endosulfa n (96 % purity ) at dose s i n die t

o f 0 , 15 , 30 , 60 , an d12 0 pp m endosulfa nfo r

2 3 week s fro mpre-layin g throug hproductio n o f egg s i n on e generation . Sixtee n mal e an d1 6 femal e bird s wer e paire dan dhouse d individually . The endpoint s monitore dinclude dclinica l sign s of toxicit y in parents , adul t

bod y weigh t

andfoo dconsumption , gros s pathologica l effect s a t sacrifice , an davariet y o f

reproductiv e measure s (numbe r o f egg s laid , numbe r

o f egg s cracked , numbe r

o f egg s set , viabl e embryos , liv e 3-wee k

embryos , numbe r

o f hatchlings , hatchlin g bod y weight , surviva l at 1 4 days , eg g shel l thickness) . Ther e wer e n o effect s o n an y reproductiv e parameter s i n th e stud y a t an y endosulfa n dose . A t

12 0 pp m endosulfa ni n th e diet , ther e wa s adecrease dfoo dconsumptio n reporte d fo r adul t birds . Th e NOAE Li n th e stud y wa s 6 0 ppm . Thes e dat a demonstrate dtha t

endosulfa n wa s no t

directl y toxi c t o endocrin e orga n system s in thi s sensitive , in viv o tes t for toxicit y durin g critica l period s of non-mammalia n development . Th e dat aprovide di n thi s avia n reproductio n study ar e functionall y equivalen t t o dat acollecte di n EDS PTie r 1 assay s tha t

examin e aspect s o f bothnon-mammalia n an dmammalia n endocrin e functio n(i.e. , 890.1100 ; 4 5Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### 890.1350 ; 890 ; 890.1400 , 890.1450 , 890.1500 , an d890.1600) . Thi s study provide s reliabl e endosulfa n OSRI . 7 . Beaver s et

a l (1987 ) Revie w o f thi s GLP-complian t

guidelin e one-generatio n reproductio n stud y i n Mallar dduck s indicate s tha t i t provide s reliabl e dat a relatin g t o EDS PTie r

1 stud y endpoint s a s show n i n Tabl e 4 above . I n thi s study , youn g adul t

Mallar dduck s wer e expose dt o endosulfa n (96 % purity ) at dose s o f 0 , 7.5 , 15 , 30 , an d6 0 pp mendosulfa n fo r 1 8 week s fro mpre-layin g throug hproductio n o f egg s i n on e generation . Sixtee n mal e an d1 6 femal e bird s wer e paire dan dhouse d individually . Th e endpoint s monitore dinclude dclinica l sign s o f toxicity i n parents , adul t

bod y weigh t

an dfoo dconsumption , gros s pathologica l effect s a t sacrifice , an davariety o f

reproductiv e measure s (number o f egg s laid , numbe r

o f egg s cracked , numbe r

o f egg s set , viabl e embryos , liv e 3-wee k

embryos , numbe r

o f hatchlings , hatchlin g bod y weight , surviva l at 1 4 days , eg g shel l thickness) . A t 6 0 pp mendosulfa n i n th e diet , ther e wa s adecrease dfoo dconsumptio n an dbod y weigh t reporte di n adul t birds , a s wel l a s adecreas e i n eg g hatchabilit y an dth e numbe r o f egg s laid . Th e NOAE Li n th e stud y wa s 3 0 ppm . Thes e dat ademonstrate dtha t endosulfa nwa s no t

directl y toxi c t o endocrin e orga n system s i n thi s i n viv o tes t fo r toxicit y durin g critica l period s o f non-mammalia n development . Th e dat aprovide di n thi s avia n reproductio n stud y ar e functionall y equivalen t to dat acollecte di n EDS PTier 1 assay s that

examin e aspect s o f bothnon-mammalia n an dmammalia n endocrin e functio n(i.e. , 890.1100 ; 890.1350 ; 890 ; 890.1400 , 890.1450 , 890.1500 , an d890.1600) . Thi s study provide s reliabl e endosulfa n OSRI .

4 6Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 8 . Dionn e (2002 ) Revie w o f

thi s GLP-complian t

guidelin e ful l lif

e cycl e stud y i n Fathea dminnow s indicate s tha t i t provide s reliabl e dat arelatin g t o EDS P Tie r 1 stud y endpoint s a s show n i n Tabl e 4 above . I n thi s study , 10 0 Fathea dminno w embryo s pe r dos e leve l were expose dt o endosulfa n (98.7 % purity ) continuousl y fo r on e complet e lif

e cycl e (26 0 days) . Th e wate r concentration s o f

endosulfa n employe dwer e 0.038 , 0.075 , 0.15 , 0.30 , an d0.6 0 n g endosulfa n pe r liter . Th e endpoint s monitore dinclude dmeasure s o f fertility , fecundity , developmen t o f fish , survival , bod y weigh t

an dlengtho f

offspring , lengtha t

maturatio n (male s an dfemales) , an ddeformitie s i n hatchlings . Th e autho r report s anon monotoni c an dsuppresse ddose-respons e effec t o n reproductiv e endpoint s at th e thre e highes t

exposur e dose s o f

endosulfa n (0.1 5 |u.g/

Lan dhigher) . Thes e thre e highes t

dos e level s were identifie da s havin g reproductiv e effect s i n Fathea dminnows . Thes e dat aar e directl y equivalen t t o dat a collecte di n th e EDS PTie r 1 890.135 0 assay (Fis hShort-term reproduction) . Althoug hth e stud y di dno t includ e measure s o f

vitellogeni n level s o r gona dsiz e o r

histopathology , th e dat aprovid e adequat e dat afo r screenin g fo r

reproductiv e hazard s t o fish . Thi s stud y provide s reliabl e endosulfa n OSRI . 9 . Heuse l (1998 ) Revie w o f

thi s GLP-complian t

stud y evaluatin g th e possible endocrin e effect s o f

endosufa n i n Fathea dminnow s indicate s tha t i t provide s reliabl e dat arelatin g to EDS PTier 1 study endpoint s a s show n i n Tabl e 4 above . I n thi s study , juvenil e Fathea dminnow s were expose dt o endosulfa n (technica l substance ) fo r 2 1 day s unde r

flow-throug h conditions . Th e wate r

concentration s o f

endosulfa n employe dwer e 0 , 0.05 , 0.1 , 0.2 , 0.40 , an d0.8 0 u g endosulfa n pe r liter . Th e endpoint s monitore dinclude dsurvival , bod y weight , bod y length , an dvitellogeni n 4 7Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 concentrations . Th e positiv e contro l compoun dteste dfo r validatio n o f

th e assay wa s 17a-ethinylestradio l (1 0 ng/ml) . Th e autho r reporte dtha t

ther e wer e n o statisticall y significan t effect s o n bod y weight , bod y length , o r vitellogeni n concentrations . Surviva l decrease dsignificantl y bu t onl y a t dos e (0. 8 u.g/L ; actua l concentratio n determine dt o b e 0.6 2 >g/L) . Th e NOE C wa s determine dt o b e 0.2 8 ug/L(correspondin g t o th e targe t dos e o f 0. 4 ug/L) . Thes e dat aindicate dtha t ther e wa s n o indicatio n o f estrogeni c effect s i n th e fish . These dat aar e directl y equivalen t t o som e o f th e dat acollecte di n th e EDS PTie r 1 890.135 0 assay (Fis hShort-term reproduction) . Thi s stud y provide s reliabl e endosulfa n OSRJ . IV . MAN A Seek s aWaive r fo r A H o f th e Tie r 1 EDS P Assay s tha t

Scree n fo r Endocrin e Disruptio n Potentia l o f Endosulfa n Ther e i s alarg e an drobus t bod y o f endosulfa n OSR I dat afo r screenin g fo r

th e potentia l endocrin e activit y o f endosulfa n i n bothmammalia n organism s an di n non mammalia n system s (birds , amphibians , andfish). The availabl e dat ainclud e in vitr o evaluation s o f

estrogen , androge n an dthyroi dhormon e bindin g and/o r function , a s wel l a s th e effect s o f

endosulfa n o n enzyme s tha t synthesiz e an dmetaboliz e estroge n an d androge n hormone s i n mammal s (Sot o e t al . (1994) ; Soto e t

al . (1995) ; Wad e e t

al . (1997) ; Ramamoorth y e t

al . (1997) ; Arcar o e t

al . (1998) ; Wilso n an dLeBlan c (1998) ; Hunte r e t al . (1999) ; Legle r

e t al . (1999) ; Won g an dMatsumur a(2000) ; Newbol de t al . (2001) ; Anderso n e t

al . (2002) ; Fan g e t

al . (2003) ; Grunfel dan dBonefeld-Jorgense n (2004) ; Bonefeld-Jorgense n e t al . (2005) ; Lemair e e t

al . (2006) ; Lavill e e t

al . (2006) ; Varayou de t

al . (2008) ; Vinggaar de t

al . (2008) ; Judso n e t

al . (2009) ; Brunell i e t

al . (2009)) . Thus , i n term s o f estrogenicity , androgenicit y an dthyroi dhormon e activity , th e availabl e dat adiscusse dabov e provid e mor e tha nadequat e screenin g fo r potentia l mode s o f actio n fo r endosulfa no n thes e endocrin e systems . Moreover , ther e ar e reliable , robus t i nviv o studie s that

directl y monitor , o r

ar e functionall y equivalen t to , al l o f th e endpoint s tha t woul db e monitore di n Tie r 1 EDS Passays . Base do n th e fac t tha t non e o f th e studie s demonstrat e tha t

endosulfa n i s adirec t

toxican t t o endocrin e system s i n organism s (discusse dabove) , ther e i s n o scientificall y defensibl e reaso n t o perfor man y o f th e Tie r 1 4 8Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3

EDS P studies , particularl y give n th e alread y extensiv e an dnumerou s studie s performe d t o date . A s aresult , MAN A i s askin g fo r awaive r o f al l eleve n o f

th e Tie r 1 studies . Th e i n vitr o studie s screenin g fo r estrogen , androge n an dthyroi dhormon e activit y indicate dthat

endosulfan , i n some instances , affecte destrogen an dandroge n function s and/o r receptors . I n vitr o studies , however , ar e onl y relevan t a s indicator s o f

th e potentia l bindin g o f

achemica l t o hormon e receptor s o r indicator s o f

th e potential t o affec t hormon e metabolism . Thi s i s becaus e th e condition s o f i n vitr o assay s d o no t

allo w fo r th e know n relationshi p o f pharmacokinetic s an dmetabolis mt o th e pharmacodynamic s o f chemica l response s an dchemica l toxicity . Further , i n th e cas e o f endocrin e syste m function , suc hisolate dassay s hav e limite dpredictiv e valu e fo r th e condition s o f

livin g organism s wher e endocrin e regulatio n i s comple x an dinterrelated . Therefore , th e i n vitr o studie s wit hendosulfa n ar e usefu l fo r identifyin g th e nee dfo r

collectio n o f

i n viv o dat a tha t shoul db e evaluate dfo r

potentia l interaction s wit hestrogen-relate dan dandrogen

relate dfunction s (e.g. , reproductiv e function) . I n th e cas e o f endosulfan , thi s i n viv o dat a wa s indee dcollecte d(th e multi-generatio n study i n rats , etc.) . It mus t als o b e remembere dtha t

th e potenc y o f

endosulfa n i n thes e i n vitr o studie s wa s ver y low , wit h potencie s i n th e rang e o f

10 5 to 10 6 time s les s tha n th e naturall y occurrin g hormones . Whe n evaluatin g th e i n viv o toxicologica l data , th e type s o f studie s an dth e dos e level s administere dmus t

bot hb e considered . Suc hi n viv o studie s ar e mor e relevan t measure s o f

endocrin e toxicit y potentia l a s metaboli c system s an dth e comple x interaction s o f

variou s hormona l system s ar e intact , unlik e th e condition s o f mos t EDS P Tie r 1 test s ( 8 o f

1 1 ar e eithe r

i n vitr o assay s o r

involv e model s wit hnon-intac t hormona l systems) . Th e endosulfa n studie s sho w tha t th e compoun ddoe s no t

appear t o targe t endocrin e organ s whe n toxicit y i s produced , a s evidence db y alac k o f effect s o n reproductiv e organ s (mal e an dfemale ) an dthyroi dglan dhistopatholog y i n al l o f th e availabl e studies . An y effect s reporte dwithendosulfa n exposure that migh t b e linke dt o endocrin e activit y wer e limite dgenerall y t o hig hdoses . Th e endosulfan guidelin e toxicolog y studies , lik e al l suc hguidelin e i n viv o toxicolog y studies , wer e designe dpurposel y t o b e valid , realisti c evaluation s o f possibl e effect s i n humans . Regulator y bodie s rel y o n suc hstudie s t o mak e decision s regardin g saf e level s fo r

huma n exposure . A s aresult , th e availabilit y o f suc hstudie s make s th e 4 9Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3



### performanc e o f

EDS PTie r

1 assay s les s relevan t

a s screenin g assay s fo r

hazard assessment . I t shoul db e clea

r base do n th e discussio n alread y provide drelatin g t o th e availabl e endosulfa n endocrin e toxicit y dat atha t non e o f th e Tie r 1 EDS Ptest s nee dt o b e conducte di f

th e goal i s t o scree n fo r

potentia l endocrin e activity . Thus , considerin g th e availabl e data , MAN A seek s awaive r fo r al l o f th e Tie r 1 EDS Pstudies . Th e rational e fo r MANA' s positio n i s outline dbelo w regardin g particular studie s that

d o no t nee dt o b e performed . A . MAN A Seek s aWaive r fo r Performanc e o f

Tier 1 I n Vitro Assay s fo r Estroge n Activit y (Estroge n Recepto r Bindin g 890.125 0 an dEstroge n Recepto r Transcriptiona l Activatio n 890.1300) EDS PTie r

1 assay s 890.125 0 an d890.130 0 ar e screenin g assay s t o identif y mode s o f

actio n fo r estrogeni c activity involvin g bindin g to an d activatio n o f

estroge n receptors . Ther e ar e thirtee n separat e studie s that providin g i nvitr o estroge n recepto r

activit y dat aavailabl e fo r

determinin g th e abprovid e adequat e OSR I fo r

determinin g th e abilit y o f

endosulfa n t o bin dt o an dactivat e estroge n receptor s i n vitro . These studie s hav e bee n publishe di n th e peer reviewe dscientifi c literatur e (Tabl e 3 ) usin g avariet y o f standar di n vitr o assay s fo r assessin g E Rbindin g potenc y an dE Ractivation . Althoug hnon e o f th e studie s i s technicall y identica l i n desig n t o th e EDS PTie r 1 E Rstudie s (890.1250 ; 890.1300) , th e availabl e studie s ar e bot hdirectl y an dfunctionall y equivalen t an d provid e dat ao n essentiall y th e sam e endpoints , includin g compariso n o f th e activit y of endosulfa nto appropriat e contro l agents . The result s hav e bee n consistent i n showin g that

endosulfan is , a

t best , awea

k estrogeni c agent , with potenc y severa l order s o f

magnitud e les s tha n positiv e control , agent s (i.e. , 17p

estradiol) ; activity o f

endosulfa n i n vitr o i s onl y see n at dose s i n th e hig h micromolar

range . Therefore , MAN A believe s ther e i s n o scientifi c basi s fo r performin g additiona l i n vitr o estroge n recepto r activit y studies , specificall y th e Estroge n Recepto r

Bindin g assay (890.1250 ) an dth e Estroge n Recepto r Transcriptiona l Activatio n (Huma n Cel l Lin e HeLa-9903 ) assay (890.1300) . Th e 5 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### availabl e dat aar e adequat e fo r assessin g th e activit y o f

endosulfa n a t

E R receptors . A s aresult , awaive r o f bot h890.125 0 (Estroge n Recepto r Bindin g assay ) an d890.130 0 (Estroge n Recepto r Transcriptiona l Activatio n assay ) i s sought . B . MAN A Seek s aWaive r fo r Performanc e o f

th e Tier 1 I n Vitr o Assay s fo r Androge n Activit y (Androge n Recepto r Bindin g 890.115 0 an dAromatas e Activity 890.1200 ) EDS PTie r

1 assay s 890.115 0 an d890.120 0 ar e screenin g assay s t o identif y mode s o f

actio n fo r androgeni c an danti-androgeni c activit y involvin g bindin g t o androge n receptor s o r alteration s i n conversio n o f androgen s t o estrogens . Ther e ar e fiv e separat e studie s providin g i n vitr o androge n recepto r activit y dat atha t

provid e adequat e OSR I fo r determinin g th e abilit y o f

endosulfa n t o bin dt o androge n receptors . Th e studie s b y Anderse n e t al . (2002) , Fan g e t

al . (2003) , Lavill e e t

al . (2006) , Vinggaar de t

al . (2008) , an dJudso n e t

al . (2009 ) provid e dat ao n bot handroge n recepto r bindin g potency , agonis t

an dantagonis t activity , a s wel l a s th e potentia l fo r endosulfa n t o inhibi t

aromatas e activit y i n vitro . Th e availabl e studie s provid e directl y an dfunctionall y equivalen t dat ao n essentiall y th e sam e endpoint s a s foun di n th e Tie r 1 EDS Passays , includin g compariso n o f

th e activit y o f

endosulfa n t o appropriat e contro l agents . Thus , ther e ar e adequat e OSR I fo r

screenin g fo r

potentia l activit y o f

endosulfa n a t

A R receptor s an do n aromatas e function , an dther e i s n o scientifi c basi s fo r performin g additiona l i n vitr o androge n recepto r activit y studies , specificall y th e Tie r 1 Androge n Recepto r

Bindin g assay (890.1150) , an dadditiona l i n vitr o aromatas e activit y studies , specificall y th e Tie r 1 Aromatas e assay (890.1200) . A waive r o f bot hth e 890.115 0 (Androge n Recepto r Bindin g assay ) stud y an dth e 890.120 0 (Aromatas e assay ) stud y wit hendosulfa ni s sought .

5 1Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### C . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Steroidogenesi s Assa y (890.1550) Th e Steroidogenesi s assay (890.1550 ) i s ascreenin g assay whos e state d inten t i s t o identif

y compound s tha t

affec t th e steroidogeni c pathwa y beginnin g wit hreaction s tha t

occu r afte r th e gonadotropi n receptor s throug hth e productio n o f testosteron e an destradiol . Thi s doe s no t includ e effect s o f

compound s o n th e hypothalamu s o r

pituitar y glands . I n thi s assay , th e level s o f estradio l an d testosteron e tha t

ar e produce di n cell s i n cultur e bot hbefor e an dafte r exposur e t o th e tes t chemica l ar e measured . Since , b y design , suc ha n i n vitr o assa y i s les s robus t an dles s relevan t

t o screenin g fo r

potentia l effect s i n intac t

organisms , an y availabl e i n viv o dat awoul db e preferre dwhe n screenin g fo r endocrin e disruptio n potentia l o f

achemical . Thus , i n viv o dat ao n se x steroi dhormon e level s an dthei r metabolis man dpharmacokinetic s i n viv o woul db e preferre dove r suc hstudie s i n cells , dat atha t

i s availabl e fo r

endosulfa n (Wilso n an dLeBlan c 1998) . Th e stud y b y Wilso n an dLeBlan c (1998 ) provide s adequat e endosulfa n OSR I fo r screenin g fo r effect s o f endosulfa n o n Steroidogenesis , sinc e th e study provide s dat ao n testosteron e metabolis man dpharmacokinetic s i n mal e an dfemal e animals . Ther e ar e als o functionall y equivalen t OSR I o nSteroidogenesi s activit y i nth e endosulfa nregistratio n studie s (e.g. , apica l effect s o n reproductiv e orga n weight s an dhistopatholog y a s wel l a s reproductiv e capacity) . Th e endosulfa n OSR I eve n include s amulti-generatio n reproductiv e toxicit y stud y wher e organism s wer e expose dt o endosulfa n a t

al l critica l stage s o f

development . Thus , ther e ar e adequat e OSR I fo r

assessin g th e activit y o f

endosulfa n o n Steroidogenesis , an d ther e i s n o scientifi c basi s fo r performin g additiona l i n vitr o Steroidogenesi s studie s whe n i n viv o dat ao n th e sam e endpoint s i s available . A s aresult , awaive r o f th e 890.155 0 (Steroidogenesi s assay ) stud y wit hendosulfa n i s sought . D . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Amphibia n Metamorphosi s Assa y (890.1100) Th e Amphibia n Metamorphosi s assa y (890.1100 ) i s describe di n th e guidelin e a s a"screenin g assa y intende dt o empiricall y identify substance s whic h 5 2Makhteshi mChemical Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### ma y interfer e wit hth e norma l functio no f th e hypothalamic-pituitary-thyroi d (HPT ) axis" . I t

i s intende dt o b e ageneralize dvertebrat e mode l tha t i s base do n th e conserve dstructure s an dfunction s o f th e HP T axi s o f

variou s species . I t

i s als o pointe dou t

tha t amphibia n metamorphosi s provide s " awell-studied , thyroid

dependen t proces s whic hrespond s t o substance s activ e withi n th e HP T axis , an d i t i s th e onl y existin g assay tha t detect s thyroi dactivit y i n a n anima l undergoin g morphologica l development" . MAN A i s seekin g t o waiv e th e requiremen t fo r performanc e o f

thi s stud y becaus e ther e ar e adequat e i n viv o dat acollecte di n guidelin e toxicolog y studie s tha t cove r al l stage s o f

mammalia n development , an d ar e consisten t

i n showin g tha t

endosulfa n ha s n o effect s o n functionall y equivalen t endpoint s i n mammalia n species . Fo r example , th e endosulfa n OSR I includ e dat afro mamulti-generatio n reproductiv e toxicit y stud y i n rats , aprenata l toxicit y stud y i n rats , an dadevelopmenta l neurotoxicit y stud y i n rats . Al l o f thes e studie s demonstrate dtha t

endosulfa ndoe s no t

induc e developmenta l an d morphologica l effects , includin g non e o f th e possibl e apical effect s that woul d result fro meithe r

increasin g o r

decreasin g thyroi dhormon e level s i n mammal s (e.g. , growth , metaboli c rat e an dfunction , feta l brai n development , an dTabl e 1 above) . Further , result s fro mth e chroni c studie s i n rat s an ddog s demonstrate dn o effect s o n thyroi dglan dhistopathology , effect s tha t woul db e expecte dt o appear i f endosulfa n wer e producin g direc t effect s o n eithe r

thyroi dhormon e synthesi s o r function . Additionally , ther e i s endosulfa n OSR I collecte di n developin g amphibian s (Brunell i e t

al . 2009 ) wher e effect s o f endosulfa n o n metamorphosi s ar e provided . Performin g th e amphibia nassa y merel y t o monito r change s i n thyroi dhormon e level s o r

se x hormon e level s i n developin g amphibians , endpoint s tha t

hav e no t bee n quantifie di n currentl y availabl e endosulfa n OSRI , i s no t scientificall y justifie dwhe n robus t i n viv o dat ai n developin g organisms , includin g tadpoles , i s alread y availabl e an dthos e dat ad o no t sho w tha t th e thyroi dhormon e syste mi s atarge t fo r endosulfa n effects . Therefore , awaive r o f 890.110 0 (Amphibia n Metamorphosi s assay ) is sough t becaus e ther e is no scientifi c basi s fo r

performin g a n additiona l stud y whe n th e availabl e endosulfa n OSR I ar e considered . 5 3Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 E . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Femal e Puberta l Assay (890.1450 ) Th e purpos e o f

th e puberta l femal e ra t

assa y (890.1450 ) i s state di n th e guidelin e t o b e identifyin g chemical s tha t hav e apotentia l t o interac t wit hth e endocrin e syste mb y identifyin g effect s o n puberta l developmen t an dthyroi d functio n i n intac t

peri-puberta l rats . Ther e ar e adequat e i n viv o OSR I availabl e fo r endosulfa n demonstratin g tha t thi s chemica l i s no t

apoten t estrogeni c o r

anti

estrogeni c agen t

i n vivo , an dha s n o identifiabl e effect s o n thyroi dfunction . Th e OSR I include s th e robust , guidelin e registratio n studie s (Tabl e 3) . Thes e i n viv o evaluation s o f

femal e reproductiv e functio nan ddevelopmenta l toxicit y i n rat s hav e provide ddat ao n exposur e o f animal s a t

al l stage s o f

developmen t

(i n utero , durin g lactation , throughou t post-nata l development , throughou t peri-puberta l development , durin g adulthood , an ddurin g senescence ) a s wel l a s providin g dat a o n man y o f

th e sam e endpoint s foun di n EDS PTie r 1 studie s (Tabl e 3) . Fo r example , i n th e Tie r

1 Femal e Puberta l assay (rat) , importan t endocrin e endpoint s monitore dinclud e identificatio n o f abnormalitie s associate dwit hse x organ s an d pubert y marker s (se e Tabl e 2 above) , a s wel l a s thyroi dtissue . Th e guidelin e toxicit y studie s i n rat s suc ha s th e two-generatio n reproductiv e toxicit y study , th e prenata l toxicit y study , th e developmenta l neurotoxicit y study , an dth e chroni c toxicit y studies , al l include dassessment s o f endosulfan' s effect s o n femal e reproductiv e tissue s an dorgans , bothi n adul t animal s an di n developin g animal s (includin g peri-puberta l animals) , includin g evaluation s o f effect s acros s generations . Thes e studie s als o include dhistopathologica l assessment s o f thyroi d glan dtissu e i n animal s expose dbot hdurin g gestatio n an ddevelopmen t an da s adults . Th e guidelin e reproductiv e toxicity an ddevelopmenta l toxicit y studie s provid e adequat e dat ao n femal e reproductiv e orga n effect s followin g endosulfa n exposure , a s wel l a s fertility , fecundity , vagina l opening , an destrou s cycling . Th e onl y endpoin t

fro mth e EDS PTie r

1 i n viv o assay i n puberta l femal e rat s tha t ha s no t bee n directl y measure di n th e currentl y availabl e endosulfa n guidelin e toxicit y studie s i s th e leve l o f thyroi dhormone s i n seru mo f animal s 5 4Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### followin g endosulfa n exposure . Give n tha t th e ultimat e apica l effect s o f eithe r increasin g o r

decreasin g thyroi dhormon e level s i n mammal s hav e bee n monitore di n th e availabl e studie s (e.g. , growth , metaboli c rat e an dfunction , feta l brai ndevelopment) , an dn o suc hapica l effect s wer e observed , ther e doe s no t appear t o b e a n adequat e scientifi c basi s fo r requirin g performanc e o f

th e Tie r 1 puberta l femal e screenin g stud y (890.1450 ) simpl y t o provid e fo r measure s o f thyroi dhormon e level s i n rats . Therefore , awaive r o f EDS PTie r

1 890.145 0 (Puberta l Femal e Ra t

Development ) i s sought . F . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Mal e Puberta l Assa y (890.1500 ) Th e purpos e o f

th e assa y (890.1500 ) i s state di n th e guidelin e t o b e identifyin g chemical s tha t

hav e apotentia l t o interac t wit hth e endocrin e syste m b y identifyin g effect s o npuberta l developmen t an dthyroi dfunctio n i nintac t peri

puberta l mal e rats . Ther e ar e ampl e i n viv o endosulfa n OSRI o n mal e reproductiv e functio n an dpatholog y t o indicat e tha t th e system s tha t

involv e androge n activit y ar e no t

uniquel y targete db y endosulfa n an dtha t the y ar e no t uniquel y sensitiv e t o th e effect s o f endosulfan . Th e endosulfa n OSR I studie s includ e th e robus t

guidelin e studie s i nrat s suc ha s th e two-generatio n reproductiv e toxicit y study , th e developmenta l toxicit y study , th e developmenta l neurotoxicit y study , an dth e subchroni c an dchroni c toxicit y studie s (Table 3) . Thes e i n viv o evaluation s o f mal e reproductiv e function , developmenta l toxicity , a s wel l a s genera l toxicit y i n rat s hav e provide ddat ao n th e sam e endpoint s a s generate di nassay s performe da s par t o f Tie r 1 i nth e EDSP , includin g th e mal e puberta l ra t

stud y (890.1500) . Fo r

example , i n th e Tie r

1 Mal e Puberta l assay (rat) , th e tes t

i s use dt o scree n fo r

androgen , anti-androgen , an dthyroi dactivit y i n mal e animal s durin g sexua l maturatio n b y examinin g abnormalitie s associate d wit hse x organ s an dpubert y marker s (se e Tabl e 2 above) , a s wel l a s thyroi d tissue . Th e guidelin e endosulfa ntoxicit y studie s i n rat s (Tabl e 3 ) al l include d assessment s o f

endosulfa n effect s o n androgen-sensitiv e mal e reproductiv e tissue s an dorgans , bot hi n adul t

animal s an di n developin g animal s (includin g peri

5 5Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 puberta l animals) , includin g assessment s o f effect s acros s generations . Thes e studie s als o include dhistopathologica l assessment s o f thyroi dglan dtissu e i n animal s expose dbot hdurin g gestatio n an ddevelopment , an da s adults . Th e onl y endpoint s fro mth e Tie r 1 i n viv o assay i n puberta l mal e rat s tha t hav e no t bee n directl y measure di n th e currentl y availabl e endosulfa n guidelin e toxicit y studie s i s th e leve l o f thyroi dhormone s i n seru mo f animal s followin g endosulfa n exposure . Th e Tie r 1 assay als o measure s level s o f th e se x hormon e testosterone . Functionall y equivalen t i n viv o endosulfa n OSR I dat ao n testosteron e level s ca n b e foun di n th e stud y b y Wilso n an dLeBlan c (1998) . Moreover , wit hrespec t

t o se x hormon e level s i n animal s (i.e. , testosterone) , th e fac t tha t non e o f th e i n viv o data , includin g th e multi-generatio n reproductiv e toxicit y stud y i n rats , indicat e toxicit y induce db y endosulfa n tha t coul db e relate d t o significan t

change s i n hormon e level s alone , ther e doe s no t appear t o b e a stron g scientifi c basi s fo r

requirin g performanc e o f EDS PTie r

1 screenin g studie s simpl y t o provid e fo r

additiona l measure s o f

se x hormon e level s i n peri-puberta l mal e rats . Further , give n tha t th e ultimat e effect s o f

eithe r increasin g o r decreasin g thyroi dhormon e level s i n mammal s hav e bee n monitore di n th e availabl e studie s (e.g. , growth , metaboli c rat e an dfunction , feta l brai n development) , an dther e i s n o evidenc e o f suc hapica l effects , ther e doe s no t appear t o b e astron g scientifi c basi s fo r requirin g performanc e o f astudy , suc ha s th e mal e puberta l assay i n rats , simpl y t o provid e fo r measure s o f

thyroi d hormon e level s i n rats . Therefore , awaive r o f EDS PTie r

1 890.150 0 (Puberta l Mal e Ra t Development ) i s sought . G . MAN A aSeek s aWaive r fo r Performanc e o f

th e Tie r 1 Uterotrophi c Assa y (890.1600 ) Th e purpos e o f

th e Uterotrophi c assa y (890.1600 ) i s state dt o b e a n i n viv o screenin g assa y providin g dat aabou t asingl e endocrin e mechanism , estrogenicity . I n th e assay , ovarectomize drat s ar e expose dfo r thre e days (subcutaneou s injection ) t o th e tes t chemical . Th e majo r

endpoin t monitore di s uterin e weigh t

changes . I n th e cas e o f

endosulfan , ther e ar e thre e endosulfan 

### 5 6Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3 

### OSR I studie s tha t

ar e actua l Uterotrophi c assay s (Wad e e t al . 1997 ; Newbol de t al . 2001 ; Varayou de t

al . 2008) . Additionally , ther e ar e adequat e endosulfa n OSR I tha t ar e directl y equivalent , monitorin g uterin e weigh t change s i n animal s o f al l stage s o f

development . Th e OSR I includ e severa l guidelin e studie s tha t involve ddosin g fo r

u p t o 2 year s (Ruckma n e t al . 1989) , a s wel l a s dosin g throughou t

al l stage s o f

developmen t

(Edward s 1983 ; MacKenzi e 1980) . A s a result , ther e i s n o scientifi c basi s fo r conductin g anothe r

Uterotrophi c assay wit h endosulfan . Therefore , awaive r o f EDS PTie r

1 890.160 0 (Uterotrophi c assay ) i s sought . H . MAN A aSeek s aWaive r fo r Performanc e o f

th e Tie r 1 Hershberge r Assa y (890.1400 ) Th e purpos e o f

th e Hershberge r

assa y (890.1400 ) i s state dt o b e a n i n viv o screenin g assa y providin g dat aabou t asingl e endocrin e mechanism , androgenicity . I n th e assay , castrate drat s ar e expose dfo r te n day s (subcutaneou s injection ) t o th e tes t

chemical . Th e majo r

endpoin t monitore di s change s i n androgen-dependen t

tissu e an dorga n weights . Clearly , ther e ar e adequat e endosulfa n OSR I tha t

ar e directl y equivalent , monitorin g weigh t change s i n a variet y o f mal e reproductiv e organ s (androgen-dependen t organs ) i n animal s o f

al l stage s o f development . Th e OSR I includ e several guidelin e studie s that involve d dosin g fo r u p t o 2 year s (Ruckma n e t al . 1989) , a s wel l a s dosin g throughou t al l stage s o f developmen t

(Edward s 1983 ; MacKenzi e 1980) . Th e multi-generatio n reproduction stud y eve n include dspermevaluation . A s aresult , ther e i s n o scientifi c basi s fo r

conductin g anothe r

i n viv o assay wit hendosulfa n wher e / 

### androgen-sensitiv e orga n weigh t change s ar e monitore da s i n th e Hershberge r assay . Therefore , awaive r o f EDS PTie r

1 890.160 0 (Hershberge r

assay ) i s sought .

5 7Makhteshit n Chemica l Works , Ltd . Study No . MA-END-EDSP-2010031 3 

### I . MAN A aSeek s aWaive r fo r Performanc e o f

th e Tier 1 Fis hShort

Ter mReproductio n Assa y (890.1350 ) Th e purpos e o f

th e assay (890.1350 ) i s state di n th e guidelin e t o b e a screenin g tes t

fo r reproductiv e fitnes s i nfathea dminno w a s ameasur e o f toxican t effects . Th e tes t

measure s avariet y o f

histologica l an dbiochemica l endpoint s tha t relat e t o disturbance s i n th e activit y o f th e hypothalamus-pituitary-gonada l (HPG ) axis . I n th e assay , mal e an dfemal e fis h(mature ) ar e expose dfo r 2 1 day s an d survival , reproductiv e behavior , an deffect s o noffsprin g ar e monitored . Ther e ar e adequat e i nviv o endosulfa nOSR I o nfis hreproductiv e functio n an deffect s o n offsprin g (Dionn e 2002 ; Gormle y an dTeathe r 2003 ; Tabl e 4) , a s wel l a s tw o reproductio n studie s i n bird s (Beaver s 1987 ; Beaver s e t al . 1987) , an dastud y evaluatin g th e potentia l endocrin e toxicity o f endosulfan i n fis h(Heusel 1999) . Th e stud y o f

Dionn e (2002 ) i s aGLP-complian t fis hful l life-cycl e stud y an db y desig n i s mor e robus t

i n term s o f

exposur e duratio n tha n th e short-ter m reproductio n stud y tha t

i s par t o f Tie r 1 i nth e EDSP . Thes e i nviv o evaluation s o f fis hreproductio n hav e provide ddat ao n mos t o f th e sam e endpoint s a s generate d i n th e EDS P Tie r

1 stud y (890.1350) . Vitellogeni n level s wer e measure di n th e 199 9 stud y b y Heusel . Th e onl y endpoint s fro mth e Tie r 1 i nviv o assay tha t

ar e no t directl y measure di nth e currentl y availabl e endosulfa n guidelin e toxicit y studie s ar e gona dsiz e an dhistolog y followin g endosulfa n exposure . However , functionall y equivalen t

i n viv o endosulfa n OSR I o n thes e endpoint s i s availabl e i n th e studie s b y Gormle y an dTeathe r (2003 ) an dDionn e e t

al . (2002) . Fo r

example , measure s o f

fertilit y an dreproductio n a s wel l a s morphologica l changes . Thus , there doe s no t

appear t o b e astron g scientifi c basi s fo r requirin g performanc e o f

a EDS PTie r

1 screenin g stud y simpl y t o provid e fo r afe w additiona l measure s i n fis hexpose dfo r

ashorte r lengtho f time . Therefore , awaive r

o f EDS PTie r

1 890.135 0 (Fis hShort-TermReproduction ) i s sought . V . Overal l Summar y Th e precedin g discussio n provide s adetaile ddescriptio n o f availabl e endosulfa n OSRI tha t i s use dt o suppor t

waiver s fo r

al l eleve n o f

th e EDS PTie r

1 screenin g assays . 5 8Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3



### Th e OSR I discusse di nthi s T O respons e includ e 2 1 publishe dstudie s an dnin e guidelin e registratio nstudies . Clearl y th e endosulfa nOSR I i s extensiv e an drich , includin g informatio nspecifi c t o screenin g fo r th e potentia l fo r

estrogen , androge n an dthyroi d hormon e syste meffect s a s wel l a s th e effect s o f endosulfa n o n specifi c endpoint s (i.e. , potential mode s o f

action) . A s aresult , ther e i s n o scientificall y defensibl e basi s fo r performin g additiona l EDS Pscreenin g studie s wit hendosulfan . Thus , MAN A i s seekin g waiver s fo r

performanc e o f

al l eleve no f

th e EDS PTie r

1 studies : 890.110 0 (Amphibia n Metamorphosis) ; 890.115 0 (Androge n Recepto r Binding) ; 890.120 0 (Aromatase) ; 890.125 0 (Estroge n Recepto r Binding) ; 890.130 0 (Estroge n Recepto r Transcriptiona l Activation) ; 890.135 0 (Fis hShort-Ter mReproductio n Assay) ; 890.140 0 (Hershberge r assay) ; 890.145 0 (Puberta l Femal e Ra t Development) ; an d890.150 0 (Puberta l Mal e Ra t Development) ; 890.155 0 (Steroidogenesis) ; an d890.160 0 (Uterotrophi c assay) .

5 9Makhteshi mChemica l Works , Ltd . Stud y No . MA-END-EDSP-2010031 3

VI . Reference s Cite d 

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Newbold , R.R. , Jefferson , W.N. , Padilla-Banks , E. , Walker , V.R. , Pena , D.S . (2001 ) Cel l respons e endpoint s enhanc e sensitivit y o f th e immatur e mous e uterotrophi c assay . Reproduct . Toxicol . 15:245-252 . Ramamorthy , K. , Wang , F. , Chen , I-C. , Norris , J.D. , McDonnell , D.P. , Leonard , L.S. , Gaido , K.W. , Bochinfuso , W. , Korach , K.S. , Safe , S . (1997 ) Estrogeni c activit y o f adieldrin/toxaphen e mixtur e i n th e mous e uterus , MCF- 7 huma n breas t cance r cells , an dyeast-base destroge n recepto r assays : n o apparen t

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Wade , M.G. , Desaulniers , D. , Leingartner , K. , Foster , W.G . (1997 ) Interaction s betwee n endosulfa n an ddieldri nn estrogen-mediate dprocesse s i n vitr o an di n vivo . Reproduct . Toxicol . 11:791-798 . Wilson , V.S. , LeBlanc , G.A . (1998 ) Endosulfa n elevate s testosteron e biotransformatio n an dclearanc e i n CD- I mice . Toxicol . Appl . Pharmacol . 148:158-168 . Wong , P.S.Y. , Matsumura , F . (2006 ) Seru mfre e BG- 1 cel l proliferatio n assay : a sensitiv e metho dfo r

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