# Vo. L DIAZINO N EDS P TES T ORDER OrderNo.EDSP-057801-3 6 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 Diazino 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 ;• . • . February 12 , 201 0

• •• • •.. • • STUD Y NO . MA-DZN-EDSP-2010021 3

Makhteshi mChemica l Works , Ltd . Beer Shev a84100 , Israe l c/o Makhteshi mAgan o f Nort h America , Inc . 451 5 Fall s o f

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TOTAL PAGES : 4 2Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3

STATEMENT O F N 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 d i 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 FIFR A Sectio n 1 0 (d ) (1 ) (A) , (B ) o r (C) . Thi s statement

override s an y confidentiality marking s withi n thi s study . Company :
Compan y Representativ e Makhteshi mChemica l Works , Ltd . Bee r Shev a84100 , Israe l

P . Leanne xPr

yiiet t Authorize d Representativ e - MC W Regulator y Produc t Manage r - MAN A

Dat e ••••• I 

## : • :Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3

GOO D LABORATOR Y PRACTIC E COMPLIANC E STATEMEN T Th e material 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 h i n 4 0 CF R160 .

Author :

Laur aM . Plunkett , Ph . D. , DABT . 

# 2-/2-20 Q

Submitter :

Dat e **• *
:•••! , P . Leann e Authorize dAgen t

(MCW ) an dRegulatory Produc t Manage r (MANA ) ••• •

Sponsor :

Dat e P . Leann e vf

u Authorize dAgen t

(MCW ) an dRegulatory Produc t Manage r (MANA )Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3

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

DiazinonOSR I 5 A . Directl y o r

Functionall y Equivalen t

Diazino n OSRJ Identifie di n th e Publishe d Literature 1 6 B . Directl y o r

Functionall y Equivalen t

Diazino n OSR

J Identifie di n Diazino n Registratio n Studie s 2 1 IV . Discussio n o f

th e EDS P Tie r

1 Test s Neede dfo r

Assessment

o f th e Endocrin e Disruptio n Potential o f

Diazino n 3 2 A . MAN A Seek s aWaive r fo r Performanc e o f

Bot h Tier 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 Transcriptional Activatio n 890.1300 ) 3 4 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 Activity (Androge n Recepto r Bindin g 890.115 0 an d Aromatas e Activity 890.1200 ) ..3 5 C . MAN A Seek s aWaive r fo r Performanc e o f

th e Tier 1 Amphibian Metamorphosi s Assa y (890.1100 ) 3 5 D . MAN A Seek s aWaive r fo r Performanc e o f

th e Tier 1 Femal e Puberta l Assay (890.1450 ) 3 6 E . MAN A Seek s aWaive r fo r Performanc e o f

th e Tier 1 Mal e Puberta l Assay (890.1500 ) 3 8 V . Overal l Summar

y 4 0 VI . Reference s Cite d4 1Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3



### I . Introductio n O n Novembe r

5 , 2009 , th e U.S . Environmenta l Protectio n Agenc y (EPA ) issue da Testin g Order

(TO ) fo r diazino n (CA S #333-41-5 ) 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 P Tier

1 testin g fo r

diazinon . Th e Tier

1 test s liste dfo r

submissio n include : 1 ) Amphibia n Metamorphosi s assa y (890.1100) ; 2 ) Androge n Recepto r Bindin g assa y (ra t prostate ) (890.1150) ; 3 ) Aromatas e assa y (human recombinant ) (890.1200) ; 4 ) Estroge n Recepto r

Bindin g assa

y (890.1250) ; 5 ) Estroge n Recepto r Transcriptional Activatio n assa

y (human cel l lin e HeLa-9903)(890.1300) ; 6 ) Fis h Short-ter m Reproductio n assa

y (890.1350) ; 7 ) Hershberge r assa y (890.1400) ; 8 ) Femal e Puberta l assa y (rat ) (890.1450) ; 9 ) Mal e Pubertal assa y (rat ) (890.1500) ; 10 ) Steroidogenesi s assa y (human cel l lin e H295R ) (890.1550) ; an d11 ) Uterotrophi c assa y (890.1600) . Thi s documen t i s arespons e t o th e TO , an dha s bee n prepare db y Makhteshim-Agan 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 dat aalread y availabl e o n th e endocrin e disruptio n potentia l o f diazinon , 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 assessment

o f man y o f th e same endpoint s include di n EDS P Tier

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

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

certai n EDS P Tie r

1 assays . II . Backgroun d EDS P Tie r

1 assay s were designe dt o provid e dat arelevan t t o assessin g th e endocrin e disruptio n potentia l o f chemical s b y identifyin g potential 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 potential mode s o f actio n assesse d 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 d aromatas e activity) , a s wel l a s i n viv o test s o f mammalia n an dfis h reproductio n involvin g bot h mal e an dfemal e organisms .Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 Befor e discussin g detail s o n th e EDS P Tie r 1 studies , i t

i s usefu l t o revie w wha

t ha s alread y bee n establishe d abou t chemically-induce d endocrin e toxicity . Ther e are well-know n biological effect s that can b e monitore d i n viv o i n anima l testin g an d use d t o identif y compound s that

ar e anti-androgens , compound s that ar e estrogenic , an d compound s that

affec t thyroi d hormon e activity . Th e biological 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 d i n Tabl e 1 are apical endpoint s that

ar e alread y routinel y monitore d i n standar d toxicolog y studie s that are part o f th e pesticid e registratio n process . Therefore , i n th e cas e o f chemical s suc h a s diazinon , whic h ar e currentl y registere d fo r us e i n th e Unite d States , there i s awealt h o f toxicolog y dat aavailabl e o n endpoint s that addres s endocrin e toxicity . Tabl e 1 Know n Chemically-Induce dEffect s o n Hormone-Relate d Apica l Biological 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 dovar

y 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 d gestatio n tim e an d tim 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 )Makhteshi mChemical Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 

### Wit h th e informatio n i n Tabl e 1 i n mind , i t i s clea r that an y discussio n o f

diazino n OSRI wil l includ e adiscussio n o f th e guidelin e toxicolog y studie s that wer e performe dt o suppor t registratio n o f

diazinon . Befor e discussio n o f eac h diazino n OSRI stud y i n some detail , i t i s importan t

to understan dwhat

specifi c endpoint s o f

endocrin e toxicit

y are bein g monitore di n EDS P Tier 1 studies . Wit h thi s approach , th e OSR I discussio n can b e clearl y focuse do n th e issu e o f th e adequac y o f

existin g diazino n OSRI t o suppor t a waive r o f certai n EDS P Tie r

1 studies . A s aresult , Tabl e 2 belo w ha s bee n constructe dt o provid e an organizational ai dthat list s th e Tier

1 EDS P assay s an deac h typ 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 h o f th e EP A tes t guideline s (890.110 0 throug h 890.1600) . I n som e cases , th e endpoin t

liste d i n Tabl e 2 ha s bee n generalize dt o includ e mor e than 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 h a s tim e t o preputia l separation , tim e t o vagina l patenc y o r

vagina l opening , tim e t o ey e opening , an dmor e subjectiv e measure s suc h a s behavioral 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 avariety o f organs , whic h may 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 mammary gland , uterus , ovaries , cervi x an dvagin ai n femal e animal s an d th e testes , epididymides , prostate , seminal 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 h a s enzym e activity measures . Th e endpoin t

liste da s "se

x 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 column o r ro w indicate s that

that particula r endpoin t i s measure di n th e that

particular EDS P Tier

1 assay , wherea s n o entry o r a"blank box " i n th e tabl e indicate s that

th e endpoin t

i s no t measure di n that

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### Inspectio n o f

th e endpoint s measure di n variou s EDS P Tie 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 stud y eac h i n fis h an damphibians . Th e amphibia n metamorphosi s assay , however , i s designe dt o b e a screenin g assa

y fo r potential effect s o n thyroi dhormon e system s i n mammals , includin g human s (see 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 Tabl e 2 wil l b e specificall y identifie di n OSRI availabl e fo r diazinon . Befor e discussin g th e OSR I fo r diazino 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 P T O documents , EP A ha s indicate dthat companie s can submi t

OSRI dat athat

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

1 , althoug h th e assa

y 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 an estroge n recepto r bindin g assa y 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 athat provide s th e same essentia l endpoint informatio n o n achemical , eve n i f method s an dprocedure s other than EDS P Tier 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 rat

reproductiv e toxicity 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 OSR I an davoid s unnecessar y additional us e o f resource s an dsacrific e o f

animals , whil e ensurin g that 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 OSRI fo r

diazinon , tw o source s wer e examined : 1 ) th e peer reviewed , publishe dscientifi c literature ; an d2 ) th e diazino n pesticid e registratio n studies . I n orde r t o ensure that

onl y reliable , scientificall y vali dOSR I were include di n thi s assessment

fo r diazinon , eac h stud y wa s analyze dusin g th e scientifi c metho dse t fort h b y Klimisc h e t

al . (1997) , whic h i s consisten t wit h th e proces s outline db 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

diazino n OSRI include didentifyin g areliabilit y indicato r fo r eac h 1 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 

### study . A s outline db y Klimisc h e t al . (1997) , reliability can b e differentiate dint o fou r categorie s a s follows , "reliabl e withou t restriction " o r

categor y 1 ; "reliabl e wit

h restrictions " o r

categor y 2 ; "no t reliable " o r

category 3 ; an d"no t

assignable " o r

categor y 4 . I n ou r evaluatio n o f

diazino n OSRI , onl y studie s that coul db e give n acategor

y ratin g o f eithe r 1 o r 2 were use di n th e OSRI evaluatio n presente dhere . Thi s means , that onl y studie s that

were GLP-compliant 1 

### o r

comparabl e t o aGLP-complian t study protoco l were give n aratin g o f

categor y 1 an dstudie s give n acategor y 2 ratin g were one s that , althoug h no t performe daccordin g t o GLP , wer e considere dt o b e well-documente dan d scientificall y acceptabl e studies . Th e diazino n OSR I wa s evaluate db y scientist s wit h trainin g i n toxicolog y an dfamiliarit y with pesticid e registratio n an dEDS P testin g protocols . Th e followin g (Sectio n II I o f thi s document ) i s adiscussio n o f th e OSRI fo r diazino n that

i s either directl y equivalen t

o r functionall y equivalen t

t o th e EDS P Tier

1 assa y endpoint s liste di n Tabl e 2 . Th e discussio n i s focuse do n describin g whic h EDS P Tier 1 endpoint s are adequatel y assesse dthroug h us e o f OSRI an dwhic h endpoint s may nee dt o b e examine dfurthe r throug h performanc e o f

EDS P Tier

1 assays , assay s tha

t wil l b e specificall y identified . 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 P Tier

1 assay s shoul db e waive dbase d o n th e availabl e OSR

I (Sectio n I V o f

thi s document) . III . Discussio n o f

Diazino n OSR

I There ar e OSR I fo r

diazino n that

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

t o dat acollecte di n EDS P Tie r 1 testing . Tabl e 3 belo w provide s a listin g o f eac h sourc e o f

OSRI fo r diazino n that

ha s bee n identified , an dorganize s th e OSRI fro mthat

sourc e base do n th e EDS P Tie r 1 endpoints , simila

r t o th e presentatio n o f th e EDS P Tie

r 1 endpoint s i n Tabl e 2 above . Followin g Tabl e 3 , th e variou s OSR I are discusse di n more detail , includin g adiscussio n o f whethe r th e OSRI are eithe r

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

1 "GLP-compliant " mean s tha t astudy wa s performe dunde r Goo dlaborator

y Practic e (GLP ) regulation s a s outline db y EPA . 1 1§

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### a

SMakhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 A . Directl y o r

Functionall y Equivalen t Diazino n OSRI Identifie di n th e Publishe dLiteratur e A searc h o f

th e publishe dscientifi c literatur e identifie dsevera l source s o f diazino n OSR I that

ar e directl y equivalen t

t o dat acollecte di n EDS P Tier 1 assays . Thes e studie s includ e Sot o e t al . (1995) ; Nishihar ae t

al . (2000) ; Kojim a e t al . (2004) ; Kojim ae t

al . (2005) ; Manab e e t

al . (2006) ; O h e t

al . (2007) ; an d Judso n e t al . (2009) . Th e followin g i s adiscussio n o f th e relevan t

OSR I fro meac h study an dastatemen t

a s t o whethe r

th e dat aar e directl y o r

functionall y equivalen t OSRI . 1 . Soto e

t a l (1995 ) Revie w o f

thi s stud y indicate s that

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

assessin g th e estroge n recepto r bindin g activit

y o f diazinon . I n thi s i n vitr o stud y o f

avariety o f

compounds , includin g diazinon , th e author s report result s o f testin g usin g th e E-SCREE N assay . Thi s i s astandardassa

y 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 (human breas t cancer cells ) i n culture . Thi s cel l lin e expresse s estrogen receptors , i.e. , estroge n receptor- a(ERa) , an d stimulatio n o f

estroge n receptor s b y compound s suc h a s estradio l lead s t o increase dproliferatio n o f

MCF- 7 cells . Th e author s repor t us e o f 17P

estradio l a s wel l a s avariety o f other know n estroge n recepto r

agonist s to validat e th e responsivenes s o f thei r assay . 17p-estradio l i s th e positiv e contro l agent

use di n EDS P Tie r

1 assays . I n Tabl e 5 fro mth e paper , th e author s repor

t finding s fro mth e E-SCREEN fo r dozen s o f

pesticides , a s wel l a s other

classe s o f

chemicals . Diazino n i s identifie da s bein g "non

estrogenic " i n th e E-SCREE N assay . Th e dat aprovide dare directl y equivalen t

t o th e typ e o f

dat acollecte di n th e EDS P Tie r 1 890.125 0 assa

y an dare reliabl e diazino n OSRI . 

### 1 6Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 

### 2 . Nishihara<*a£(2000 ) Revie w o f thi s stud y indicate s that

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

assessin g th e estroge n recepto r transactivatio n potential o f

diazinon . I n thi s i n vitr o study , ayeas t two-hybri dassa

y (ER arecepto r

an d TIF 2 co-activator , S . cerevisiae Y19 0 cells ) wa s use dto scree n fo r th e ability o f agrou p o f

50 0 individua l tes t

chemical s t o activate th e estroge n receptor . Th e assa

y use dwa s base do n th e ligand-dependen t interactio n o f tw o proteins , th e hormon e recepto r an dit s co-activator , wit

h hormonal activit y identifie db y detectio n o f th e presenc e o f

p-galactosidase . Diazino n wa s on e o f

th e 50 0 tes t

chemical s screene dfo r

estroge n activity i n thi s assay . O f

50 0 tes t chemicals , onl y 6 4 were foun dt o b e "positive " a s havin g estrogeni c activity . Diazino n wa s negative , showin g n o significan t estrogeni c activity a

t dose s up t o th e highes t

tes t dos e o f 10 0 u M ( 1 x 10" 4 

### M) . Th e author s reporte daRECi o > 1 x 10" 4 

### M fo r diazinon , wher e RECi o i s th e concentratio n o f

tes t chemical showin g 10 % o f th e agonis t

activit y o f th e positiv e control , l?p-estradiol . 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 P Tier 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 , an dare reliabl e diazino n OSRI . 3 . Kojimae/a/ . (2004 ) Revie w o f thi s study indicate s that

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

assessin g th e estroge n an dandroge n recepto r transactivatio n potential o f diazinon . I n thi s study , 20 0 individua l pesticide s were screene dfo r thei r potential agonis t

an dantagonis t

activity at tw o huma n estroge n recepto r subtype s (ER aan dER0) , an dahuman androge n receptor , i n sensitiv e transactivatio n assay s i n Chines e hamste r ovar y cells . Diazino n wa s on e o f th e pesticide s tested . Th e assay s measure dfirefl y luciferas e gen e expression . Th e activity o f th e pesticides , includin g diazinon , wa s 1 7Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 

### measure di n al l thre e assay s an dreporte drelativ e t o th e activit y o f positiv e contro l agents . Th e author s reporte dthat diazino n wa s no t

show n t o hav e activit y i n an y o f

th e thre e systems , a

t dose s teste dfro m10" 8 

### t o 10~ 5 

### M . Th e dat aprovide di n thi s stud y are directl y equivalen t t o th e typ e o f

dat a collecte di n th e EDS P Tie

r 1 890.130 0 assay , an dare functionall y equivalen t t o th e dat acollecte di n th e 890.125 0 assa y an dth e 890.115 0 assay . Thus , thes e dat aals o provid e reliabl e diazino n OSRI . 4 . Kojim ae t

al . (2005 ) Revie w o f thi s study indicate s that

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

assessin g th e estroge n recepto r transactivatio n potentia l o f

diazinon . I n thi s study , BG 1 huma n ovarian carcinom acell s wer e transfecte d with luciferas e reporte r

plasmid . Thi s luciferas e gen e expressio n assa y wa s use dt o asses s estrogeni c activit y o f diazinon , where luciferas e expressio n i s unde r

estrogen-mediate dcontrol . Diazino n wa s weakl y estrogenic , wit

h effect s seen onl y at

ados e o f 46 0 u M diazinon , o r

4. 6 x 10" 4 

### M diazinon . I t

shoul db e note dthat

th e potenc y o f

diazino n i n thi s assa y wa s simila

r to th e potenc y reporte db y Nishihar ae t al . (2000) . 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 P Tie 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 diazino n OSRI . 5 . Manabee/a/ . (2006 ) 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

assessin g th e estroge n recepto r bindin g potentia l o f diazinon . I n thi s i n vitr o study , th e author s repor t result s fo r

si x pesticide s teste dindividuall y usin g arat pituitar y tumo r

cel l line , MtT/Se , whic h expresse s both ER aan dERp . Thi s i s astandar dassa y employe di n man y 1 8Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 

### laboratorie s an dmeasure s th e comparativ e potenc y o f tes t compound s t o induc e proliferatio n o f

thes e cell s i n culture . Stimulatio n o f thes e receptor s b y compound s suc h a s estradio l lead s t o increase dcel l proliferation . Th e author s repor

t us e o f l?p-estradio l a s an agonis t an d4-hydroxytamoxife n a s an antagonis t

t o validate th e responsivenes s o f thei r assay . I n Tabl e 2 fro mth e paper , th e author s report finding s fo r

th e si x pesticides . Diazino n i s identifie da s bein g awea k estrogeni c compound . Th e reporte dRECio , whic h i s th e dos e equivalen t t o 10 % o f th e maximu mestradio l respons e (RECio) , wa s 9.7xlO~ 5 

### M , o r 9 7 \i M diazinon . Again , i t i s note dthat

th e potenc y o f

diazino n i n thi s assa

y wa s simila

r t o th e potenc y seen b y Nishihar ae t

al . (2000 ) an dKojim ae t

al . (2005) . Th e dat aprovide di n thi s stud y are directl y equivalen t t o th e typ e o f

dat acollecte di n th e EDS P Tier 1 890.125 0 assay , an dar e functionall y equivalen t t o th e dat acollecte di n th e 890.130 0 assay . Thus , thes e dat aals o provid e reliabl e diazino n OSRI . 6 . O h et

a L (2007 ) Revie w o f thi s study indicate s that

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

assessin g th e estroge n recepto r bindin g potentia l o f diazinon . I n thi s i n vitr o study , th e author s report result s fo r

avariet y o f pesticide s teste dindividuall y usin g th e E-SCREEN assay . Thi s i s a standar dassa

y employe di n man y laboratorie s an dmeasure s th e comparativ e potenc y o f

tes t compound s t o induc e proliferatio n o f MCF- 7 cell s i n culture . Thi s huma n breas t cancer cel l lin e expresse s estroge n receptors , i.e. , estroge n receptor- a(ERa) , an dstimulatio n o f thes e receptor s b y compound s suc h a s estradio l lead s t o increase dproliferatio n o f MCF- 7 cells . Th e author s report us e o f 17p-estradio l t o validat e th e responsivenes s o f

thei r assay . I n Figur e 2 fro mth e paper , th e author s repor t finding s fro mth e E-SCREEN fo r al l teste dcompounds . Diazino n i s identifie da s bein g non-estrogeni c i n th e E-SCREE N assay . Th e dat a
1 9Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 provide dar e directl y equivalen t t o th e typ e o f

dat acollecte d i n th e EDS P Tier 1 890.125 0 assay , an d are reliabl e diazino n OSRI . 7 . Judso n et

al . (2009 ) Th e citatio n reference d here refer s t o th e publishe d pape r an d th e accompanyin g file s foun do n th e EP A website fo r th e ToxCas t

program 2

. Revie w o f

th e dat aindicate s that

i t provide s reliabl e dat afo r assessin g estroge n recepto r

binding , androge n recepto r binding , estroge n recepto r transactivation , aromatas e activity , an d thyroi d hormon e bindin g potentia l o f diazinon . I n apape r publishe d onlin 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 diazino n an d diazoxon . Diazino n an d it s majo r metabolit e diazoxon , a s wel l a s th e al l o f th e other compounds , were teste d fo r estroge n recepto r

binding , androge n recepto r binding , estroge n recepto r transactivation , aromatas e activity , an d thyroi d hormon e bindin g potential , amon g avariet y o f other endpoints . Th e specifi c test s performe d include dth e following : bindin g t o bovin e estroge n receptor s fro muterin e membran e (bER) ; bindin g t o huma n estroge n receptor s fro mbreas t cance r 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 rat androge n receptor s (rAR ; recombinant) ; bindin g t o huma n androge n receptor s fro mLnCA P cell s (hAR) ; bindin g t o rat thyrotropin-releasin g hormon e recepto r fro mforebrai n membrane s (rTRH) ; bindin g t o huma n thyroi d hormon e receptor-alph a(hTRa ; recombinant) ; activit

y to inhibi t

th e aromatas e enzyme ; estrogen receptor transactivation ; androge n recepto r transactivation ; an d thyroi d hormon e recepto r transactivation . 2 Th e ToxCas t

progra mi s aprogramwithi 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 away 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 d i n th e programar e i n vitr o assay s examinin g th e ability o f chemical s t o affec t

estrogen , androgen , an dthyroi d hormon e systems . Detail s o f th e programan d th e dat acan b e foun dat

www.epa.gov/ncct/toxcast . 2 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 

### Th e dat afro mthes e specifi c test s 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 Tier 1 assays . Th e result s were a s follows . I n th e cell-fre e screenin g assays , diazino n teste da

t asingl e initia l concentratio n o f 2 5 u 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 a t thi s dose , n o othe r

dose s were tested . Mor e specifically , diazino n di dno t

sho w evidenc e o f

bindin g t o bovin e o r

human estroge n receptors , bovin e o r

huma n progesteron e receptors , ra t o r huma n androge n receptors , rat

thyrotropin-releasin g hormon e receptor , o r huma n thyroi d hormon e receptor-a . I n cell-base dassays , diazino n di dno t activate huma n androge n receptors , huma n ERa , o r human thyroi dhormon e receptor-(3 , an dals o di dno t

bloc k activity o f

establishe dligand s at

thes e recepto r

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

activit y demonstrated) . I n th e hormon e recepto r transactivatio n assays , diazino n di dno t activate th e huma n androge n receptor , huma n estrogen-relate dreceptor-a , huma n estrogen-relate d receptor-y , o r

thyroi dhormon e receptor-a . Diazino n showe dwea k activit y i n ahuma n estroge n receptor- atransactivatio n assay , with activity seen at concentration s o f

betwee n 1 9 an d3 8 uM . I t i s note dthat

th e positiv e contro l compound , 17p-estradiol , hadactivit y i n thi s sam

e assa y i n th e lo w nanomola r range . Th e majo r

diazino n metabolite , diazoxon , wa s teste di n al l o f thes e sam e system s an di t

wa s uniforml y negativ e i n al l systems . 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 P Tie 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 an endpoin t

o f EDS P Tier

1 assay s 890.1100 , 890.1450 , an d890.1500 . Thes e dataare reliabl e diazino n OSRI . B . Directl y o r

Functionall y Equivalen t Diazino n OSRI Identifie di n Diazino n Registratio n Studie s A searc h o f

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

diazino n identifie dsevera l source s o f

diazino n OSRI that

are 2 1Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 directl y o r functionall y equivalen t

t o endpoints/dat acollecte di n EDS P Tier 1 assays . Thes e studie s includ e th e followin g (se e Tabl e 3 above) : asubchroni c toxicity stud y i n rat s (Sing h 1988) , achroni c toxicity study i n rat s (Kirchne r e t al . 1991) , achroni c toxicity stud y i n dog s (Rudzki e t al . 1991) , amulti-generatio n reproductiv e toxicit y stud y i n rat s (Gikni s 1989) , aprenata l developmenta l toxicity study i n rat s (Infurn a1985) , an dadevelopmental neurotoxicit y stud y i n rat s (Mandell a2003) . Th e followin g i s adiscussio n o f th e relevan t

OSR I fro m eac h stud 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 . Sing h (1988 ) Revie w o f

thi s GLP-compliant , guidelin e subchroni c toxicit y study i n rat s indicate s tha t

i t provide s reliabl e dat afo r assessin g avariety 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 . I n thi s guidelin e subchroni c toxicity stud y i n rats , youn g adul t mal e an dfemal e Sprague-Dawle y (CD ) rat s wer e administere d87.7 % pur e diazino n i n die t

a t dose s o f 0 , 0.5 , 5 , 25 0 an d250 0 pp mdiazino n fo r 9 0 days . I t

shoul db e note dthat

adietary route o f

administratio n i s more relevan t t o th e expecte dhuma n exposur e t o diazino n tha n dat athat 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 , intraperitoneal dosing , subcutaneou s dosing) . A s i n an y standardguidelin e roden t

subchroni c toxicit y study , endpoint s relevan t t o endocrin e activity were assesse d (se e Tabl e 3) . Th e endpoint s measure di n th e subchroni c stud y that

ar e directl y equivalen t

t o endpoint s monitore di n EDS P Tie r

1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival , body weight , clinica l chemistry , organ weight s (adrenal , thyroid , pituitary , kidney , liver) ; reproductiv e organ weight s (ovaries , uterus , femal e mammar y gland , testes , epididymides , prostate , seminal vesicle) ; organ histopatholog y (adrenal , thyroid , pituitary , kidney , liver) ; an dreproductiv e organ histopatholog y (femal e 

### 2 2Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 

### mammary gland , ovaries , uterus , testes , epididymides , prostate , seminal vesicle) . I t

shoul db e note da s wel l that th e subchroni c ra

t stud y involve s repeate ddosin g fo r

9 0 days , atim e perio dmuc h longe r tha n an y tim e perio do f exposur e i n an EDS P Tier 1 assa y (i.e. , maximu mtim e o f exposure o f

les s tha n on e month) . Th e developmental perio dassesse di n th e subchroni c study i s youn g adult , fo r

bot h mal e an dfemal e animals . Th e mos t sensitiv e no observe dadvers e effec t

leve l (NOAEL ) i n th e study wa s 0. 5 pp man dwa s base do n decrease dre dbloo dcel l acetylcholinesteras e level s a t th e 5 pp m exposure level . A s show n i n Tabl e 3 , th e 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 were n o effect s o n organ weight s o f an y organ relevan t to endocrin e activity , except fo r a n increase dliver

weight i n femal e rat s only . N o effect s o n organ histopatholog y wer e reporte dexcep t fo r centrilobula

r hypertroph y i n femal e rat s at dose s greate r

tha n 25 0 pp m diazinon . Bod y weigh t

decrease di n male s an dfemale s bu t onl y at th e highes t tes t dos e (250 0 pp mdiazinon) . Thes e dat ademonstrat e that diazino n wa s no t

toxi c t o endocrin e organ s system s i n thi s stud y i n rats . Th e dat aprovide di n thi s 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 , 890.1600 . Th e dat aprovid e informatio n o n i n viv o estrogen , androgen , an dthyroi dhormon e syste m toxicity o f diazino n i n youn g adul t

animals . Thes e dat aar e reliabl e diazino n OSRI . 2 . KirchnerefaM1991 ) Revie w o f thi s GLP-complian t

guidelin e chroni c ( 1 an d2 yea r sacrifices ) toxicit

y study i n rat s indicate s tha t i t provide s reliabl e datafo r assessin g avariety o f

EDS P Tier

1 study endpoint s a s show n i n Tabl e 3

2 3Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 above , endpoint s relatin g t o estrogen , androge n an d thyroi d hormon e syste mactivity . I n thi s guidelin e stud y i n rats , youn g adul t mal e an dfemal e Sprague-Dawle y (CD ) rat s were administere d 87.7 % pur e diazino n i n die t a t dose s o f 0 , 0.1,1.5 , 12 5 an d25 0 pp mdiazino n fo r 5 4 o r 9 9 week s ( 1 o r 2 years) . I t

shoul d b e note d that

adietary rout e o f

administratio n i s mor e relevan t t o th e expecte d huma n exposur e t o diazino n than dat athat migh t b e collecte d usin g other

route s o f administratio n i n animal s (e.g. , ora l gavag e dosing , intraperitoneal dosing , subcutaneou s dosing) . A s i n an y standar d guidelin e roden t chroni c toxicit y study , endpoint s relevan t t o endocrin e activity were assesse d (se e Tabl e 3) . Th e endpoint s measure d i n th e chroni c stud y that

ar e directl y equivalen t

t o endpoint s monitore d i n EDS P Tie r

1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival , bod y weight , clinica l chemistry , organ weight s (adrenal , thyroid , pituitary , kidney , liver) ; reproductiv e organ weight s (ovaries , uterus , femal e mammary gland , testes , epididymides , prostate , seminal vesicle) ; organ histopatholog y (adrenal , thyroid , pituitary , kidney , liver) ; an d reproductiv e orga n histopatholog y (femal e mammary gland , ovaries , uterus , vagina , testes , epididymides , prostate , seminal vesicle) . I t

shoul d b e note d a s wel l that th e chroni c rat

stud y involve s repeate d dosin g fo r 1 t o 2 years , atim e perio d muc h longe r than an y tim e perio d o f

exposur e i n EDS P Tie r

1 assays , an d coverin g a developmenta l perio d no t covere d b y EDS P Tier

1 studies , i.e. , senescence . Th e developmenta l period s assesse d i n th e chroni c study includ e youn g adult , adul t

an d senescence , i n bot

h mal e an d femal e animals . Th e mos t sensitiv e NOAE L i n th e study wa s 1. 5 pp man d wa s base d o n decrease d re d bloo d cel l acetylcholinesteras e level s at th e 25 0 pp m exposur 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 d thyroid-responsiv e tissue s (e.g. , thyroid , brain) , wer e examine d histopathologicall y i n thi s 

### 2 4Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2 0 1 00 2 1 3 

### study . There wer e n o effect s o n orga n weight s o f

an y organ relevan t

t o endocrin e activity , an dn o effect s o n organ histopatholog y were reported , includin g endocrin e organ s (i.e. , thyroid , pituitary , an dvariou s reproductiv e organ s i n male s an dfemales) . Body weigh t increase di n male s an dfemale s bu t

onl y at th e highes t

tes t dos e (25 0 pp mdiazinon) . Thes e dat ademonstrat e that diazino n wa s no t

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

t o specifi c dat acollecte di n th e followin g EDS P Tier 1 assays : 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 . Th e dat aprovid e informatio n o n estrogen , androgen , an dthyroi dhormon e syste mtoxicit y o f

diazinon . Thes e dat aare reliabl e diazino n OSRI . 3 . 

### Revie w o f

thi s GLP-complian t

guidelin e chroni c ( 1 year ) toxicity study i n dog s indicate s that i t provide s reliabl e dat afo r

assessin g avariety o f EDS P Tier

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 . 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 d87.7 % pur e diazino n i n die t a t dose s o f 0 , 0.1 , 0.5 , 15 0 an d22 5 pp mdiazino n fo r 1 year . I t shoul db e note dthat

a dietary rout e o f

administratio n i s more relevan t t o th e expecte dhuma n exposur e t o diazino n tha n dat athat migh t b e collecte dusin g othe r

route s o f administratio n i n animal s (e.g. , oral gavag e dosing , intraperitoneal dosing , subcutaneou s dosing) . A s i n an y standar dguidelin e do g chroni c toxicity study , 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 chroni c stud y that ar e directl y equivalen t t o endpoint s monitore di n EDS P Tier 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival , body weight , clinical chemistry , organ weight s (adrenal , thyroid , pituitary , kidney , liver) ; reproductiv e organ weight s (ovaries , uterus , vagina , testes , 2 5Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 

### epididymides , prostate) ; organ histopatholog y (adrenal , thyroid , pituitary , kidney , liver) ; an dreproductiv e orga n histopatholog y (femal e mammary gland , ovaries , uterus , vagina , testes , epididymides , prostate) . I t shoul db e note da s wel l that

th e chroni c do g stud y involve s repeate ddosin g fo r 1 year , atim e perio dmuc h longe 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 d i n th e chroni c study includ e youn g adul t

an dadult , i n bot

h mal e an dfemal e animals . Th e mos t sensitiv e NOAE L i n th e study wa s 0. 5 pp man dwa s base do n decrease d re dbloo dcel l acetylcholinesteras e level s a t th e 15 0 pp mexposure level . There were n o effect s o n orga n weight s o f

an y organ 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 ademonstrate that diazino 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 study 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 Tier 1 assays : 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 . Th e dat aprovid e informatio n o n estrogen , androgen , an dthyroi dhormon e syste mtoxicit y o f

diazinon . Thes e dat aare reliabl e diazino n OSRI . 4 . GiknistVa/ . (1989 ) Revie w o f thi s GLP-complian t

guidelin e multi-generatio n (two ) reproductiv e toxicity stud y i n rat s indicate s that i t provide s reliabl e dat a fo r assessin g avariety o f

EDS P Tie r

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 study ha s th e mos t extensiv e coverag e o f

EDS P Tier 1 endpoint s o f

an y singl e diazino n study an dcover s al l developmental period s covere db y EDS P Tier 1 assay s (i n utero , lactation , post-natal , peri-pubertal , youn g adult , an dadult) . 2 6Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 

### I n thi s guidelin e study i n rats , mal e an dfemal e Charle s Rive r C D rat s wer e administere d94.9 % pur e diazino n i n die t a t dose s o f 0 , 10 , 10 0 an d50 0 pp mdiazino n fo r

1 0 week s pre-matin g (PI) , 3 week s matin g (PI) , an d1 1 week s pre-matin g (Fl) . Pup s o f th e F l generatio n were , therefore , expose di n tttero , durin g lactation , an dthe n place do n diazino n i n die t immediatel y at

weanin g throug h th e post-natal perio dan dthroug h matin g an dsubsequen t

parturition , deliver y o f

th e F 2 generation . Thus , thi s study include dexposur e o f

F l animal s o f

bot h sexe s durin g peri-puberty , an importan t consideratio n fo r

late r discussio n o f

th e puberta l rat

EDS P Tier 1 assays . A s with th e other 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 exposure t o diazino n than dat athat

migh t b e collecte dusin g other

route s o f administratio n i n animal s (e.g. , oral gavag e dosing , intraperitoneal dosing , subcutaneou s dosing) . Th e multi-generatio n reproductiv e toxicit y i s expecte dt o b e a critical EDS P Tier

2 study . Althoug h th e diazino 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 toxicity 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 study wa s performed . I t

i s an OP P 83- 4 study design , an dlik e th e mos t curren t guidelin e stud y desig n i t

wa s designe dt o examin e potentia l effect s o f diazino n o n endocrin e organs , i n particular th e estroge n an dandroge n system s that

ar e critical t o reproductiv e function . A s aresul t o f bein g a reproductiv e toxicit y study , man y endpoint s relevan t t o endocrin e activity were assesse d (se e Tabl e 3) . Th e endpoint s measure di n th e multi

generatio n reproductiv e toxicit y study tha

t ar e directl y equivalent

to endpoint s monitore di n EDS P Tier 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival ; bod y weight (bot h adult s an dpups) ; developmental measure s (matin g inde x i n bot h sexes , gestatio n index , gestatio n length , se x rati o o f tw o successiv e generations , malformation s i n bot

h generations , postnatal developmen t measure s i n F l 2 7Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 pups) ; reproductiv e organ weight s (ovarie s an d testes) ; reproductiv e orga n histopatholog y (femal e mammary gland , ovaries , uterus , vagina , cervix , testes , epididymides , prostate , seminal vesicle , coagulatin g gland) ; fertilit y indices ; fecundit y indices ; an d indirec t measure s o f

estrou s cyclicity . Th e mos t sensitiv e NOAE L i n th e stud y wa s 1 0 pp man d wa s base d o n adecrease d F l materna l an d pu p bod y weigh t an d decrease d pu p survival a t th e 10 0 an d 50 0 pp mexposure levels . There were n o effect s o n endocrin e orga n weight s excep t fo r adecrease d teste s weight at th e highes t tes t dos e onl y (50 0 pp mdiazinon) , ados e leve l tha t wa s als o associate d wit

h materna l toxicity . There were n o effect s o n endocrin e orga n histopatholog y a

t an y dos e leve l teste d (e.g. , thyroid , pituitary , an d variou s reproductiv e organ s i n male s an d female s expose d eve n durin g sensitiv e developmenta l periods) . Ther e were n o statisticall y significan t effect s o n reproductiv e indice s i n eithe r generatio n (al l monitore d endpoints) . There were n o reproductiv e syste mabnormalitie s note d i n pup s o f either generation . There wa s atren d toward s aprolonge d gestatio n i n bot h generation s at

th e hig h dos e leve l (50 0 ppm) , bu t th e effec t wa s no t statisticall y significant . I n th e F l generation , atren d als o wa s observe d fo r adecrease d numbe r

o f pregnancie s an d th e numbe r o f viabl e newborns , a s wel l a s advers e effect s o n matin g an d fertility , agai n onl y at th e tes t dos e that produce d sign s o f

materna l toxicity (50 0 pp mdiazinon) . Thes e dat a demonstrat e that

diazino 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 critical period s o f development . Th e dat aprovide d i n thi s multi-generatio n reproductiv e toxicity study ar e directl y and/o r

functionall y equivalent

to dat acollecte d i 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 d 890.1600) . Th e dat a provid e informatio n o n estrogen , androgen , an d thyroi d hormon e syste m toxicity o f

diazino n i n viv o an d are mor e than adequat e t o asses s th e



### 2 8Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 endocrin e toxicity potentia l o f diazinon . Moreover , i n conjunction wit h th e othe r availabl e publishe d toxicit y data , th e multi-generatio n reproductiv e stud y dat aar e als o directl y applicabl e t o identifyin g actual mode s o f actio n fo r diazino n (se e Tabl e 1) . Fo r example , th e lac k

o f effect s o n fertilit y an d femal e an d mal e endocrin e orga n histopatholog y i n th e multi

generatio n stud y i s consisten t wit h finding s i n vitr o that

diazino n i s no t

a poten t estrogeni c agent , i s no t

an androge n recepto r

bindin g agent , an d ha s n o effect s o n aromatas e activit y (e.g. , Soto e t al . 1995 ; Nishihar ae t

al . 2000 ; Kojim ae t

al . 2004 ; Kojim ae t

al . 2005 ; Manab e e t

al . 2006 ; Judso n e t al . 2009) . Further , th e lac k o f effect s o n thyroi d organ histopatholog y an dultimat e effect s o f

alterin g thyroi d hormon e activit y i n th e multi

generatio n study ar e consisten t wit h th e lac k o f thyroi d hormon e recepto r bindin g b y diazino n (Judso n e t al . 2009 ) 5 . Infurn a198

5 Revie w o f

thi s GLP-complian t

guidelin e prenatal (teratology ) toxicit y stud y i n rat s indicate s that i t provide s reliabl e dat afo r assessin g a variety o f EDS P Tie r

1 study endpoint s a s show n i n Tabl e 3 above . Thi s stud y cover s aspecifi c developmenta l perio d no t examine d i n mammal s i n th e EDS P Tie 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 d t o assay s conducte d with amphibian s an d fish . I n thi s guidelin e stud y i n rats , femal e Charle s Rive r C D rat s were administere d diazino n o f

unspecifie dpurit y b y gavag e a t dose s o f 0 , 10 , 20 , an d10 0 m g diazinon

/ k g bod y weigh t

fro mgestatio n day si x (GD6 ) throug h GDIS . I t

shoul d b e note d that

th e gavag e route o f

administratio n i s no t a s relevan t

fo r huma n healt

h ris k assessment

bu t i s th e mos t

routin e route o f exposure fo r

teratolog y studies . Lik e al l guidelin e prenata l toxicity studies , thi s stud y wa s designe d t o examin e th e teratogeni c potential o f diazinon . 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 d i n th e prenatal toxicit y stud y that

ar e directl y 

### 2 9Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 equivalen t t o endpoint s monitore di n EDS P Tier 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival ; bod y weigh t (bot h adult s an dpups) ; developmenta l measure s (fetotoxicity ; skeletal an d sof t tissu e malformations) ; reproductiv e orga n weight s (gravi duterus ; testes , ovary , uterus) ; an dfertilit y indice s (survival , pre - an dpost implantatio n loss) . Th e mos t sensitiv e NOAE L i n th e stud y wa s 2 0 mg/kg/day an d wa s base do n decrease dmaterna l an dfeta l bod y weigh t a t th e 10 0 mg/k g exposur e level . There were n o reporte dteratogeni c effect s i n pup s at an y dos e level . 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 d female s expose deve n durin g development) . Ther e were n o statisticall y significan t effect s o n fertilit y indices . Thes e dat ademonstrate dthat diazino n wa s no t

toxi c t o endocrin e organ system s i n thi s sensitive , i n viv o tes t fo r toxicity durin g critical period s o f developmen t

i n utero . Th e dat aprovide di n thi s prenatal toxicity stud y ar e functionall y equivalen t t o dat acollecte di n EDS P Tier 1 assay s that

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 diazino n i n vivo , an d are reliabl e diazino n OSRI . 6 . Mandell a200 3 Revie w o f

thi s GLP-complian t

guidelin e developmenta l neurotoxicit

y study i n rat s indicate s that i t provide s reliabl e dat afo r assessin g avariety o f

EDS P Tier

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 P Tier 1 assays , i n uter o exposure , a s wel l a s postnata l developmen t

i n rats . Thi s study 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 vaginal openin g i n females . 3 0Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 

### I n thi s guidelin e stud y i n rats , femal e Sprague-Dawle y rat s were administere d92.9 % pur e diazino n i n die t a t dose s o f 0 , 0.3 , 30 , an d30 0 pp mdiazino n fro mGD 6 throug h PND2 1 (weaning) , wit h F l pup s evaluate dat

PND60 . I t

shoul db e note dthat

adietary rout e o f administratio n i s mor e relevan t t o th e expecte dhuma n exposure t o diazino n tha n dat athat

migh t b e collecte dusin g other

route s o f administratio n i n animal s (e.g. , ora l gavag e dosing , intraperitoneal dosing , subcutaneou s dosing) . A s aresul t

o f bein g adevelopmenta l toxicit y study , certai n endpoint s relevan t

t o endocrin e activity were assesse d(se e Tabl e 3) . Th e endpoint s measure di n th e developmental neurotoxicit y study tha t ar e directl y equivalen t

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

(bot h adult s an dpups) ; an ddevelopmental measure s (fetotoxicity , preputia l separation , vagina l opening . Th e mos t sensitiv e NOAE L i n th e stud y wa s 0. 3 pp mdiazino n an d wa s base do n decrease dmaterna l acetylcholinesteras e level s i n bloo dan d brain . N o gros s lesion s o f

an y kin dwere reported , includin g lesion s i n endocrin e organs . N o effect s o n an y reproductiv e indice s were reported . There wer e n o observe deffect s o n pu p bod y weigh t o r lactatio n o r

nursin g behavio r an dn o effect s o n pu p survival . Pu p growt h wa s decrease dat

th e highes t tes t dos e o f 30 0 pp mdiazino n at

bot h PND 7 an dPND21 . Vagina l openin g an dpreputia l separatio n i n pup s were delaye db y les s tha n 2 day s bu t onl y a t th e highes t

tes t dos e (30 0 ppm) , ados e that wa s associate d wit h materna l toxicit y a s wel l a s significan t clinical sign s o f

toxicity (inhibitio n o f

acetylcholinesteras e activity i n bloo dan dbrai n o f pups) . Thes e dat ademonstrate dthat diazino 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 tes t fo r toxicity 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 prenatal toxicity study ar e functionall y equivalen t t o dat acollecte di n EDS P Tier 1 assay s that

examin e aspect s o f 3 1Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 mammalian endocrin e functio n (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , an d890.1600) . Th e dat ao n vagina l openin g an dpreputia l separatio n ar e endpoint s that ha dno t bee n include di n other

guidelin e diazino n toxicity studie s an d are monitore di n EDS P Tie r 1 assay s (890.145 0 an d 890.1500) . Thi s study provide s reliabl e diazino n OSRI . IV . Discussio n o f

th e EDS P Tie r

1 Test s Neede dfo r

Assessmen t

o f th e Endocrin e Disruptio n Potentia l o f

Diazino n I n Septembe r

2008 , th e Agenc y fo r

Toxi c Substance s an dDiseas e Registry (ATSDR ) publishe dan updat e t o th e diazino n toxiciologica l profil e documen t that include dadiscussio n o f

th e potentia l endocrin e toxicity o f diazino n base darevie w o f th e publishe ddat ao n diazinon . I n that document , i t

i s state dthat

"Result s o f

availabl e animal studie s indicat e tha

t th e reproductiv e syste mi s no t particularl y sensitiv e t o diazino n toxicity. " (se e pag e 11 0 o f ATSDR . 2008 . lexicologica l Profil e fo r

Diazinon . September) . Th e documen t als o state s "Result s o f

availabl e i n vitr o assessment s o f diazino n estrogenicity indicat e apotentiall y wea k estrogeni c effec t

at best. " (se e pag e 11 1 o f ATSDR . 2008 . Toxicologica l Profil e fo r

Diazinon . September) . Revie w o f th e informatio n presente di n Tabl e 3 (above ) i s consisten t wit h th e statement s foun di n th e 200 8 toxicologica l profil e fo r diazinon . I n th e cas e o f

diazinon' s potentia l fo r

estrogeni c activit y i n vitro , th e compoun d ha s demonstrate donl y limited , wea k estrogeni c activit y i n vitr o i n som e publishe d studie s (e.g. , Kojim ae t

al . 2005 ; Manab e e t

al . 2006) , wit

h th e majorit y o f

th e diazino n OSRI demonstratin g that

th e compoun dwoul dno t ac t throug h estrogeni c o r

anti

estrogeni c mechanism s t o produc e toxicity . Th e OSR I studie s wit h diazino n provid e bot

h directl y equivalen t

an d functionall y equivalen t measure s o f

th e potenc y o f

diazino n t o bin dt o ER s an daffec t

th e functio n o f

ERs . Whe n th e concentration s o f diazino n show n t o hav e effect s i n thes e studie s ar e considere d(concentratio n i n th e micromola r rang e o r > 1 x 10" 4 M) , i t

i s unlikel y that

E Rbindin g assay s fro mEDS P Tie r 1 testing , i f performed , woul dindicat e that diazino n i s apoten t

o r biologicall y relevan t

E Ragonis t

o r antagonist . Th e i n viv o dat afo r diazino n relevan t

t o estrogeni c activity i s consisten t i n showin g that

diazino n doe s no t

affec t femal e reproductiv e functio n i n mammals . 32Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 

### With respec t

t o androge n activit

y i n vitro , th e recen t

publicatio n o f

dat acollecte d a s part o f th e ToxCas t

program(Judso n e t

al . 2009 ) ha s demonstrate dthat diazino n wa s inactiv e a s either

an A Ragonis t

o r antagonist , an dha dn o activity t o affec t aromatas e function , th e enzyme that

convert s androgen s to estrogen s (anti-androgeni c activity) . Moreover , ther e ar e avariety o f i n viv o studie s availabl e examinin g th e effect s o f diazino n o n androge n system s i n mammals . Whe n considere dtogether , th e dat ad o no t indicat e that

diazino n i s apoten t

reproductiv e toxican t

that produce s androgen-specifi c effects . Revie w o f

th e diazino n i n viv o guidelin e registratio n studies , b y themselves , confirm s th e finding s o f

th e ATSD R(2008 ) that

diazino n i s no t

apoten t reproductiv e o r developmental toxicant . Suc h i n viv o studie s are mor e relevan t measure s o f

endocrin e toxicity potentia l a s metaboli c system s an dth e comple x interaction s o f variou s hormona l system s are intact , unlik e th e condition s o f mos t EDS P Tier

1 test s ( 8 o f

1 1 are either

i n vitr o assay s o r

involv e model s wit

h non-intac t

hormonal systems) . Th e diazino n studie s sho w tha t th e compoun ddoe s no t

appea r 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 d thyroi dglan dhistopatholog y i n al l o f th e availabl e studies . An y effect s reporte dwit h diazino n exposure that

migh t b e linke dt o endocrin e activity were limite dgenerall y t o hig h dose s (10 0 pp mdiazino n i n die t an dgreater) . Thes e effect s include ddecrease d pu p surviva l an dgrowt

h i n th e two-generatio n reproductiv e toxicity stud y (a t dose s o f 10 0 pp mdiazino n an dgreater ) an ddelay s i n preputia l separatio n an dvaginal openin g i n th e developmenta l neurotoxicity study (a t dose s o f 30 0 pp mdiazinon) . Th e diazino n guidelin e toxicolog y studies , lik e al l suc h guidelin e i n viv o toxicolog y studies , were designe dpurposel y t o b e valid , realisti c evaluation s o f possibl e effect s i n humans . Regulatory bodie s rel y o n suc h studie s t o mak e decision s regardin g saf e level s fo r

human exposure . A s aresult , th e availability o f suc h studie s make s th e performanc e o f

EDS P Tier

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 diazino n endocrin e toxicity dat athat mos t o f th e Tie r 1 EDS P test s nee dno t

b e conducte di f

th e goal i s t o scree n fo r

potentia l endocrin e activity . However , ther e ar e 3 3Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3

certai n endpoint s o f

EDS P Tier

1 testin g that

hav e no t bee n specificall y measure di n availabl e diazino n studies . Considerin g th e availabl e data , MAN A propose s that onl y th e followin g Tier

1 studie s b e conducte da s part o f th e EDS P testin g progra mfo r diazinon : Fis h Short-TermReproductio n (890.1350) ; Hershberge r assa y i n rat s (890.1400) ; Uterotrophi c assa

y i n rat s (890.1600) ; an dSteroidogenesi s (Human Cel l Lin e H295R ) (890.1500) . Th e rational e fo r MANA' s positio n i s outline dbelo w regardin g particular studie s tha t

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

Bot h Tie r 1 I n Vitro Assay s fo r

Estroge n Activity (Estroge n Recepto r Bindin g 890.125 0 an d Estroge n Recepto r

Transcriptiona l Activatio n 890.1300 ) EDS P Tier

1 assay s 890.125 0 an d 890.130 0 are screenin g assay s t o identif y mode s o f

actio n fo r

estrogeni c activity involvin g bindin g t o an d activatio n o f

estroge n receptors . There are adequat e i n vitr o estroge n recepto r activity dat aavailabl e fo r

determinin g th e ability o f diazino n t o bin dt o an d activat e estroge n receptor s i n vitro . Thes e 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 h non e o f th e studie s i s technicall y identical i n desig n to th e EDS P Tier 1 E Rstudie s (890.1250 ; 890.1300) , th e availabl e studie s ar e bot h directl 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 activity o f diazino n t o appropriat e contro l agents . Th e result s hav e bee n consistent i n showin g tha t

diazino n is , at

best , awea

k estrogeni c agent , with potenc y severa l order s o f magnitud e les s than positiv e contro l agent s (i.e. , l?p-estradiol) ; activity o f diazino n i n vitr o i s onl y see n a t dose s i n th e hig h micromola r range . Th e majo r metabolit e o f

diazinon , diazoxo n wa s show n t o b e inactiv e i n al l E Rassay s i n whic h i t ha s bee n teste d(Judso n e t al . 2009) . Therefore , MAN A believe s there i s n o scientifi c basi s fo r

performin g additional i n vitr o estroge n recepto r activit y studies , specificall y th e Estroge n Recepto r Bindin g assa

y (890.1250 ) an dth e Estroge n Recepto r

Transcriptional Activatio n (Human Cel l Lin e HeLa-9903 ) assa y (890.1300) . Th e availabl e dat aare adequat e fo r assessin g th e activity o f 3 4Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 diazino n at

E Rreceptors . A s aresult , awaiver o f bot h 890.125 0 (Estroge n Recepto r Bindin g assay ) an d890.130 0 (Estroge n Recepto r Transcriptional 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 Vitro Assay s fo r Androge n Activity (Androge n Recepto r Bindin g 890.115 0 an d Aromatas e Activity 890.1200 ) EDS P Tie r 1 assay s 890.115 0 an d 890.1200 ar e screenin g assay s to identif y mode s o f

actio n fo r

androgeni c an danti-androgeni c activity 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 . There ar e adequate i n vitr o androge n recepto r activit y dat aavailabl e fo r determinin g th e ability o f

diazino n t o bin d t o androge n receptors . Th e stud y b y Judso n e t al . (2009 ) provid e dat ao n both androge n recepto r bindin g potency , agonis t an dantagonis t

activity , a s wel l a s th e potential fo r diazino n t o inhibi t aromatas e activity i n vitro . Diazino n an dit s majo r metabolite , diazoxon , wer e inactiv e i n al l thes e assays . Further , th e availabl e studie s provid e directl y an d functionall y equivalen t

dat ao n essentiall y th e same endpoints , includin g compariso n o f

th e activity o f

diazino n t o appropriat e contro l agents . Thus , ther e are adequat

e OSR I fo r assessin g th e activit

y o f diazino n a

t A Rreceptor s an do n aromatas e function . Thus , ther e i s n o scientifi c basi s fo r performin g additional i n vitr o androge n recepto r

activity studies , specificall y th e Tie r 1 Androge n Recepto r Bindin g assa

y (890.1150) , a s wel l a s n o scientifi c basi s fo r performin g additional i n vitr o aromatas e activity studies , specificall y th e Tier 1 Aromatas e assa y (890.1200) . A s aresult , awaive r o f bot h 890.115 0 (Androge n Recepto r Bindin g assay ) an d890.120 0 (Aromatas e assay ) fo r diazino n i s sought . C . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Amphibia n Metamorphosi s Assay (890.1100 ) Th e Amphibia n Metamorphosi s assa y (890.1100 ) i s describe d i n th e guidelin e a s a"screenin g assa y intende d t o empiricall y identif y substance s whic h may interfer e wit

h th e normal functio n o f

th e hypothalamic-pituitary-thyroi d 

### 3 5Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 (HPT ) axis" . I t

i s intende d t o b e ageneralize d vertebrat e mode l that i s base d o n th e conserve d structure s an d function s o f th e HPT axi s o f

variou s species . It

i s als o pointe d ou t

that amphibian metamorphosi s provide s " awell-studied , thyroid

dependen t proces s whic h respond 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 assa

y that detect s thyroi d activity i n an animal 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 study becaus e ther e are adequat e i n viv o dat acollecte d i n guidelin e toxicolog y studie s that cover al l stage s o f

mammalian development , an d ar e consistent

i n showin g that

diazino n ha s n o effect s o n functionall y equivalen t endpoint s i n mammalian species . Fo r example , th e diazino n OSRI includ e dat a fro mamulti-generatio n reproductiv e toxicity stud y i n rats , aprenatal toxicit y stud y i n rats , an d adevelopmenta l neurotoxicity stud y i n rats . Al l o f thes e studie s demonstrate d that

diazino n doe s no t

induc e developmental an d morphologica l effects , includin g non e o f th e possibl e apical effect s tha t woul d resul t

fro meither increasin g o r

decreasin g thyroi d hormon e level s i n mammal s (e.g. , growth , metaboli c rate 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 d n o effect s o n thyroi dglan d histopathology , effect s that woul d b e expecte d t o appea r i f diazino n were producin g direc t

effect s o n eithe r

thyroi d hormon e synthesi s o r function . Performin g th e amphibian assa y merel y t o monito r

change s i n thyroi d hormon e level s o r se x hormon e level s i n developin g amphibians , endpoint s that hav e no t bee n quantifie d i n currentl y availabl e diazino n OSRI , i s no t scientificall y justifie d whe n robus t

i n viv o dat ai n developin g organism s i s alread y availabl e an ddoe s no t sho w that

th e thyroi d hormon e syste mi s atarge t fo r diazino n effects . Therefore , awaive r

o f 890.110 0 (Amphibia

n Metamorphosi s assay ) i s sought becaus e ther e i s n o scientifi c basi s fo r performin g an additiona l stud y whe n th e availabl e diazino n OSR I are considered . I t shoul d als o b e pointe d ou t that i n th e discussio n belo w o f

th e waiver s bein g sough t fo r th e pubertal assay s i n rat s (bot h mal e an d female) , MAN A i s proposin g t o ad d measuremen t o f thyroi d hormon e level s t o th e Uterotrophi c an d Hershberge r assay s tha

t wil l b e performe d wit

h


### 3 6Makhteshi mChemical Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 diazinon . Thus , direc t

effect s o f diazino n t o affec t

thyroi dhormon e synthesi s throug h action s i n th e brai n an dth e thyroi dglan dwil l b e measure di n vivo . D . MAN A Seeks aWaive r fo r Performanc e o f

th e Tier 1 Femal e Puberta l Assa

y (890.1450

) Th e purpos e o f

th e pubertal femal e rat

assa y (890.1450 ) i s state di n th e guidelin e t o b e identifyin g chemical s that hav e apotentia l t o interac t wit h th 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 . There are adequat e i n viv o OSRI availabl e fo r diazino n demonstratin g that thi s chemical i s no t

apoten t estrogeni c o r

anti

estrogeni c agent

i n vivo . Th e OSR I include s th e robust , guidelin e registratio n studie s (Tabl e 3) . Thes e i n viv o assessment s o f femal e reproductiv e functio n an d developmental toxicit y i n rat s hav e provide ddat ao n exposure o f animal s at

al l stage s o f developmen t

(i n utero , durin g lactation , throughou t post-natal development , throughou t

peri-puberta l development , durin g adulthood , an d durin g senescence ) a s wel l a s providin g dat ao n man y o f th e same endpoint s foun di n EDS P Tier

1 studie s (Tabl e 3) . Fo r

example , i n th e Tier

1 Femal e Puberta l assa

y (rat) , importan t

endocrin e endpoint s monitore dinclud e identificatio n o f

abnormalitie s associate dwit h se x organ s an dpubert y marker s (se e Tabl e 2 above) , a s wel l a s thyroi dtissue . Th e guidelin e toxicity studie s i n rat s suc h a s th e two-generatio n reproductiv e toxicity study , th e prenatal toxicity study , th e developmental neurotoxicit y study , an dth e subchroni c an dchroni c toxicity studies , al l include dassessment s o f diazinon' s effect s o n femal e reproductiv e tissue s an dorgans , bot

h i n adul t animal s an di n developin g animal s (includin g peri-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 dboth durin g gestatio n an ddevelopmen t an da s adults . Th e guidelin e reproductiv e toxicit y an ddevelopmenta l toxicity studie s provid e adequate dat ao n femal e reproductiv e organ effect s followin g diazino n exposure , a s wel l a s fertility , fecundity , vaginal opening , an destrou s cycling .
3 7Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 

### Th e onl y endpoin t

fro mth e EDS P Tier

1 i n viv o assa

y i n puberta l femal e rat s that ha s no t bee n directl y measure di n th e currentl y availabl e diazino n guidelin e toxicity studie s i s th e leve l o f thyroi dhormone s i n seru mo f animal s followin g diazino n exposure . Give n that th e ultimat e apical effect s o f either 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) , ther e doe s no t appea r t o b e astron g scientifi c basi s fo r requirin g performanc e o f

th e Tier 1 puberta l femal e screenin g study (890.1450 ) simpl y t o provid e fo r

measure s o f

thyroi dhormon e level s i n rats . Therefore , MAN A propose s that

monitorin g o f

thyroi dhormon e level s b e adde da s an endpoin t fo r th e Uterotrophi c assay , wher e rat s d o no t hav e an intac t

HP G axis . I n thi s way , th e potentia l mod e o f actio n o f diazino n t o affec t

thyroi dhormon e level s wil l b e directl y addressed . MAN A propose s t o conduc t th e Tie r 1 Uterotrophi c assa y i n rat s becaus e th e tes t

follow s uterin e weigh t

change s i n ovarectomize d femal e rat s a s ascree n fo r

estrogeni c activity , amode l no t currentl y covere dwith diazino n OSRI , whil e OSR I directe dt o diazino n an destroge n activity ar e availabl e i n peripuberta l femal e rat s (multi-generatio n reproductiv e toxicity study) . Therefore , awaive r o f EDS P Tie r

1 890.145 0 (Pubertal Femal e Rat Development ) i s sought . E . MAN A Seek s aWaive r fo r Performanc e o f

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

th e puberta l mal e rat

assa y (890.1500 ) i s state di n th e guidelin e t o b e identifyin g chemical s that hav e apotentia l t o interac t wit h th 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 ampl e i n viv o diazino n OSRI o n mal e reproductiv e functio n an dpatholog y t o indicat e that th e system s that

involv e androge n activity ar e no t

uniquel y targete db y diazino n an dthat the y ar e no t uniquel y sensitiv e to th e effect s o f diazinon . Th e diazino n OSRI studie s includ e th e robus t guidelin e studie s i n rat s suc h a s th e two-generatio n reproductiv e toxicit y study , th e developmenta l toxicity study , th e developmental neurotoxicity 3 8Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3 

### study , an dth e subchroni c an dchroni c toxicit y studie s (Tabl e 3) . Thes e i n viv o assessment s o f

mal e reproductiv e function , developmenta l toxicity , a s wel l a s general toxicit y i n rat s hav e provide ddat ao n th e same endpoint s a s generate di n assay s performe da s part

o f Tie r 1 i n th e EDSP , includin g th e mal e pubertal rat study (890.1500) . Fo r

example , i n th e Tier

1 Mal e Puberta l assa

y (rat) , th e tes t

i s use dt o screen fo r

androgen , anti-androgen , an dthyroi dactivit y i n mal e animal s durin g sexual maturatio n b y examinin g abnormalitie s associate dwit h se x organ s an dpubert y marker s (se e Tabl e 2 above) , a s wel l a s thyroi dtissue . Th e guidelin e diazino n toxicit y studie s i n rat s (Tabl e 3 ) al l include dassessment s o f diazinon' s effect s o n androgen-sensitiv e mal e reproductiv e tissue s an dorgans , bot h i n adul t animal s an di n developin g animal s (includin g peri-puberta l animals) , includin g assessment s o f

effect s acros s generations . Thes e studie s als o include d histopathological assessment s o f thyroi dglan dtissu e i n animal s expose dbot h durin g gestatio n an ddevelopment , an da s adults . Th e onl y endpoint s fro mth e Tier 1 i n viv o assa

y i n puberta l mal e rat s that hav e no t bee n directl y measure di n th e currentl y availabl e diazino n guidelin e toxicity studie s are th e level s o f se x hormone s (i.e. , testosterone ) an dth e leve l o f thyroi dhormone s i n seru mo f animal s followin g diazino n exposure . I n th e cas e o f measurement

o f se x hormon e level s i n animal s (i.e. , testosterone) , th e fac t that 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 diazino n that coul db e relate dt o significan t change s i n hormon e level s alone , ther e doe s no t appea r t o b e astron g scientifi c basi s fo r

requirin g performanc e o f

EDS P Tie r

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

measure s o f

se x hormon e level s i n mal e rats . Further , give n that th e ultimat e effect s o f

either 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) , ther e doe s no t appea r t o b e astron g scientifi c basi s fo r

requirin g performanc e o f

astudy , suc h a s th e mal e puberta l assa y i n rats , simpl y t o provid e fo r measure s o f

thyroi dhormon e level s i n rats . A s aresult , MA N A propose s that monitorin g o f

thyroi dhormon e level s b e adde da s a n endpoint fo r th e Hershberge

r assay , where rat s d o no t

hav e a n intac t HP G axis . 3 9Makhteshi mChemica l Works , Ltd . Study No . MA-DZN-EDSP-2010021 3 I n thi s way , th e potential mod e o f actio n o f diazino n t o affec t

thyroi dhormon e level s wil l b e directl y addressed . MAN A propose s t o conduc t th e Hershberge r assa y becaus e th e tes t

follow s change s i n castrate dmal e rat s a s ascree n fo r androge n activity effects , amode l no t currentl y covere dwit

h diazino n OSRI , whil e OSRI directe dt o diazino n an dandroge n activity ar e availabl e i n peripuberta l mal e rat s (multi-generatio n reproductiv e toxicity study) . Therefore , a waive r o f EDS P Tier

1 890.150 0 (Pubertal Mal e Ra t Development ) i s sought . V . Overal l Summar y Th e precedin g discussio n provide s adetaile ddescriptio n o f availabl e diazino n OSRI tha t can b e use dt o suppor

t waiver s fo r

certai n EDS P Tier

1 screenin g assays . Th e diazino n OSR I i s ric h i n informatio n includin g informatio n specifi c t o assessin g th e potentia l fo r

estrogen , androge n an dthyroi dhormon e syste meffect s a s wel l a s th e effect s o f diazino n o n specifi c endpoint s (i.e. , potentia l mode s o f action) . MAN A i s seekin g waiver s fo r

performanc e o f

seve n 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 Transcriptional Activation) ; 890.145 0 (Puberta l Femal e Rat Development) ; an d890.150 0 (Pubertal Mal e Ra t Development) . MAN A als o propose s to ad d measurement o f thyroi dhormon e level s t o th e protocol s fo r

bot h th e Uterotrophi c assa

y i n rat s (890.1600 ) an dth e Hershberge r assa y i n rat s (890.1400) . Th e additio n o f measuremen t

o f thyroi dhormon e level s t o thes e tw o assay s wil l provid e dat ao n th e potential direc t effect s o f diazino n t o affec t

thyroi d hormon e releas e o r

synthesi s i n th e brai n an dpituitary gland . MAN A propose s t o conduc t th e Steroidogenesi s assa

y (890.1550 ) i n orde r

t o collec t dat ao n th e potential fo r

diazino n t o affec t

synthesi s o f

steroi dhormones , bot

h androgen s an destrogens . Additionally , MAN A propose s t o conduc t th e Fis h Short-Ter

m Reproductio n assa

y (890.1350 ) i n order

t o scree n fo r

potential endocrin e toxicity o f diazino n i n fish .

4 0Makhteshi mChemica l Works , Ltd . Stud y No . MA-DZN-EDSP-2010021 3

VI . Reference s Cite d 

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