N R 481420-0 1 

## -2.32 ,



# GLYPHOSATE : A Comprehensiv e Revie w an dEvaluatio n o f Othe r Scientificall y Relevan t

Informatio n (OSRI ) tha t Addresse s th e Requirement s fo r

Tier 1 Screenin g Unde r

th e Endocrin e Disrupte r Screenin g Progra m Prepare do n behal f

o f th e 

## Join t Glyphosat e Tas k

Force , LL C

Jun e 22 , 201 0

• • • • • • • ••••• • • • •

••• • • • • 

### • • • • • • • c • • • • •

•

### Executiv e Summar y

Introductio nan dBackgroun d I nJun e 2007 , EP A publishe di n th e Federa l Registe r aNotic e announcin g th e Draf t Lis t o f Initia l Pesticide Activ e Ingredient s an dPesticide Inert Ingredien t

t o b e Considere dfo r

Screenin g unde r th e Federa

l Food , Dru g an dCosmeti c Ac t [7 2 F R33486] . Th e initia l chemical s t o b e teste d unde r th e EP A Endocrin e Disrupte r Screenin g Progra m(EDSP ) wer e selecte dbase do n fou r huma nexposur e pathway s describe di n detai l i n aSeptembe r 2005 Federa l Registe r

notice , namel y foo dconsumption , drinkin g wate r consumption , residentia l us e exposur e an d occupationa l exposur e (contac t wit hpesticide-treate dsurfaces ) [7 0 F R56449] . Th e highes t priorit y fo r

inclusio no n th e initia l lis t wa s fo r substances , suc ha s glyphosate , havin g potentia l exposur e throug hal l fou r

pathways . Throughou t

th e selectio nprocess , EP A clearl y state dtha t "[t]hi s lis t shoul dno t

b e construe da s alis t

o f know no r likely endocrin e disruptors . Nothin g i n th e approac hfo r

generatin g th e initia l lis t provide s abasi s t o infe r

tha t b y simpl y bein g o n th e lis t thes e chemical s are suspecte dt o interfer e withth e endocrin e system s o f huma no r othe r species , an di t

woul db e inappropriat e to d o so. " Th e Offic e o f

Managemen t

an dBudge t

state di n it s "Term s o f

Clearance " relate dt o EPA' s issuanc e o f

th e firs t se t o f testin g order s tha t

"EPA shoul dpromot e an dencourag e tes t orde r recipient s t o submi t

OSRI i n lie u o f

performin g al l o r

som e o f th e Tie r I assays , an dEP A shoul daccep t

OSRI a s sufficien t

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

exten t possible " (OMB , 2009) . Thi s documen t

provide s acomprehensiv e overvie w o f Othe r Scientificall y Relevan t

Informatio n (OSRI ) t o asses s endocrine-relate dendpoint s tha t provid e functionall y equivalen t informatio nt o th e propose dTie r

1 screenin g battery . OSR I i s define db y EP A a s informatio ntha t inform s th e determinatio na s t o whethe r th e substanc e ma y hav e simila r effec t t o asubstanc e tha t

interact s wit hestrogen , androge no r thyroi dsystems . Functionall y equivalent , i nthi s context , refer s t o dat ao f asuitabl e natur e an dqualit y t o provid e th e sam e essentia l predictiv e informatio no n a chemica l eve ni f

differen t method s an dprocedure s ma y hav e bee nuse dfo r obtainin g th e data . Specifically , EP A ha s characterize dOSRI a s bein g either : (1 ) functionall y equivalen t t o informatio nobtaine dfro mth e Tie r 1 assay s o r

(2 ) dat afro massay s tha t

perfor mth e sam e functio na s EDS PTie r

1 Screens . OSR I fo r

glyphosat e i s draw nfro mbotho f thes e categories . Factor s include di nthi s evaluatio no f OSR I ar e functiona l equivalenc e o f th e endpoints , thei r sensitivity , thei r

specificit y fo r

aparticula r

mod e o f action , confidenc e i nth e conclusion s an d applicabilit y acros s taxa . Factor s als o includ e rout e an dduration/frequenc y o f administration, * • dos e level s tested , ag e a t

exposure , species , numbe r o f tes t units , variability , arjd * fjhethe r tht'dat S provide s abasi s fo r

conclusion s fo r

potentia l interactio nwit hth e endocrin e syStern " i & mammalia nan dnon-mammalia nsystems . OSRI t o provid e informatio nt o bypasf»Tje r 1 ; \. € 

### screenin g ca nbroadl y includ e th e followin g stud y types : structur e activit y relationsbfps , i n assays , metabolism , subchronic , chronic , reproductiv e an ddevelopmenta l studrasjG§R I for** ' glyphosat e primaril y include s informatio nfro mtoxicit y test s carrie dou t t o inteirii^ikflial regulator y guideline s tha t

include s an dassessmen t

o f reproduction , fertilit y an ddevelopmwitei * benchmark s predictiv e o f

endocrin e modulatin g potential . Additionally , i n man y o f thes e la.. , thes e studie s histopatholog y i s typicall y carrie dou t on :• Endocrin e tissue s (pituitary , thyroid , parathyroid , pancreas , adrenal , ovar y an dtestis )

• Hormon e sensitiv e mal e tissue s (prostate , epididymides , semina l vesicles )

• Hormon e sensitiv e femal e tissue s (mammary , uteru s an dreproductiv e tract ) Othe r relevan t scientifi c informatio npresente dher e fo r glyphosat e i s dat aobtaine dfro mstud y protocol s tha t

focu s o n reproduction , fertilit y an ddevelopmen t fo r mammalia n specie s an dothe r taxa , suc ha s bird s an dfish . Th e Tie r 1 screenin g batter y i s comprise do f 1 1 assays , fiv e o f

whic har e i n vitr o an d6 o f whic h ar e i n viv o assays . Th e i n vitr o screenin g assay s includ e th e estroge n recepto r (ER ) competitiv e bindin g assay , th e E Rtranscriptiona l activatio n assay wit hstabl y transforme dcel l line , th e androge n recepto r

(AR ) competitiv e bindin g assay , th e aromatas e inhibitio nassa y an dth e steroidogenesi s assay i n H295 Rcells . Th e i n viv o screenin g assay s includ e th e uterotrophi c assay , th e Hershberge r

assay , th e mal e an dfemal e puberta l assays , th e amphibia n metamorphosi s assay , an dth e short-ter mfis hreproductio n assay . Glyphosat e (CA S numbe r 1071-83-6 ) [N-(phosphonomethyl ) glycine ] i s a n herbicid e belongin g t o th e phosphon o amin o aci dclas s o f pesticides . Th e herbicida l propertie s o f glyphosat e wer e discovere db y Monsant o i n1970 , an dth e firs t commercia l formulation s wer e introduce di n197 4 unde r th e RoundupTM ® bran dname . Glyphosat e i s afoliar , non-selective , systemi c herbicid e widel y use dt o contro l weed s i n agricultura l crop s an dnon-agricultura l sites . Glyphosat e i s a specifi c inhibito r

o f th e enzym e 5-enolpyruvylshikimat e 3-phosphat e synthas e (EPSPS) . Thi s enzym e i s th e sixt henzym e o f th e shikimat e pathwa y an di t i s essentia l fo r

th e biosynthesi s o f aromati c amin o acid s (i.e. , tyrosine , tryptopha n an dphenylalanine ) an dth e othe r aromati c compound s i nalga e an dhighe r plants , bacteria , fung i an dsom e parasites . Inhibitio no f thi s enzym e lead s t o plan t

cel l death . I t

als o importan t

t o poin t ou t tha t th e shikimat e pathwa y i s absen t i n animals . Becaus e o f

it s ver y specifi c mod e o f action , glyphosat e exhibit s lo w toxicit y t o man y taxonomi c group s o f non-targe t

organism s includin g mammals , birds , fish , aquati c invertebrate s an dterrestria l invertebrates . Importan t consideration s i n evaluatin g potentia l t o interac t wit hth e endocrin e syste minclud e absorption , distribution , metabolis man dexcretio n (ADME) . Lo w absorption , limite d distributio nan dmetabolism , an drapi deliminatio n greatl y diminis hth e potentia l fo r acompoun d t o interac t wit hth e endocrin e system . Glyphosat e ha s bee n show n i n multipl e studie s conducte d o n multipl e specie s t o hav e lo w G I trac t an dderma l absorptio n (Ridle y an dMirly , 1988 ; B o Nielse ne t

al. , 2009) , limite ddistributio n (Colvi n an dMiller , 1973) , almos t n o mammalia n metabolis m(Ridley , 1983) , an drapi dexcretio n (Wester , 1991) . Therefore , th e potentia l fo r systemi c exposure s t o endocrin e tissue s i s extremel y lo w fo r glyphosat e an dtransformatio n products . Glyphosat e ha s undergon e acomprehensiv e batter y o f i n viv o toxicolog y an decotoxicolog y testin g tha t

cove r abroa dspectru mo f

endocrin e endpoints . Thi s testin g include s reproductive , developmental , subchroni c an dchroni c test s tha t hav e robus t

experimenta l designs , follo w internationall y accepte dprotocols , hav e ahig hleve l o f replicatio n an dalon g histor y o f us e i n hazar didentificatio nan dris k assessment . Thes e i n viv o studie s thoroughl y asses s endocrin e function s tha t

ar e require dfo r

reproduction , development , an dchroni c health . I t i s noteworth y tha t th e tw o recen t

ra t multigenerationa l studie s hav e bee n conducte dtha t confor mt o th e 199 8 U S EP A guidelin e (on e o f whic hals o conform s t o th e 200 1 OEC D 41 6 guideline) , includin grigorou s investigation s o f endocrin e sensitiv e endpoints . I n man y o f th e chroni c studie s n o effect s and/o r

n o effect s relate dt o endocrin e activit y wer e detecte da t hig hconcentration s an da t highes t concentratio n tested . Thi s provide s stron g evidenc e tha t glyphosat e doe s no t

hav e th e potentia l t o b e a n endocrin e activ e compound . Additionally , th e endocrine-modulatin g potentia l o f glyphosat e ha s bee n evaluate di n avariet y o f i n vitr o assays , whic hals o demonstrat e tha t glyphosat e doe s no t

hav e endocrin e activity . A n overvie w o f th e OSRJ t o mee t

al l o f thes e Tie r 1 screenin g requirement s i s summarize dbelow . E Rbindin g an dtranscriptio n activation : Assay s tha t provid e dat atha t

ar e functionall y equivalen t

t o dat agenerate db y th e ERa/ p competitiv e bindin g assay usin g ra t uterin e cytoso l an dth e ER atransactivatio n assay hav e alread y bee n conducted . Pee r reviewe darticle s b y Kojim ae t al . (2004 ) an dPeti t

e t al . (1997 ) presen t

scientifi c informatio nfro mi n vitr o tes t system s tha t

ar e ver y comparabl e i n methodology , sensitivit y an dspecificit y t o th e tw o Tie r 1 E Rbindin g assays . Th e result s fro mthes e assay s consistentl y demonstrat e tha t glyphosat e i s no t a n E Ragonis t

o r antagonist . Furthermore , astructur e activit y relationshi p (SAR ) analysi s wit h methodolog y develope db y EPA' s Offic e o f Researc han dDevelopmen t clearl y show s tha t glyphosat e doe s no t

hav e th e chemica l structur e o f atypica l molecul e wit hestrogeni c potential . Th e i n vitr o dat ademonstratin g tha t o f glyphosat e doe s no t

hav e E Rbindin g potentia l i s consisten t wit hth e comprehensiv e i n viv o dat aavailabl e fo r glyphosate . Th e i n viv o dat a demonstrate s n o effect s o n an y endpoin t tha t i s sensitiv e an dspecifi c t o compound s tha t hav e estrogeni c o r

anti-estrogeni c activity . A detaile drevie w o f th e i n viv o dat ause dt o evaluat e estrogenicit y i s discusse di nth e section s fo r th e uterotrophic , femal e puberta l assa y an dfis h screenin g assays . Base do n th e weigh t o f evidence , i t

ca n b e conclude dtha t

n o additiona l Tie r

1 i nvitr o screenin g i s require dt o evaluat e th e potentia l fo r E Ragonis man dantagonism . A Rbinding : Assay s tha t provid e dat atha t

i s functionall y equivalen t t o dat agenerate db y th e A R competitiv e bindin g assa y usin g ra t prostat e cytoso l hav e als o alread y bee n conducted . Th e pee r reviewe darticl e b y Kojim ae t al . (2004 ) present s scientifi c dat ausin g a n i n vitr o tes t syste man d methodolog y tha t

i s functionall y equivalen t t o th e methodolog y fo r

th e A Rbindin g assay i nth e Tie r 1 screenin g batter y tha t

demonstrate s tha t

glyphosat e i s no t

a n A Ragonis t

o r antagonist . Furthermore , th e SA Ranalysi s agai n indicate s tha t glyphosat e doe s no t

hav e th e chemica l structur e o f

atypica l molecul e tha t

exhibit s androgeni c potential . Lac k o f A Rbindin g i s confirme db y th e comprehensiv e i n viv o dat aavailabl e fro mchroni c studie s o n glyphosate . Th e i nviv o dat ademonstrate s n o effec t o n endpoint s tha t

ar e sensitiv e an dspecifi c t o compound s tha t hav e androgeni c o r

anti-androgeni c activity . A detaile drevie w o f th e i n viv o dat atha t

confirm s glyphosate' s i s no t

androgeni c o r

anti-androgeni c i s extensivel y reviewe di n th e section s fo r th e Hershberger , mal e puberta l assay an dfis hscreenin g assay . Base do n th e weigh t o f evidenc e i t ca n b e conclude dtha t

n o additiona l i n vitr o screenin g i s require dt o evaluat e th e potentia l fo r A R agonis man dantagonism . Aromatas e Inhibition : Althoug hi n vitr o dat ai s no t availabl e t o asses s aromatas e inhibition , OSRJ o f th e sam e natur e an dqualit y i s availabl e fro mexistin g i n viv o chroni c studies . N o treatmen t relate deffect s wer e observe di n thes e studie s o n sensitiv e an dspecifi c endpoint s tha t woul db e indicativ e o f

aromatas e inhibition , whic hprovide s stron g an dconsisten t evidenc e tha t glyphosat e i n no t

a n inhibito r

o f aromatas e activity . Scientificall y relevan t informatio n relate dt o steroidogenesi s i s comprehensivel y discusse di n th e followin g sectio n an dals o applie s t o aromatas e inhibition . Consequently , th e requiremen t fo r performin g th e i n vitr o aromatas e assayha s bee n me t b y providin g OSR I fro mstandar dregulator y i n viv o studie s tha t hav e th e abilit y t o detec t effect s o f

inhibitio no f

aromatas e activity . Steroidogenesis : I n vitr o dat aassessin g potentia l impact s o n th e complet e steroidogeni c pathwa y ar e no t

availabl e fo r

glyphosate ; however , dat atha t i s functionall y equivalen t t o th e i n vitr o Steroidogenesi s assay i s provide db y endpoint s include di n ra t multigenerationa l studies . Specifically , i t

wa s confirme di n th e mos t recen t multigenerationa l stud y tha t ther e wa s n o treatmen t relate deffec t

a t hig hdietar y exposur e level s o n endpoint s tha t woul db e impacte db y a n alteratio n o f

Steroidogenesis , e.g. , th e weigh t o f testes , caud aepididymides , epididymides , semina l vesicle s an dcoagulatin g glands , uterus , ovar y an dlive r o f th e F O an dF l paren t

animal s (Moxon , 2000 ; Dhins ae t

al. , 2007) . Additionally , ther e wa s n o treatmen t relate deffec t

u p t o th e highes t dos e teste d(HDT ) o n th e histopatholog y o f lef t testis , lef t epididymis , prostate , semina l vesicle s wit hcoagulatin g gland , uterus , lef t ovary , vagina , cervix , an doviducts . Furthermore , ther e wa s n o treatmen t

relate deffec t

u p t o th e HD T o n mal e an dfemal e reproductiv e indice s tha t include dmating , fertility , conception , gestation , lactatio n ag e an dweigh t a t VO , ag e a t

PPS , testicula r descent , ovaria n evaluatio n fo r follicles , parturition , normalit y o f externa l genitali aan d numbe r o f implantatio n sites . Th e i n viv o informatio n fro mthes e studie s provide s compellin g evidenc e o f

n o impac t

o f glyphosat e o n Steroidogenesis . Uterotrophic : A stron g weigh t o f evidenc e ha s bee n provide di n Sectio n 3. 1 o f thi s document , wit hfunctionall y equivalen t informatio nsupportin g alac k o f estrogeni c activit y o f

glyphosat e i n viv o fo r fou r multigenerationa l studies , subchroni c an dchroni c studie s i n rats . Th e result s o f th e i nviv o studie s ar e consisten t wit hresult s fro mi n vitr o assay s tha t demonstrat e glyphosat e i s no t a nE Rligand . Functionall y equivalen t dat a(uterin e weights ) o f

th e sam e natur e an dqualit y t o dat agenerate dwit hth e Uterotrophi c assay (uterin e weight s an dhistopathology ) demonstrat e tha t glyphosat e doe s no t

posses s estrogeni c activity . Hershberger : A stron g weigh t o f evidenc e ha s bee n presente di n Sectio n 3. 3 o f thi s document , usin g functionall y equivalen t informatio n supportin g alac k o f androge n recepto r

agonis man d antagonis man d5a-reductas e inhibitio no f glyphosat e i n viv o wit hfou r multigenerationa l studies , subchroni c an dchroni c studie s i nrats . Thi s functionall y equivalen t dat afro mmultigenerationa l studies , subchroni c an dchroni c studie s i n rat s demonstrat e tha t glyphosat e i s no t

a n androge n recepto r agonis t o r antagonis t

o r a5a-reductas e inhibitor . Femal e Pubertal : Functionall y equivalen t dat at o th e femal e puberta l endpoint s ar e describe d i nSectio n 3. 3 o f

thi s document , wit hacomprehensiv e overvie w o f endpoint s addresse di n th e multigenerationa l reproductive , subchroni c an dchroni c ra t studies . Endpoint s i nth e femal e puberta l assay ar e largel y asubse t o f th e multigenerationa l studie s performe do n glyphosat e an d consequentl y provid e dat ao f th e sam e natur e an dqualit y t o dat adevelope di n th e femal e puberta l assay o f

th e Tie r 1 batter y o f

th e Endocrin e Disrupte r

Screenin g Program . Mal e Pubertal : Lik e th e femal e puberta l assay , functionall y equivalen t dat at o th e mal e puberta l endpoint s ar e describe di n Sectio n 3.4 , wit hacomprehensiv e overvie w o f endpoint s addresse di n th e multigenerationa l reproductive , subchroni c an dchroni c ra t studies . Endpoint s i nth e mal e puberta l assay ar e als o essentiall y asubse t o f endpoint s fro mth e multigenerationa l studie s performe dfo r

glyphosat e an dconsequentl y provid e dat ao f th e sam e natur e an dqualit y t o dat adevelope di n th e mal e puberta l assay require dt o b e conducte di n th e EDSP . Amphibia n Metamorphosis : Th e amphibia n metamorphosi s assa y ha s bee n include di n th e Tie r 1 batter y a s ageneralize dmode l fo r impact s o n vertebrat e thyroi dfunctio nbecaus e th eamphibia nthyroi dsyste mi s regulate dan drespond s i n asimila r manne r t o perturbatio na s tha t

o f othe r tetrapods , includin g mammals . Therefore , combine dresult s fro mexistin g glyphosat e studie s provid e informatio ntha t i s functionall y equivalen t t o tha t generate di n th e amphibia n metamorphosi s assay . Thyroi dhistopatholog y ha s bee nevaluate di n standar dmammalia n toxicolog y studie s an ddemonstrate s n o impac t o n thyroi dstructur e an dfunction . Supportin g informatio ni s draw nfro mth e fis hful l life-cycl e stud y tha t show s n o impac t

o ngrowt han d development , whic har e sensitiv e measuremen t endpoint s fo r

impact s o n thyroi dstructur e an d function . Give nal l o f

thi s availabl e informatio no nglyphosate , th e amphibia nmetamorphosi s screenin g assa y doe s no t

offe r an y ne w relevan t

informatio nt o th e toxicolog y dat ase t fo r Tie r 1 screenin g purposes . FishShort-ter mReproduction : Th e methodolog y an dinformatio ngenerate dfro mafis hful l life-cycl e stud y provide s acomprehensiv e evaluatio no f potentia l modulatio no f

endocrin e activit y i nfis han dprovide s functionall y equivalen t informatio nt o informatio nfro mth e short

ter mfis hreproductio nassay . Th e fis hful l life-cycl e stud y i s th e highes t Tie r offis h ecotoxicolog y testin g an dtypicall y performe dwit hth e fathea dminno w (Pimphale s promelas ) a s afreshwate r

tes t species . I n th e study , th e entir e life-cycl e i s exposed ; therefore , d e fact o th e mos t sensitiv e life-stag e t o an y toxican t (includin g endocrin e activ e compounds ) i s encompasse d b y th e tes t

design . Although , endpoint s tha t ar e potentiall y influence db y endocrin e activity , suc ha s gonada l histopatholog y an dsteroi dhormon e production , ar e no t typicall y measure di n th e ful l life-cycle , afis hful l life-cycl e stud y provide s acomparabl e investigatio nt o th e tes t propose di nth e highe r

Tie r 2 testin g fo r

fish . Th e fis hful l life-cycl e stud y fo r glyphosat e demonstrate dn o treatment-relate dtreatmen t relate deffect s o n th e survival , growthan deg g productio no f

firs t generatio nfis ho r

o n hatchability , a s wel l a s surviva l an dgrowt ho f second

generatio negg s an dfr y ove r a n 8 mont hexposur e perio dt o concentration s a s hig ha s 25. 7 mg/L . Furthermore , i t

ha s bee nshow ni n viv o tha t glyphosat e doe s no t

induc e productio no f vitellogenin , whic hi s i nagreemen t wit hSA Ranalysi s o f

glyphosate . Th e result s fro mth e fis h full-lif e cycl e stud y ar e consisten t wit hth e result s for msubchroni c an dchroni c tes t wit h mammalia nspecies , whic hdemonstrat e n o change s i nendocrin e function , endocrin e orga n histopathology , an dal l reproductiv e parameters . Additionally , th e result s fro mavia none generatio nreproductio nstudie s demonstrat e n o effec t o n an y o f th e measure dendpoint s a t hig h tes t concentrations . Base do n thi s OSRI , ther e i s sufficien t informatio nt o mee t

th e requirement s fo r performin g afis hshor t

ter mreproductio nstudy . 

### Publishe dGlyphosat e Review s fo r Endocrin e Activit y Severa l in-dept hreview s o n th e safet y o f glyphosat e hav e bee nconducte db y severa l regulator y agencie s an dscientifi c institution s worldwid e hav e conclude dtha t ther e i s n o indicatio ntha t glyphosat e ha s potentia l endocrin e activit y o r wil l resul t

i n advers e effect s o n endocrin e system s i nhuman s o r

othe r mammals . Th e followin g point s summariz e th e conclusion s o f thes e reviews . • Th e U.S . EP A (1998 ) reviewe dth e subchroni c an dchroni c mammalia nstudie s fo r glyphosat e an dconclude dtha t ther e wa s n o evidenc e t o sugges t tha t glyphosat e produce s endocrine-modulatin g effects . • I n acomprehensiv e revie w o f th e standar dmammalia ntoxicolog y studies , William s e t al. , (2000 ) conclude dtha t

glyphosat e doe s no t

hav e th e potentia l t o produc e advers e effect s o n endocrin e system s i n human s o r othe r mammals .• A repor t prepare dfo r

th e USD A fores t

servic e b y Syracus e Environmenta l Researc h(SERA ) conclude dtha t

extensiv e testin g i nexperimenta l animal s an dwildlif e provide dreasonabl y stron g evidenc e tha t

glyphosat e i s no t

a n endocrin e disrupte r

(SERA , 2002) . • Th e lis t

o f purporte dendocrin e disrupter s compile db y th e Institut e o f Environmen t

an d Healt h(IEH , 2005 ) list s glyphosat e a s asubstanc e wit hn o evidenc e o f potentia l endocrine

disruptin g effects . • Followin g arevie w o f th e standar dmammalia n an dwildlif e toxicolog y studies , i t wa s conclude di n arecen t

ECETO C guidanc e documen t o n identifyin g endocrin e disruptin g effect s tha t

glyphosat e i s no t

a n endocrin e disrupto r

usin g th e internationall y accepte d Weybridg e definitio nfo r endocrin e disruptio n (ECETOC , 2009) . Summar y an dConclusion s Th e endocrine-modulatin g potentia l o f glyphosat e ha s bee n evaluate di n avariet y o f studie s includin g i n vitr o assay s an dstandar di n viv o toxicolog y studie s tha t ar e functionall y equivalen t t o th e dat agenerate di n th e Tie r 1 screenin g battery . Th e i n viv o studie s comprehensivel y asses s endocrin e function s tha t

ar e require dfo r

reproduction , developmen t an dchroni c healt han dsho w n o indicatio n o f

endocrin e disruptio n i nmammal s an dwildlif e species . Glyphosat e ha s demonstrate dn o (anti- ) estrogeni c o r (anti- ) androgeni c potentia l i n i n vitr o assays , an dther e wa s n o indicatio n o f

change s i nendocrin e functio no r endocrin e orga n histopatholog y i nan y o f th e i n viv o studies . Singl e effect s wer e bot hmino r an dno t consisten t

withi n o r betwee n studie s o r wer e relate dt o genera l toxicity . Additionally , structur e activit y model s predic t tha t glyphosat e doe s no t

hav e estrogeni c o r

androgeni c activity . Base do n thi s weight-of-evidenc e analysi s fro mglyphosate' s extensiv e toxicologica l database , ther e i s sufficien t

scientificall y relevan t

informatio n o f

th e sam e natur e an dqualit y a s tha t develope di n th e Tie r

1 screenin g batter y t o conclud e tha t glyphosat e doe s no t

hav e endocrine

modulatin g potential . Therefore , th e requirement s o f Tie r 1 testin g hav e bee n satisfie dan d additiona l testin g unde r

th e Endocrin e Disruptio n Screenin g an dTestin g Progra mi s no t warranted .

1 Weybridg e (1996 ) "A nendocrin e disrupte r i s a nexogenou s substanc e tha t cause s advers e healt heffect s i na n intac t organism , o r

it s progeny , secondar y (consequent ) t o change s i n endocrin e function . A potentia l endocrin e disrupte r i s asubstanc e tha t

possesse s propertie s tha t migh t b e expecte dt o leadt o endocrin e disruptio n i n a n intac t organism. "

## Content s

Executiv e Summar y 2 Introductio nan dBackgroun d2 Publishe dGlyphosat e Review s fo r Endocrin e Activit y 6 Summar y an dConclusion s 7 1. 0 Introductio n1 1 1. 1 Consideration s fo r

Dos e Settin g fo r

Endocrin e Screenin g an dTestin g 1 5 2. 0 I n Vitr o Assay s 1 6 2.1 Estroge nCompetitiv e Recepto r Bindin g Assa y (OPPT S 890.1250 ) andER Transcriptiona l Activatio nAssa y (OPPT S 890.1300 ) 1 6 2.1. 1 Structur e Activit y Relationshi p 1 6 2.1. 2 Estroge nRecepto r (ER ) Bindin g an dTransactivatio nAssay s 1 6 2.1. 3 Conclusio n-Estroge n Recepto r Agonis man dAntagonis m2 1 2. 2 Androge nRecepto r (AR ) Bindin g Assa y (OPPT S 890.1150 ) 2 2 2.2. 1 Structur e Activit y Relationshi p (SAR ) Predictio no f A RBindin g Potentia l 2 2 2.2. 2 Androge nRecepto r (AR ) Bindin g Assa y 2 2 2.2. 1 Conclusio n- Androge nRecepto r Agonis man dAntagonis mo f Glyphosat e 2 4 2. 3 Aromatas e Assa y (OPPT S 890.1200 ) 2 5 2.3. 1 Conclusio n- Aromatas e Inhibitio n2 6 2. 4 Steroidogenesi s Assa y (OPPT S 890.1550 ) 2 7 2.3. 1 Conclusio n- Steroidogenesi s 2 9 3. 0 Mammalia nI n Viv o Assay s 2 9 3. 1 Uterotrophi c Assa y (OPPT S 890.1600 ) 3 0 3.1. 1 Discussio n3 1 3.1. 2 Conclusion s - Uterotrophi c Assa y 3 4 3. 2 Hershberge r

Bioassa y (OPPT S 890.1400 ) 3 5 3.2. 1 Prostat e /

Ventra l Prostat e 3 6 3.2. 2 Semina l Vesicle s 3 6 3.2. 3 Levato r

An i Bulbocavernosu s (LABC ) 3 7 3.2. 4 Bulbourethra l (Cowper's ) Glan d3 7 3.2. 5 Glan s Peni s Weigh t 3 8 3.2. 6 Live r

Weigh t 3 8 3.2. 7 Luteinizin g Hormon e (LH ) 3 9 3.2. 8 Testosteron e 3 93.2. 9 Othe r endpoint s o f

relevanc e t o th e Hershberge r Assa y 3 9 3.2.1 0 Conclusio n- Hershberge r Assa y 4 0 3. 3 Puberta l Developmen t an dThyroi dFunctio ni n Intac t Juvenil e /

Peripuberta l Femal e Rat s (OPPT S 890.1450 ) 4 0 3.3. 1 Growth , Bod y Weigh t 4 1 3.3. 2 Ag e a t

Vagina l Openin g 4 2 3.3. 3 Bod y Weigh t

a t Vagina l Openin g 4 2 3.3. 4 Uteru s Weigh t

an dHistopatholog y 4 2 3.3. 5 Estru s Cyclicit y 4 3 3.3. 6 Ovarie s Weigh t

an dHistopatholog y 4 3 3.3. 7 Thyroi dWeigh t

an dHistopatholog y 4 3 3.3. 8 Thyroi dStimulatio nHormon e (TSH ) 4 4 3.3. 9 Thyroxin e (T 4 ) 4 4 3.3.1 0 Kidne y Weigh t

an dHistopatholog y 4 5 3.3.1 1 Live r

Weigh t 4 5 3.3.1 2 Adrenal s Weigh t 4 5 3.3.1 3 Pituitar y Weigh t

4 6 3.3.1 4 Clinica l Chemistr y 4 6 3.3.1 5 Conclusio n- Femal e Puberta l Assa y 4 6 3. 4 Puberta l Developmen t an dThyroi dFunctio ni n Intac t Juvenil e /

Peripuberta l Mal e Rat s (OPPT S 890.1500 ) 4 7 3.4. 1 Growth , Bod y Weigh t 4 8 3.4. 2 Ag e a t

Preputia l Separatio n4 9 3.4. 3 Bod y Weigh t

a t Preputia l Separatio n4 9 3.4. 4 Teste s Weigh t

an dHistopatholog y 5 0 3.4. 5 Epididymide s Weigh t an dHistopatholog y 5 0 3.4. 6 Prostat e Weigh t

5 0 3.4. 7 Semina l Vesicle s Weigh t 5 1 3.4. 8 Testosteron e 5 1 3.4. 9 Thyroi dweigh t

an dHistopatholog y 5 1 3.4.1 0 Thyroi dSimulatio nHormon e (TSH ) 5 1 3.4.1 1 Thyroxin e (T 4 ) 5 2 3.4.1 2 Kidne y Weigh t

an dHistopatholog y 5 2 3.4.1 3 Live r

Weigh t 5 2 3.4.1 4 Adrenal s Weigh t 5 23.4.1 5 Pituitar y Weigh t 5 3 3.4.1 6 Clinica l Chemistr y 5 3 3.4.1 7 Conclusio n- Mal e Puberta l Assa y 5 3 3. 5 Summar y o f

I n Viv o OSR I fo r

Mammalia nSpecie s 5 5 4. 0 Non-Mammalia nI n Viv o Assay s 5 5 4. 1 Amphibia nMetamorphosi s Assa y (AMA , OPPT S 890.1100 ) 5 5 4.1. 1 Conclusio n- Amphibia nMetamorphosi s Assa y (AMA ) 5 7 4. 2 Fis hShort-ter mReproductio nAssa y (OPPT S 890.1350 ) 5 8 4.2. 1 Result s Summary-Fis hFul l Lifecycl e Stud y 6 1 4.2. 2 Structur e Activit y Relationshi p Indicate s Lac k o f Estrogenicit y 6 1 4.2. 3 Glyphosat e doe s no t induc e productio no f

vitellogeni n6 1 4.2. 4 Avia none-generatio nreproductio nassay s 6 2 4.2. 5 Conclusio n- Fis hshort-ter mreproductio nassay 6 2 5. 0 Conclusio n- EDS P Tie r 1 OSR I Overvie w 6 4 5. 1 Glyphosat e Review s fo r Endocrin e Activit y 6 7 5. 2 Summar y an dConclusion s 6 7 Reference s 6 8 Appendi x 1 7 5 Appendi x 2 7 6 Appendi x 3 8 1



### 1 0

### 1. 0 Introductio n

I nJun e 2007 , EP A publishe di n th e Federa l Registe r aNotic e announcin g th e Draf t Lis t o f Initia l Pesticide Activ e Ingredient s an dPesticide Inert s t o b e Considere dfo r Screenin g unde r

th e Federal Food , Drug , an dCosmeti c Ac t [7 2 F R33486] . Th e initia l chemical s t o b e teste dunde r th e EP A Endocrin e Disrupte r Testin g Progra mwer e selecte dbase do n fou r huma n exposur e pathway s describe di ndetai l i na nearlie r Federa l Registe r

notice , namely , foo dconsumption , drinkin g wate r

consumption , residentia l us e exposure , an doccupationa l exposur e (contac t wit h pesticide-treate dsurfaces ) [7 0 F R56449] . Th e highes t priorit y fo r

inclusio n o n th e initia l lis t wa s fo r substances , lik e glyphosate , tha t hav e potentia l exposur e throug hal l fou r pathways . Throughou t

th e selectio n process , EP A clearl y state dtha t "ftjhi s lis t

shoul dno t

b e construe da s alis t o f know n o r likely endocrin e disruptors . Nothin g i n th e approac h fo r generatin g th e initial lis t provide s abasi s t o infe r

tha t b y simpl y bein g o n th e lis t thes e chemica l are suspecte dt o interfer e withth e endocrin e system s o f huma no r othe r species , an di t

woul db e inappropriat e t o do so. " Glyphosat e (N-(phosphonomethyl)glycine , CA S numbe r 1071-83-6 ) i s a n herbicid e belongin g t o th e phosphon o amin o aci dclas s o f pesticides . Glyphosat e i s afolia r applied , non-selective , systemi c herbicid e widel y use dt o contro l weed s i n agricultura l crop s an do n non-agricultura l sites . Glyphosat e i s aspecifi c inhibito r o f th e sixt henzym e o f

th e shikimat e pathway , 5

- enolpyruvyl-shikimat e 3-phosphat e synthas e (EPSPS) , whic hi s essentia l fo r th e biosynthesi s o f aromati c amin o acid s (i.e. , tyrosine , tryptopha n an dphenylalanine ) an dothe r aromati c compound s i n alga e an dhighe r plants , bacteri aan dfungi . Inhibitio n o f thi s enzym e lead s t o plan t cel l death . Sinc e th e shikimat e pathwa y and , therefore , th e EPS Psynthas e targe t site , i s foun donl y i nplants , bacteri aan dfungi , an dno t i nanimals , glyphosat e generall y exhibit s lo w toxicit y t o highe r

organisms , includin g mammals , birds , fish , aquati c invertebrate s an dterrestria l invertebrates . Th e Offic e o f

Managemen t

an dBudge t

state di n it s "Term s o f

Clearance " regardin g EPA' s issuanc e o f

th e initia l se t

o f tes t order s fo r

th e Endocrin e Disrupto r

Screenin g Progra m(EDSP ) tha t "EP A shoul dpromot e an dencourag e tes t order recipient s t o submi t

OSR I i n lieu o f performin g al l o r

som e o f th e Tier I assays , an dEP A shoul daccep t OSR I a s sufficien t

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

exten t possible " (OMB , 2009) . Othe r scientificall y relevan t

informatio n (OSRI ) i s define db y EP A a s "informatio n tha t inform s th e determinatio n a s t o whethe r th e substanc e ma y hav e asimila r effec t t o asubstanc e tha t interact s wit hestrogen , androge n an dthyroi dsystems . " Functionall y equivalent , i n thi s context , refer s t o dat ao f

asuitabl e natur e an dqualit y tha t provide s th e sam e essentia l predictiv e informatio no n achemica l eve n i f differen t method s an dprocedure s ma y hav e bee n use dfo r obtainin g th e data . Specifically , EP A ha s characterize dOSR I a s bein g either , (1 ) functionall y equivalen t

t o informatio n obtaine dfro mth e Tie r 1 assays , o r

(2 ) dat afro massay s tha t

perfor m th e sam e functio na s EDS PTie r 1 Screens . Thi s documen t

provide s scientifi c informatio n regardin g endocrine-relate dendpoint s fo r glyphosat e tha t

i s functionall y equivalen t t o th e informatio n propose dt o b e obtaine db y th e Tie r 1 Screenin g (T1S ) batter y o f th e EDSP . Factor s include di n thi s evaluatio n o f othe r scientificall y relate dinformatio n wer e functiona l equivalenc e o f th e endpoints , sensitivit y o f th e test , specificit y fo r

aparticula r

mod e o f action , confidenc e i n th e conclusion s an dapplicabilit y acros s taxa . Othe r

factor s wer e rout e an dduration/frequenc y o f administration , dos e levels , ag e a t 1 1exposure , species , numbe r o f tes t units , variability , an dwhethe r o r no t th e dat aprovide dabasi s fo r conclusion s relate dt o th e potentia l interactio n o f glyphosat e wit hth e endocrin e syste mi n mammalia n an dnon-mammalia n systems . I t i s plausibl e tha t

OSRI fo r Tie r 1 assay s ca nbroadl y includ e anumbe r o f stud y types , including , structur e activit y relationship s (SAR ) analyses , mechanisti c i n vitr o assay s an di n viv o metabolism , subchronic , chronic , reproductiv e an ddevelopmenta l studies . Th e majorit y o f th e OSR I presente dher e fo r

glyphosat e i s fro mtoxicit y test s conducte daccordin g t o internationa l regulator y guidelines . Thi s dat aprimaril y consist s o f developmenta l benchmark s predictiv e o f endocrin e disruptio n potentia l an dhistopatholog y o n ke y tissues , including :

• Endocrin e tissue s (pituitary , thyroid , parathyroid , pancrea s adrenal , ovar y an dtestis )

• Hormon e sensitiv e mal e tissue s (prostate , epididymides , an dsemina l vesicles )

• Hormon e sensitiv e femal e tissue s (mammary , uteru s an dovaries ) Othe r relevan t scientifi c informatio n presente dher e fo r glyphosat e i s dat aobtaine dfro mstud y protocol s tha t

focu s o n reproduction , fertilit y an ddevelopmen t fo r mammalia n specie s an dothe r taxa , suc ha s bird s an dfish . Th e endocrine-modulatin g potentia l o f glyphosat e ha s bee n evaluate di n avariet y o f studies , includin g i n vitr o assay s an dstandar di n viv o toxicolog y studies . Glyphosat e ha s demonstrate d n o estrogenic , anti-estrogenic , androgeni c o r anti-androgeni c potentia l i n an y i n vitr o assays . Ther e wer e als o n o definitiv e finding s o f endocrine-modulatin g effect s i nth e glyphosat e subchronic , chronic , developmental , o r reproductiv e toxicit y studie s (se e Table s 3 an d4 fro m William s e t

al . 2000 ; Moxon , 2000 ; Dhins ae t al , 2007) . Histopathologica l observation s o f endocrin e an dreproductiv e tissue s fro manimal s i nchroni c an dmultigenerationa l toxicit y studie s indicat e tha t glyphosat e exposur e ha dn o advers e histologica l consequenc e o n an y reproductiv e o r endocrin e tissu e fro meithe r mal e o r femal e rat s eve n a t exaggerate ddosag e levels . Also , n o advers e result s wer e obtaine di n adominan t letha l stud y conducte da t

ver y hig hdose s o f

glyphosate . Whil e thi s latte r tes t i s typicall y use dt o asses s geneti c toxicity , substance s tha t affec t mal e reproductiv e functio nthroug hendocrin e modulatin g mechanism s ca n als o produc e effect s i n thi s typ e o f study ; suc hreproductiv e indice s includ e mating , fertilit y an dconception . Additionally , n o effect s indicativ e o f endocrin e activit y wer e detecte di nafis hful l life-cycl e stud y an dsevera l avia n one-generatio n reproductio n studie s performe da t

exaggerate ddosag e levels . I nsummary , n o endocrine-relate deffect s wer e observe di nfou r independent , multigenerationa l reproductio n studie s conducte da t severa l dose s rangin g fro mlo w t o thos e exceedin g expecte d huma nglyphosat e exposur e b y a t leas t severa l order s o f

magnitude , afis hful l life-cycl e stud y an dsevera l avia n reproductio n studies . I t ca n b e concluded , therefore , tha t asufficien t

batter y o f studie s ha s alread y bee n conducte dt o evaluat e th e endocrine-modulatin g potentia l o f glyphosat e an dtha t th e result s fro mal l thes e studie s demonstrat e tha t glyphosat e i s no t

areproductiv e toxican t an ddoe s no t

pertur b th e endocrin e system . In-dept hreview s o n th e safet y o f glyphosat e hav e bee n conducte db y severa l regulator y agencie s an dscientifi c institution s worldwide , an dal l hav e conclude dtha t ther e i s n o indicatio n tha t glyphosat e ha s potentia l endocrin e activit y o r wil l resul t

i nadvers e effect s o nendocrin e system s i nhuman s o r

othe r mammals . Th e conclusion s o f thes e review s ar e summarize dbelow . 1 2• Th e U.S . EP A (1998 ) reviewe dth e subchroni c an dchroni c mammalia n studie s fo r

glyphosat e an dconclude dtha t ther e wa s n o evidenc e t o sugges t tha t glyphosat e produce s endocrine-modulatin g effects . • I n acomprehensiv e revie w o f th e standar dmammalia n toxicolog y studies , William s e t al. , (2000 ) conclude dtha t

glyphosat e doe s no t

hav e th e potentia l t o produc e advers e effect s o n endocrin e system s i n human s o r othe r mammals . • A repor t

submitte dt o th e USD A Fores t Servic e b y Syracus e Environmenta l Researc h (SERA ) conclude dtha t

extensiv e testin g i n experimenta l animal s an dwildlif e provide d reasonabl y stron g evidenc e tha t glyphosat e i s no t

a n endocrin e disrupte r

(SERA , 2002) . • Th e lis t

o f purporte dendocrin e disrupter s compile db y th e Institut e o f Environmen t

an d Healt h(IEH , 2005 ) list s glyphosat e a s asubstanc e wit hn o evidenc e o f potentia l endocrine

disruptin g effects . • Followin g arevie w o f th e standar dmammalia n an dwildlif e toxicolog y studies , i t wa s conclude di narecen t

ECETO C guidanc e documen t o nidentifyin g endocrin e disruptin g effect s tha t

glyphosat e i s no t

a n endocrin e disrupte r

usin g th e internationall y accepte d Weybridg e definition 2 fo r endocrin e disruptio n (ECETOC , 2009) . Importan t consideration s i n evaluatin g th e potentia l o f achemica l t o interac t

wit hth e endocrin e syste minclud e absorption , distribution , metabolis man dexcretio n (ADME) . Lo w absorption , limite ddistributio n an drapi dexcretio n o f achemica l fro mth e bod y limit s it s potentia l t o interac t wit hth e endocrin e system . Metabolite s ca n hav e agreate r o r lesse r potentia l t o interac t

wit hth e endocrin e system , dependin g o n th e natur e o f th e metabolit e relativ e t o th e paren t molecule . I t ha s bee n show n i nmultipl e studie s conducte di nmultipl e specie s tha t glyphosat e exhibit s lo w G I trac t an dderma l absorptio n (Ridle y an dMirly , 1988 ; B o Neilse n e t al , 2009) , limite d distributio ni n th e bod y (Colvi n an dMiller , 1973) , almos t n o mammalia n metabolis m(Ridley , 1983) , an drapi dexcretio n fro mth e bod y (Wester , 1991) . Therefore , th e potentia l fo r systemi c exposur e o f

endocrin e tissue s t o glyphosat e metabolite s i s extremel y low . OEC D membe r

countrie s hav e lon g recognize dth e potentia l o f structur e activit y relationship s (SAR ) analysi s a s a nai di nconductin g initia l hazar dassessment s fo r chemicals , includin g potentia l endocrin e activity . I n Augus t 2009 , th e EPA' s midcontinen t Divisio n i n Dulut h presente dastructur e activit y mode l develope dt o evaluat e estroge n recepto r (ER ) bindin g potential . Thi s mode l i s a n hypothesis-drive n approac hbase do n wha t i s describe da s 'Exper t Rules ' t o assig n E Rbindin g potentia l an dho w th e systemprovide s informatio n supportin g th e assignment . EPA' s FIFR A Scientifi c Advisor y Panel , wit hth e hel p o f th e Foo dQualit y Protectio n Ac t

Scienc e Revie w Board , endorse dth e mode l a s atoo l t o predic t whethe r a chemica l ca n bin dt o th e E Ran deventuall y t o hel p prioritiz e chemical s fo r Tie r 1 screenin g unde r th e EDSP .

2 Weybridg e (1996 ) "A n endocrin e disrupte r i s a n exogenou s substanc e tha t cause s advers e healt heffect s i n a n intac t organism , o r

it s progeny , secondar y (consequent ) t o change s i n endocrin e function . A potentia l endocrin e disrupte r i s asubstanc e tha t

possesse s propertie s tha t migh t b e expecte dt o lea dt o endocrin e disruptio n i n a n intac t organism. " 1 3Thi s mode l predict s tha t achemica l wil l hav e lo w potentia l fo r interactio n wit hth e E Rif , 1 . th e chemica l belong s t o agrou p wher e testin g showe dn o evidenc e o f E Rinteractio n (relativ e bindin g affinit y les s tha n 0.00001%) , a s wit hacycli c molecule s o r cycli c molecule s tha t

d o no t contai n alikel y H-bondin g group , an d 2 . th e lo g KO Wofth e chemica l i s les s tha n 1.3 . Molecule s tha t

hav e bee n show n t o hav e E Rbindin g activit y ar e cycli c o r aromatic , havin g a n overal l rin g structur e a s describe db y Blai r e t al. , 2000 ; Nishihar ae t

al. , 2000 ; Kojim ae t

al , 2004 . Glyphosat e i s a n acycli c molecule , a s illustrate dbelow , and , therefore , doe s no t contai n th e cor e structur e o f

compound s predicte dt o hav e estrogeni c activity . Glyphosat e als o ha s alo g K ow o f muc hles s tha n 1. 3 (lo g K o w = -3.2) . Applyin g th e expert rule s o f th e mode l t o glyphosate , arelativ e bindin g affinit y (RBA ) o f <0.00001 % ca n b e assigned , indicatin g essentiall y n o potentia l t o interac t wit hth e ER . 

# 1 * Glyphosat e lo g Ko w = - 3. 2

Commo n structur e i n estrogeni c compounds . R= hydrophobi c moiet y an dB = bulk y moiet y Althoug hSA Rmodel s ar e no t develope dt o th e sam e degre e fo r androgen s a s the y ar e fo r estrogens , man y o f

th e sam e principal s tha t

appl y t o assessin g estrogeni c potentia l appl y t o assessin g androgeni c an danti-androgeni c potential . Base do n it s chemica l structure , glyphosat e i s predicte dt o no t

hav e androgeni c activit y (Deviller s e t al. , 2009 ; Panay e e t

al. , 2008) . Th e repea t

dos e i n viv o toxicolog y studie s require db y th e U.S . EP A an dothe r regulator y agencie s worldwid e detec t modulatio n o f

endocrin e systemactivit y (Carne y e t al. , 1997 ; Steven s e t al , 1997 , 1998 ; Harve y an dJohnson , 2002) . Thes e i n viv o studie s ar e mor e predictiv e tha n i n vitr o screenin g assay s a s the y asses s avariet y o f endocrine-sensitiv e endpoint s i n animal s tha t ar e capabl e o f

metaboli c activatio n and/o r detoxification . Thes e studie s als o us e extende d exposur e period s encompassin g variou s stage s o f endocrin e developmen t

i n intac t organisms . Endocrine-activ e substance s affectin g asingl e o r multipl e endocrin e targe t site s invariabl y initiat e direc t

o r compensator y biochemical , cellular , and/o r histopathologica l processe s tha t wil l 

### 1 4

### b e detectedi nstandar dtoxicolog y studie s require dfo r pesticid e registratio ni nCanada , Europe , Japa nan dth e Unite dStates . A comprehensiv e histopathologica l assessmen t o f endocrin e tissue s combine dwit hgros s orga npatholog y an dorga nweigh t dat aallow s detectio no f

al l advers e endocrinopathies . Th e standar dtoxicolog y an decotoxicolog y studie s tha t provid e valuabl e informatio no n potentia l endocrine-modulatin g effect s includ e subchronic , chronic , an ddevelopmenta l an d reproductio nstudie s (Tabl e 1) . Th e multigenerationa l ra t reproductio nstud y i s currentl y th e mos t comprehensiv e an ddefinitiv e stud y fo r evaluatin g th e potentia l o f substance s t o produc e endocrin e modulatin g effect s i nhuman s an dothe r mammals . Thi s stud y evaluate s effect s o n gonada l developmen t

an dfunction , estrou s cycles , matin g behavior , fertilization , implantation , i n uter o development , parturition , lactation , an dth e abilit y o f offsprin g t o survive , develo p an d successfull y reproduce . A histopathologica l assessmen t o f relevan t orga nsystem s i s aprominen t featur e o f thes e studies . Developmenta l toxicit y studie s evaluat e effect s o nman y o f thes e sam e processes , whil e subchroni c an dchroni c studie s incorporat e numerou s direc t an dindirec t evaluation s o f

endocrin e an dreproductiv e tissues , suc ha s targe t orga nweight s an da comprehensiv e assessmen t o f endocrin e orga npathology . Tabl e 1 : I n viv o ecotoxicolog y guidelin e tests tha t provid e endpoint s capabl e o f detectin g endocrin e effect s an dacompariso n t o th e ra t multigenerationa l reproductio n stud y Endpoin t Fertilizatio nSucces s Hatchabilit y Birt ho r hatchin g weigh t Morphologica l abnormalitie s Viabilit y Behavio r Growt h Onse t o f maturit y (o r puberty ) o r

reproductio n Fecundit y o f

offsprin g Se x rati o o f

offsprin g Interse x i noffsprin g Eg g shel l thinnin g Fis hFul l Lif e Cycl e Tes t (OPPT S 850.1500) Ye s Ye s N o Ye s Ye s Ye s (mating ) Ye s Ye s N o N o N o N A Avia n Reproductio n Tes t

(OEC D 206/OPPT S 850.2300 ) Ye s Ye s Ye s Ye s Ye s N o Ye s Ye s N o N o N o Ye s Ra t

Multigenerationa l Reproductio n Tes t Ye s N A Ye s Ye s Ye s Ye s (mating ) Ye s Ye s Ye s Ye s Ye s N A 

### 1. 1 Consideration s fo r Dos e Settin g fo r

Endocrin e Screenin g an dTestin g On e o f th e mos t critica l issue s face di nendocrin e testin g i s settin g th e appropriat e dos e level s o r concentration s a t

whic ht o conduc t

th e screenin g assays . Appropriat e dos e settin g i s critica l t o no t confus e systemi c toxicit y wit hgenuin e endocrin e mediate deffects . Therefore , dos e settin g take s o nconsiderabl y greate r importanc e i nth e testin g fo r advers e effect s o nth e endocrin e 1 5system s tha n i n traditiona l toxicit y testing . Th e purpos e o f screenin g i s t o identify th e potentia l t o interac t

wit hth e endocrin e syste mvi aa n estrogenic , androgeni c o r thyrogeni c mod e o f

action , no t t o merel y identify advers e effects . Thus , i t i s critica l tha t

dose s ar e se t

i nth e rang e wher e effect s o n th e measure dendpoint s ar e no t indirectl y impacte db y genera l mechanism s o f toxicity , suc ha s live r

toxicit y o r

bod y weigh t

changes , o r

produc e a n indirec t

effec t a s th e resul t

o f over t toxicit y o r

agenera l stres s response . A novervie w o f

eac ho f th e eleve n assay s i n th e Tie r 1 batter y i s provide di n th e introductio n t o eac hsectio n below . Additionally , abrie f tabula r summar y describin g eac ho f th e assay s i s presente di nAppendi x 1 . 

### 2. 0 I n Vitr o Assay s 2. 1 Estroge n Competitiv e Recepto r Bindin g Assa y (OPPT S 890.1250 ) an dE R Transcriptiona l Activatio n Assa y (OPPT S 890,1300 ) Functionall y equivalen t

dat at o tha t

whic hca n b e obtaine db y performin g th e E Rbindin g assa y an dth e E Rtranscriptiona l activatio n assa y ar e availabl e fo r glyphosat e i n thre e independen t studie s reporte di n peer-reviewe djournals . Thes e studie s clearl y demonstrat e tha t glyphosat e doe s no t posses s estrogeni c o r

anti-estrogeni c potentia l an dwer e conducte dusin g comparabl e an dfunctionall y equivalen t methodolog y t o OPPT S guideline s fo r th e Tie r 1 E Rcompetitiv e bindin g assay an dE Rtranscriptiona l activatio n assay . Thi s OSR I demonstrate s tha t glyphosat e doe s no t posses s estrogeni c o r

anti-estrogeni c potential . Thi s sectio n primaril y review s OSR I tha t assesse s th e potentia l fo r direc t interactio n o f

glyphosat e wit hth e estroge n recepto r usin g i n vitr o tes t system s simila r

t o thos e define di n th e EDSP . Thi s sectio n als o include s adiscussio n o n th e us e o f structur e activit y relationshi p model s t o evaluat e achemical' s estroge n recepto r bindin g potentia l an dho w i t ca n b e use dt o validat e th e result s o f th e estroge n recepto r

bindin g an dtranscriptiona l activatio n studie s fo r glyphosate . 2.1. 1 Structur e Activit y Relationshi p Ther e ar e tw o approache s t o asses s th e potentia l fo r achemica l t o interac t

wit hth e estroge n receptor . On e approac hi s t o utiliz e predictiv e structur e activit y relationshi p (SAR ) models . Membe r countrie s o f

th e OECD , o f

whic hth e U.S . i s one , hav e lon g recognize dth e potentia l o f SA Ranalysi s a s a n ai di n conductin g initia l hazar dassessment s o f chemicals . A summar y o f EPA' s recen t

approac ht o assessin g potentia l endocrin e activit y base do n structur e wa s summarize di n th e introductio n o f thi s document . Th e othe r approac ht o asses s th e potentia l fo r achemica l t o interac t

wit hth e estroge n recepto r i s t o conduc t

i n vitr o assays , whic har e discusse di nth e followin g sections . 2.1. 2 Estroge n Recepto r (ER ) Bindin g an dTransactivatio n Assay s Th e tw o i n vitr o tes t

system s tha t

asses s interactio n wit hth e estroge n recepto r i n th e Tie r

1 screenin g batter y o f

th e EDS Par e th e ERa/p competitiv e radioligan dbindin g assay (OPPT S 890.1250 ) an dth e ER p transactivatio n assay (OPPT S 890.1300) . ER amediate s th e classi c estrogeni c respons e an dmodel s currentl y bein g develope dt o measur e interactio nwit hth e estroge n recepto r mainl y us e th e ER aisoform . Th e ERa/ p bindin g assay utilize s ra t

uterin e cytoso l rathe r

tha n arecombinan t

ER . EP A an dOEC D ar e currentl y developin g a n E Rbindin g assay tha t utilize s recombinan t

form s o f th e estroge n recepto r

rathe r 1 6tha nra t uterin e cytoso l becaus e th e ra t uterin e cytoso l preparatio ni s unstabl e an dagreate r potentia l fo r

assay-to-assa y variatio ni s realize dwhe nusin g ra t uterin e cytosol . Bindin g assay s wit hrecombinan t

endocrin e receptor s wil l yiel dmor e consisten t result s an deliminat e th e nee dt o us e larg e number s o f

animal s t o obtai nth e ra t

uterin e cytoso l substrate . Th e ICs o valu e fo r

estradio l i nE Rcompetitiv e bindin g assay s an dtransactivatio nassay s ar e comparabl e t o th e ICs o valu e fo r th e OPPT S 890.125 0 E Rcompetitiv e bindin g assay an dth e OPPT S 890.130 0 transcriptiona l activatio nassays , respectively . Th e measuremen t endpoint s fo r thes e tw o assay s ar e highl y correlate dan ddemonstrat e simila r sensitivit y an dspecificit y (Sonnevel de t

al. , 2006 ; Takeyoshi , 2006 ; Law s e t al. , 2006) . Thi s resul t

i s plausibl e an dno t unexpecte dsinc e bothassay s ar e measurin g th e sam e functiona l endpoint , receptor-ligan d binding . E Rbindin g an dtransactivatio nassay s hav e bee nshow nt o b e goo dpredictor s o f i n viv o estrogeni c activit y (Sonnevel de t al. , 2006 ; Takeyoshi , 2006) . Thi s i s particularl y tru e fo r compound s wher e metabolis mstudie s i n rodent s hav e show ntha t the y ar e poorl y metabolized , ar e no t substantiall y absorbe dwhe ningested , ar e rapidl y excreted , an dd o no t accumulat e i n th e bod y (EPA , 1993 ; Willia me t al. , 2000 , Ridle y an dMirly , 1988) , a s i s th e cas e fo r glyphosate . Competitiv e radioligan dbindin g E Rassay s hav e bee nuse dfo r decade s an dar e on e o f

th e "gol d standards " fo r

identifyin g tes t

chemical s tha t

bin dt o th e E R(Clar k

an dGorski , 1969 ; O'Keef e an dHanda , 1990 ; Law s e t al . 2006) . Th e E Rbindin g assay , measure s th e abilit y o f a radiolabele dligan d(l?p-estradiol ) t o interac t wit hth e E Ri n th e presenc e o f increasin g concentration s o f

ates t chemical . Th e methodolog y i n EPA' s estroge nrecepto r bindin g assay OPPT S 890.125 0 tes t

guidelin e i s essentiall y th e sam e a s th e methodolog y outline db y Law s e t al . (2006) . Ra t

uterin e cytoso l (RUC) , containin g th e ER , i s incubate di n tes t tube s wit h increasin g concentration s o f th e tes t substanc e an da n aliquo t o f radiolabele d[ 3 H]-estradiol . I f th e tes t substanc e interact s wit hth e receptor' s bindin g domain , les s radioligan dca n bind , s o a n activ e competito r

produce s adescendin g concentration-respons e plot . Similarly , tes t substance s tha t d o no t displac e radiolabeled-estradio l fro mth e estroge nrecepto r woul dno t

hav e a n affinit y fo r estrogen-binding . Th e assay can , therefore , b e use dt o determin e th e relativ e bindin g affinitie s o f

environmenta l chemical s fo r th e estroge nrecepto r

b y compariso nwit hth e abilit y o f IVp-estradio l t o bin dt o th e estroge nreceptor . Althoug hth e E Rbindin g assay detect s both agonist s an dantagonists , i t canno t distinguis hbetwee nthem . Th e criterio nfo r apositiv e interactio nwit hth e E Rreceptor s i s >50 % displacemen t o f th e radioligan di n 2 o f

thre e replicat e assays . Th e ER atransactivatio nassa y utilize s ERa-HeLa-990 3 immorta l cel l lin e i n ametho dvalidate d b y th e Japanes e Chemical s Evaluatio nan dResearc hInstitut e throug hth e OEC D validatio n process . Ther e ar e als o ongoin g effort s t o develo p additiona l E Rtransactivatio nassay s (e.g. , LUMI-CELL ) t o asses s agonis man dantagonism . I n agonis mstudies , cell s ar e treate dwit ha tes t substanc e an dth e inductio no f th e reporte r

gen e produc t

i s utilize dt o measur e th e response . Fo r a n assessmen t

o f relativ e potency , th e respons e o f th e tes t substanc e ca n b e compare dt o th e inductio nb y areferenc e estrogen . Alternatively , whe ndose-respons e dat aar e generated , th e EC5 0 fo r th e tes t substanc e ca n b e determine dan dcompare dwit htha t fo r th e referenc e estrogen . I nantagonis mstudies , th e cell s ar e expose dsimultaneousl y t o th e referenc e estroge nan dth e tes t substance , whil e contro l cell s ar e expose dt o th e referenc e estroge nonly . Usually , aconstan t concentratio no f

referenc e estroge ni s use dan dth e declin e o f it s transcriptiona l activit y wit hth e 

### 1 7tes t

substanc e i s monitored . Th e differenc e i ninductio n o f th e reporte r

gen e produc t

i nth e presenc e an dabsenc e o f

th e tes t substanc e i s use da s ameasur e o f estroge n antagonism . E Rtransact!vatio n assay s demonstrat e ahig hleve l o f concordanc e fo r

sensitivit y an dspecificit y wit hth e EDSP' s E Rbindin g assa y an dimmatur e ra t uterotrophi c assay (Takeyoshi , 2006) . Therefore , result s fro mstabl y transfecte dE Rtransactivatio n assay s provid e reliabl e informatio n abou t th e biologica l effec t

o f chemical s mediate db y ER-ligan dinteraction s (Takeyoshi , 2006) . Th e criterio n fo r

apositiv e interactio n wit hth e E Rreceptor s i n th e ERa-HeLa-990 3 immorta l cel l lin e use di n th e OPPT S 890.130 0 guidelin e i s calculatio n o f aP C 1 0 value , whic hrepresent s 10 % o f th e maximu minducin g potentia l o f 1 n M l?p-estradiol . Th e consensu s o f OECD' s independen t

pee r revie w pane l wa s tha t

th e PC1 0 valu e i s no t

a n appropriat e criterio n fo r a positiv e response . Th e Peer-Revie w Panel' s justificatio n fo r thi s recommendatio n wa s tha t th e P C 1 0 represent s amargina l respons e o f doubtfu l predictiv e valu e an dwil l likel y generat e a n unacceptabl y hig hrat e o f fals e positive s (OECD , 2007) . Th e fold-inductio nfo r 17- 0 estradio l a t aconcentratio n o f

1 n M ha s bee n show n t o rang e fro m4

- t o 30-fol dan dtypicall y resul t i n approximatel y a10-fol dinduction . Four-fol dinductio n represent s awea k respons e wit h1 n M 17- Pestradio l an ddemonstrate s th e potentia l fo r aver y smal l dynami c rang e fo r th e assay o n a give nday . Wit honl y afour-fol dmaxima l induction , th e PC5 0 woul drepresen t approximatel y a 2-fol dinductio n an daP C 1 0 woul db e significantl y les s tha n atwo-fol dinduction . I t i s routinel y understoo db y expert s tha t les s tha n a2-fol dinductio n i n reporte r gen e assay s i s no t

considere da positiv e response . Indeed , th e result s fro mth e validatio n stud y hav e a n unacceptabl y hig hfalse

positiv e rat e whe n assessin g th e P C 1 0 valu e a s a n endpoint . Ther e wer e fals e positive s observe d i n1 o f th e 3 replicat e assay s fo r 3 o f th e 9 tes t substance s i n 3 o f

th e lab s (se e tabl e 6. 2 o f

th e validatio nrepor t

o f Takeyoshi , 2006) . Therefore , th e result s ar e equivoca l fo r nearl y one-thir d o f th e substance s teste di n th e validatio n whe n usin g th e P C 1 0 endpoint . Thi s resul t i s afunctio n o f th e variabilit y o f

th e tes t syste ma t lo w inductio n level s an ddemonstrate s th e lac k o f validit y o f th e P C 1 0 valu e a s th e criterio n fo r apositiv e response . Th e Pane l strongl y recommende dthat a n intermediat e valu e betwee n th e P C 1 0 an dPC5 0 (e.g. , PC25 ) shoul db e use da s th e criterio n fo r apositiv e respons e (OECD , 2007 ; ICCVAM , 2007) . Th e EDSP' s E Rtransactivatio n assay wa s develope db y stabl y transformin g th e HeLa-990 3 cel l lin e wit hafirefl y luciferas e reporte r vecto r activate db y avitellogeni n estrogen-responsiv e elemen t drive n b y amous e metallothionei n promote r TAT A element . Stabl y transfecte dline s hav e th e obviou s advantag e ove r transien t transfection s o f

no t havin g t o continuousl y prepar e multipl e batche s o f

transientl y transfecte dcells . However , transien t transfection s hav e th e advantag e ove r

stabl y transforme dline s i n tha t th e leve l o f

E Ra t ahighe r leve l wit htransien t transfection s an dca n provid e greate r sensitivity . Th e ke y functiona l differenc e betwee n th e bindin g an dtransactivatio n assay s i s tha t th e transactivatio n assay ha s th e potentia l t o differentiat e betwee n agonis man dantagonism . However , th e ER atransactivatio n assay currentl y i n th e Tie r

1 screenin g batter y doe s no t provid e methodolog y t o detec t ER aagonists . Th e estrogeni c an danti-estrogeni c potentia l o f glyphosat e ha s bee n studie di n vitr o b y independen t

researcher s an dpublishe di npeer-reviewe djournals . Th e methodolog y use di n thes e studie s (Kojim ae t

al . 2004 ; Peti t

e t al . 1997 ; Xi e e t

al . 2005 ) i s functionall y equivalen t t o th e E Rbindin g an dtransactivatio n assay s define di n th e Tie r 1 batter y o f

th e EDSP . Eve n thoug hth e i n vitr o dat awer e generate dwit hdifferen t experimenta l methods , i t i s o f suitabl e natur e an dqualit y t o provid e th e sam e essentia l informatio n t o evaluat e potentia l estrogeni c an d anti-estrogeni c potentia l fo r glyphosate . Th e result s fro mthes e thre e investigation s provide s

1 8stron g evidenc e tha t glyphosat e i s no t

a n ERa/ Pagonis t

o r antagonist . A comprehensiv e revie w o f eac ho f thes e assay s follows . Kojima , H. , Katsura , E. , Takeuchi , S. , Niiyami , K. , an dKobayashi , K . (2004) . Screenin g fo r estroge nan dandroge nrecepto r activitie s i n 20 0 pesticide s b y i n vitr o reporte r gen e assay s usin g Chines e hamste r

ovar y cells . Environ . Healt hPerspect . 1 12:524-531 . Th e abilit y o f

glyphosat e t o ac t

a s agonis t

an dantagonis t

o f th e huma nER qan dhuma n isoform s wa s investigate db y Kojim ae t al . usin g Chines e hamste r ovar y (CHO-K1 ) cell s tha t wer e transientl y transfecte dwit ha n estroge nresponsiv e reporte r vector . Th e estroge n responsiv e elemen t

containin g reporte r

plasmi dwa s constructe da s describe db y Kojim ae t al. , (2003). Fo r

detectio no f

ER qan dER p activity , cell s wer e transfecte dwit h5 n g o f ER qo r ER p expressio nvector s an d5 0 n g o f th e E Rreporte r

construct . Additionally , apRL-SV4 0 construc t containin g th e Renill aluciferas e gen e wa s co-transfecte dt o evaluat e transfectio nefficienc y an d t o discriminat e betwee nspecifi c dow nregulatio nfro mcytotoxicity . Thre e hour s post-transfection , cell s wer e dose dfo r 2 4 hour s wit hconcentration s o f glyphosat e rangin g fro m10" 5 t o 10" 8 M (i.e. , 0.0 1 u M t o 1 0 uM) . Th e 24-hou r exposur e duratio nfo r

thi s assay i s th e sam e a s tha t

require db y th e OPPT S 890. 1 30 0 E Rtransactivatio nguideline . Transfecte dcell s wer e culture di n complet e media , containin g penicillin-streptomyci nan d10 % charcoal-dextran-treate dfeta l bovin e seru ma t 37° C i n a n atmospher e o f 5 % CO2/95 % ai r

unde r saturate dhumidity . Th e approximat e ECs o value s fo r ER qan dER p wer e 1 an d1 0 pM , respectively , whic har e 6 t o 7 order s o f magnitud e greate r

tha nth e highes t

glyphosat e concentratio ntested . Paralle l assay s wer e performe dt o full y characteriz e th e concentratio n effec t relationshi p wit hestradio l fo r ER qan dER p (Figur e 1) .

. s

S * ] 

# •;

-1 1 -1 3 -1 2 -1 1 -1 0 -8 - 7 Figur e 1 . Concentration-respons e curve s fo r estradio l i n th e ER qan dER p assay s i n Chines e hamste r ovary cell s transientl y transfecte dwit hhER a o r hERp , th e E Rluciferas e reporte r vecto r an da constitutivel y activ e Renill aluciferas e expressio n reporte r (transfectio n an dtoxicit y control) . Value s represen t th e mea n ± S D o f

thre e independen t experiment s an dar e presente da s mea n n-fol d inductio nover

th e vehicl e control .

Th e sensitivit y o f

thi s assay an ddynami c rang e ar e comparabl e t o th e sensitivit y an ddynami c rang e o f othe r E Rreporte r

assay s reporte di n th e ope nliteratur e (Legle r e t al. , 1999 ; Roger s e t al. , 2000 ; Wilso ne t

al. , 2004 ; Gordo nan dClark , 2005 ) an dt o tha t o f th e OPPT S 890.130 0 E R transactivatio nguidelin e (Takeyoshi , 2006) . Also , th e ICs o valu e reporte db y Kojim ae t al . fo r th e transactivatio nassay fo r glyphosat e i s comparabl e t o th e ICs o valu e reporte db y Law s e t al . usin g th e OPPT S 890.125 0 E Rcompetitiv e bindin g assay (Law s e t al. , 2006) . Thi s i s plausibl e 1 9an dno t unexpecte dsinc e bothassay s ar e measurin g th e sam e functiona l endpoint , receptor ligan dbinding . Agonis t activit y o f

glyphosat e wa s evaluate db y relativ e activit y expresse da s th e 20 % relativ e stimulator y concentratio n (EC2o) ; tha t is , th e concentratio n o f

glyphosat e showin g 20 % o f th e activit y o f maximall y inducin g concentration s o f 10" 1 0 M estradio l fo r

hER aan d10~ 9 M estradio l fo r ER p activity , respectively . Base do n thi s criterion , glyphosat e wa s no t identifie da s a chemica l havin g ER aan dER p antagonis t activit y (Kojim ae t

al , 2004) . Peti t F. , LeGof

f P. , Craved ! J.P. , Valotair e Y. , F . Pakdel . (1997) . Tw o complementar y bioassay s fo r

screenin g th e estrogeni c potenc y o f xenobiotics : recombinan t yeas t fo r trou t estroge n recepto r

an dtrou t hepatocyt e cultures . J . Molecular Endocrinology . 19 : 321-335 . Peti t e t al . (1997 ) screene dglyphosat e fo r estrogeni c potenc y usin g a n i n vitr o recombinan t yeas t syste mexpressin g th e rainbo w trou t estroge n receptor . Yeas t

cell s containin g alac Z reporte r gen e linke dt o tw o estrogen-responsiv e element s wer e treate di n cultur e a t 10" 8 t o 10" 4 M (0.0 1 t o 10 0 uM ) glyphosat e fo r

fou r hours . Fo r

thes e assays , 17p-estradio l wa s use da s th e positiv e contro l an dproduce dgoo dresponsivenes s wit happroximatel y a100-fol dinductio no f P

galactosidas e activity . T o ensur e tha t th e absenc e o f

arespons e wa s no t

du e t o toxicity , cel l densit y measurement s wer e mad e befor e an dafte r treatment . Th e estradio l concentratio n require dt o produc e maxima l activatio n wa s 10 " M an dwa s reporte da s 100 % activity . Basa l inductio nrepresente d1 4 t o 18 % o f th e maximu mactivit y obtaine dwit hth e recepto r i n th e absenc e o f

ligand . Th e reporte dactivit y fo r glyphosat e o n estroge n dependen t gen e transactivatio n wa s reporte dt o b e 15.84% , wel l withi n th e rang e o f basa l activity , indicatin g n o evidenc e o f

estrogeni c activity . Xi e L. , Thrippleto n K. , Irwi n M.A. , Siemerin g G.S. , Mekebr i A. , Cran e D. , Berr y K. , Schlen k D . (2005) . Evaluatio n o f estrogeni c activitie s o f

aquati c herbicide s an dsurfactant s usin g arainbo w trou t

vitellogeni n assay . Toxico l Sci . 87:91-8 . I nXi e e t al . (2005) , th e estrogeni c potenc y o f glyphosat e wa s evaluate dusin g vitellogeni n a s a biomarke r

o f exposur e fo r

juvenil e rainbo w trou t

(standar dlength : 11. 5 ± 2. 2 cm ) expose dt o a mea n measure dconcentratio n o f 0.1 1 mg/Lglyphosate . Th e analytica l metho duse dfo r measurin g th e concentratio n o f glyphosat e i n wate r

wa s EP A Metho d54 7 wit halimi t o f detectio n o f

0.005 0 ug/ Lan drecover y rangin g fro m80-87% . Rainbo w trou t

wer e expose dfo r

7 day s a t 1 6 ± 1° C i n 20- Ltank s unde r static-renewa l an d receive dwate r

exchange s daily . I n addition , rainbo w trou t wer e expose dt o fiv e concentration s o f 17p-estradio l t o characteriz e th e concentratio n effec t relationshi p fo r

17p-estradiol . Eac h concentratio n o f

chemica l (treatmen t

o r control ) ha dthre e replicate s wit htw o fis hi n eac htank . Th e tes t wate r i n eac hindividua l tan k

wa s monitore ddail y fo r

wate r chemistr y afte r

ever y wate r renewal . Th e hardnes s o f

th e tes t wate r range dfro m14 2 t o 16 2 mg/ 1 (a s CaCOs) ; th e fre e chlorin e wa s <0. 2 mg/1 . Alkalinit y range dfro m14 8 t o 18 0 mg/1 , an dammoni a(a s N-NFh ) wa s <0.0 2 mg/1 . Dissolve doxyge n average d94.6 % o f th e ai r saturatio n value , an dp H value s range d fro m6. 0 t o 8. 2 mg/1 . Fis hwer e fe dtrou t cho w equivalen t

t o approximatel y 1 % o f

thei r bod y weigh t durin g th e exposur e an dhel dunde r a1 6 h: 8 h(light:dark ) photoperiod . Th e result s o f thi s stud y demonstrat e tha t glyphosat e di dno t

induc e productio n o f

vitellogenin , providin g additiona l evidenc e tha t

glyphosat e doe s no t

hav e estrogeni c activit y (Figur e 2) . 2 0O )



## I 9 0 

## - g > 8 0 - 

## 7 T7 0 - § 6 0 0 5 0 | 40

- § > 3 0 ' 

### 0 2 0 I 1 0 - > 0 - 

# Pis TMLJ»r 

### ,._.,,, T 

### ,,O. i:: 

### , o o o o o 

### 3 . 3 . 2 . 2 .

—* K ) Figur e 2 . Plasm avitellogeni n juvenil e rainbo w trou t

expose d level s i n t o glyphosat e (G ) fo r 1 days . Contro l 1 an dcontro l 2 represen t negativ e controls .

2.1. 3 Conclusio n - Estroge n Recepto r Agonis man dAntagonis m Dat agenerate db y Kojim ae t al . (2004) , Peti t

e t al . (1997 ) an dXi e (2005 ) i n peer-reviewe d journal s clearl y an dconsistentl y demonstrat e tha t glyphosat e doe s no t

hav e a n E Rbindin g potential , eithe r

a s a n agonis t

o r antagonist . Th e methodologie s employe db y Kojim aan dPeti t i nthes e i n vitr o tes t

system s ar e comparabl e t o th e overal l approac ho f th e tw o Tie r

1 E Rbindin g assay s o f th e EDSP—th e ERa/ Pcompetitiv e radioligan dbindin g assay usin g ra t uterin e cytoso l (OPPT S 890.1250 ) an dth e ER atransactivatio n assay (OPPT S 890.1300) . Specifically , transcriptiona l activatio n assay s performe db y Kojim ae t al . (2004 ) ar e functionall y equivalen t t o th e ERa/ Pcompetitiv e radioligan dbindin g assay (OPPT S 890.1250 ) an dth e ER p transactivatio n assay (OPPT S 890.1300 ) fo r th e followin g reasons :

• Kojim ae t

al . (2004 ) evaluate dth e abilit y o f glyphosat e t o agoniz e an dantagoniz e ER aan d ER p binding , whic hi s th e purpos e o f th e ERa/ Pcompetitiv e radioligan dbindin g assay (OPPT S 890.1250) . Th e methodolog y o f Kojim ae t

al . wa s capabl e o f

differentiatin g betwee n ERa/p agonist s an dantagonists , whic hth e ERa/Pcompetitiv e radioligan dbindin g an dtranscriptiona l activatio n assay s ar e not . Overall , E Rtransactivatio n assay s demonstrat e ahig hleve l o f

concordanc e (sensitivit y an dspecificity ) wit hth e EDSP' s E Rbindin g assay . Thi s i s plausibl e an dno t

unexpecte dsinc e bot hassay s ar e measurin g th e sam e functiona l endpoint , ER-ligan dbinding . Therefore , result s fro mE Rtransactivatio n assay s provid e reliabl e an dequivalen t

informatio n t o E Rcompetitiv e radioligan dbindin g assays . • Th e ERa/p transactivatio n assay performe db y Kojim ae t al . (2004 ) evaluate dth e sam e ke y biochemica l mechanism s a s th e ER p transactivatio n assay (OPPT S 890.1300) . These step s includ e E Rligan dbinding , nuclea r translocation , bindin g t o th e DN A responsiv e element , an dreporte r

gen e transactivation . Additionally , th e sensitivit y o f th e assay an ddynami c rang e o f Kojim ae t

al . ar e comparabl e t o th e sensitivit y an ddynami c rang e o f othe r E R reporte r gen e assay s i n th e ope n literatur e an dth e OPPT S 890.130 0 E Rtransactivatio n assay use dt o develo p th e OPPT S guideline . • Th e i n vitr o E Rtransactivatio n assay performe db y Peti t e t al . (1997 ) als o provide s dat ao f th e sam e natur e an dqualit y a s OPPT S 890.130 0 E Rtransactivatio n guideline . Thi s assay 

### 2 1assesse s th e ke y step s evaluate db y OPPT S 890.125 0 an dOPPT S 890.130 0 tha t includ e E R bindin g an dth e othe r

ke y step s o f

DN A bindin g an dgen e transactivation . • Th e i n viv o assay reporte db y Xi e e t al . (2005 ) provide s anothe r confirmator y an dsensitiv e mechanisti c assessmen t

o f E Rbindin g potential . These i n vitr o an di n viv o mechanisti c finding s tha t demonstrat e n o E Ragonisti c o r antagonisti c bindin g potentia l ar e furthe r supporte db y aSA Ranalysi s usin g methodolog y develope db y EPA' s Offic e o f

Researc han dDevelopment . SA Ranalysi s clearl y show s n o estrogeni c potentia l fo r glyphosat e an dth e comprehensiv e i n viv o dat aavailabl e fo r glyphosat e demonstrat e n o effect s o n an y endpoin t

tha t i s sensitiv e an dspecifi c t o compound s tha t hav e estrogeni c o r

anti

estrogeni c activity . A detaile drevie w o f th e i n viv o dat ause dt o evaluat e estrogenicit y i s reviewe di n th e section s fo r th e uterotrophic , puberta l assay s an dfis hscreenin g assays . Th e dat agenerate db y Kojim ae t al . (2004 ) an dPeti t

e t al . (1997 ) ar e functionall y equivalen t t o bot hth e Estroge n Competitiv e Recepto r Bindin g (OPPT S 890.1250 ) an dth e E RTranscriptiona l Activatio nAssa y (OPPT S 890.1300 ) define di n th e EDS PTie r 1 tes t battery . Consequently , n o additiona l i n vitr o screenin g shoul db e require dt o evaluat e th e potentia l fo r E Ragonis man d antagonism . 2. 2 Androge n Recepto r (AR ) Bindin g Assa y (OPPT S 890.1150 ) Th e EDS PTie r

1 screenin g batter y include s on e i n vitr o assay t o asses s th e potentia l fo r a chemica l t o interac t

wit hth e androge n recepto r (AR) . A n independen t

stud y tha t

i s functionall y equivalen t

t o th e A RBindin g Assa y o f th e EDS P(OPPT S Guidelin e 890.1150 ) an dpublishe di n Environmenta l Healt hPerspective s b y Kojim ae t al . i n 200 4 clearl y demonstrate s th e lac k o f an y antagonisti c androge n recepto r activit y fo r

glyphosate . Thi s sectio n review s currentl y availabl e scientificall y relevan t

information , generate dusin g a n i n vitr o tes t syste msimila r

t o th e A R bindin g assay (OPPT S 890.1150 ) define di n th e EDSP , tha t ca n b e use dt o asses s th e potentia l fo r glyphosat e t o directl y interac t wit hth e androge n receptor . Thi s sectio n als o discusse s th e us e o f structur e activit y relationshi p (SAR ) model s t o evaluat e a chemical' s androge n recepto r bindin g potentia l an dho w i t ca n b e use dt o validat e th e result s o f th e assay fo r

glyphosate . 2.2. 1 Structur e Activit y Relationshi p (SAR) Predictio n o f A RBindin g Potentia l Althoug hSA Rmodel s ar e no t develope dt o th e sam e degre e fo r androgen s a s the y ar e fo r estrogens , man y o f

th e sam e principal s tha t

appl y t o evaluatin g glyphosat e fo r estrogeni c potentia l appl y t o a n assessmen t o f androgeni c potential . Base do n it s structure , glyphosat e i s predicte dno t

t o hav e androgeni c activit y (Deviller s e t al. , 2009 ; Panay e e t

al. , 2008) . Th e conclusio ntha t

glyphosat e doe s no t

hav e androgeni c potentia l ha s bee n empiricall y confirme db y anumbe r o f i n vitr o assay s an di n viv o tests . A revie w o f th e i n vitr o an di n viv o dat atha t ha s evaluate dpotentia l androgeni c an danti-androgeni c activit y o f glyphosat e i s discusse di n th e followin g sections . 2.2. 2 Androge n Recepto r (AR ) Bindin g Assay Th e androge n recepto r

(AR ) bindin g assay i s ver y simila r t o th e E Rcompetitiv e ligan dbindin g assay i nit s genera l procedure . Th e A Rbindin g assay i nth e Tie r 1 batter y o f

th e EDS Putilize s ra t prostat e cytoso l fro manima l tissu e (ra t prostrate ) rathe r

tha n arecombinan t

androge n receptor . EP A an dOEC D are , however , currentl y developin g a n A Rbindin g assay tha t utilize s 2 2recombinan t form s o f th e androge n recepto r

because , a s wit hth e estroge n recepto r bindin g assay , th e ra t

prostat e cytoso l preparatio n i s unstabl e an dit s us e exhibit s agreate r potentia l fo r assay-to-assa y variation . Bindin g assay s wit hrecombinan t AR s demonstrat e mor e consisten t result s an deliminat e th e nee dt o sacrific e larg e number s animal s t o obtai n th e prostrat e cytoso l preparation . Overall , \C$ Q value s wit handrogeni c compound s fo r A Rbindin g an dtransactivatio n assay s ar e comparabl e t o th e ICs o value s fo r th e OPPT S 890.115 0 A Rcompetitiv e bindin g assay (Sonnevel de t

al. , 2006 ; Bec k

e t al. , 2007) . Thi s i s plausibl e an dno t unexpecte dsinc e bot h assay s ar e measurin g th e sam e functiona l endpoint , AR-ligan dbinding . Additionally , A R bindin g an dtransactivatio n assay s hav e bee n show n t o b e goo dpredictor s o f i n viv o androgeni c activit y (Sonnevel de t

al. , 2006) . Competitiv e radioligan dbindin g A Rassay s hav e bee n use dfo r decade s an dar e on e o f

th e standard s fo r

identifyin g tes t

chemical s tha t

bin dt o A R(Kelc e e t

al , 1995) . Th e A Rbindin g assay measure s th e abilit y o f aradiolabele dligan d([ 3 H]-R1881 ) t o interac t wit hth e A Ri n th e presenc e o f

increasin g concentration s o f ates t chemical . Th e methodolog y i n EPA' s A Rbindin g assay (OPPT S 890.1150 ) i s essentiall y th e sam e standar dmethodolog y outline db y Kelc e e t al . (1995 ) an di n aserie s o f

publication s publishe db y Kelc e e t al . Ra t uterin e prostat e containin g A Ri s incubate di n tes t

tube s wit hincreasin g concentration s o f th e tes t substanc e an da n aliquo t o f radiolabele d[ 3 H]-R1881 . I f th e tes t substanc e interact s wit hth e receptor' s bindin g domain , les s radioligan dca n bind , s o a n activ e competito r produce s adescendin g concentration-respons e plot . Similarly , tes t

substance s tha t

d o no t displac e th e radiolabele dligan dfro mA Rwoul dno t hav e a n affinit y fo r

AR-binding . Th e assay can , therefore , b e use dt o determin e th e relativ e bindin g affinitie s o f

environmenta l chemical s fo r th e androge n receptor , b y compariso n wit hth e abilit y o f [ 3 H]-R188 1 t o bind . Althoug hth e A Rbindin g assay detect s bot hagonist s an d antagonists , i t

canno t distinguis hbetwee n them . Th e criterio n fo r apositiv e interactio n wit hth e androge n recepto r

i s >50 % displacemen t

o f th e radioligand . Th e androgeni c potentia l o f glyphosat e ha s bee n studie di n vitr o b y independen t researcher s an d publishe di n apeer-reviewe djournal . Th e methodolog y use di n thi s stud y (Kojim ae t al . 2004 ) i s functionall y equivalen t

t o th e A Rbindin g assa

y define di n th e Tie r

1 batter y o f

th e EDSP . Th e conclusio no f

th e researcher s i n thi s stud y i s tha t glyphosat e doe s no t

exhibi t androge n recepto r antagonis t activity . A comprehensiv e revie w o f thi s assay follows . Kojima , H. , Katsura , E. , Takeuchi , S. , Niiyami , K. , an dKobayashi , K . (2004) . Screenin g fo r estroge n an dandroge n recepto r activitie s i n 20 0 pesticide s b y i n vitr o reporte r gen e assay s usin g Chines e hamste r

ovar y cells . Environ . Healt hPerspect . 112:524-531 . Th e abilit y o f

glyphosat e t o ac t

a s agonis t

an dantagonis t

o f th e huma n A Rwa s investigate d usin g Chines e hamste r

ovar y (CHO-K1 ) cell s tha t wer e transientl y transfecte dwit ha n androge n responsiv e reporte r

vecto r (Kojim ae t

al. , 2004) . Th e androge n responsiv e elemen t containin g reporte r plasmi dwa s constructe da s describe di n Kojim ae t al. , (2003) . T o evaluat e agonis t an d antagonis t effect s o f

A Ractivity , cell s wer e transfecte dwit h2. 5 n g o f androge n recepto r expressio n vector s an d5 0 n g o f th e A Rreporte r

construct . Additionally , apRL-SV4 0 construc t containin g th e Renill aluciferas e gen e wa s co-transfecte dt o evaluat e transfectio n efficienc y an d t o distinguis hbetwee n specifi c dow n regulatio n fro mcytotoxicity . 

### 2 3A t

thre e hour s post-transfection , cell s wer e dose dfo r 2 4 hour s wit hconcentration s o f glyphosat e rangin g fro m10" 5 t o 10~ 8 M . Thi s 24-hou r exposur e i s th e sam e a s tha t require db y th e A R transactivatio n guidelin e OPPT S 890.1150 . Transfecte dcell s wer e culture di n complet e medi a containin g penicillin-streptomyci n an d10 % charcoal-dextran-treate dfeta l bovin e seru ma t 37° C i na natmospher e o f

5 % CC>2/95 % ai r

unde r saturate dhumidity . Th e approximat e ICs o valu e fo r th e A Rwa s approximatel y 6 order s o f magnitud e greate r

tha n th e highes t

glyphosat e concentratio n tested . Paralle l assay s wer e performe dt o full y characteriz e th e concentratio n effec t

relationshi p wit hestradio l fo r A R(Figur e 3) . 

### I

-1 4 -1 3 -1 2 -1 1 -1 0 -8 LoglM ! DH T Figur e 3 . Concentration-respons e curv e fo r 5a

dihydrotestosteron e an dandroge n recepto r (AR ) i n Chines e hamste r

ovar y cell s transientl y transfecte d wit hth e A Ran dth e A Rluciferas e reporte r vecto r an daconstitutivel y activ e Renill aluciferas e expressio n reporte r

(transfectio n an dtoxicit y control) . Value s represen t th e mea n ± S D o f

thre e independen t

experiment s an dar e presente da s mea n n-fol dinductio n ove r

th e vehicl e control .

Th e sensitivit y o f

thi s assay an ddynami c rang e i s comparabl e t o th e sensitivit y an ddynami c rang e o f othe r standar dA Rreporte r

assay s i n th e ope n literatur e (Bec k e t al. , 2008) . Additionally , th e \C$ o valu e fo r th e transactivatio n assay conducte db y Kojim ae t al . i s comparabl e t o th e ICs o valu e fo r th e A Rcompetitiv e bindin g assay conducte daccordin g t o OPPT S guidelin e 890.115 0 (Arak i e t al. , 2005 ; Sonnevel de t

al. , 2005 ; Bec k

e t al. , 2008) . Thi s i s plausibl e an dno t

unexpecte dsinc e bot hassay s ar e measurin g th e sam e functiona l endpoint , receptor-ligan dbinding . Agonis t activit y wa s evaluate db y relativ e activit y expresse da s th e 20 % relativ e stimulator y concentratio n (ICio) ; tha t

is , th e concentratio n o f

glyphosat e showin g 20 % o f th e activit y o f maximall y inducin g concentration s o f 10"' ° M dihydrotestosterone . Base do n thi s criterion , glyphosat e wa s no t

identifie da s achemica l havin g androge n recepto r antagonis t

activit y (Kojim ae t

al , 2004) . 2.2. 1 Conclusio n - Androge n Recepto r Agonis man dAntagonis mo f Glyphosat e Dat agenerate db y Kojim ae t al . (2004 ) clearl y an dconsistentl y demonstrat e tha t glyphosat e i s no t a n androge n recepto r

agonis t o r antagonist . Thi s peer-reviewe darticl e describe s a n i n vitr o tes t syste man dmethodolog y tha t i s comparabl e an dfunctionall y equivalen t t o th e methodolog y fo r th e A Rbindin g assay i nth e Tie r 1 screenin g battery describe di nOPPT S Guidelin e 890.1150 . Th e rational e fo r functiona l equivalenc e i s provide dbelow . • Specifically , Kojim ae t al . evaluate dth e abilit y o f glyphosat e t o agoniz e A Rbindin g o r antagoniz e radiolabele d[ 3 H]-R18 8 bindin g t o th e AR , whic hi s th e sol e purpos e o f th e A Rcompetitiv e radioligan dbindin g assay . 

### 2 4• Th e methodolog y o f Kojim ae t

al . wa s capabl e o f

no t onl y detectin g A Ragonist s an d antagonist s bu t

als o t o differentiat e betwee n A Ragonist s an dantagonists . Th e A R competitiv e radioligan dbindin g assay i s no t capabl e o f

makin g thi s differentiation . • Overall , ICs o value s wit handrogeni c compound s fo r A Rbindin g an dtransactivatio n assay s ar e comparabl e t o th e ICs o value s fo r th e OPPT S 890.115 0 A Rcompetitiv e bindin g assay . Thi s i s plausibl e an dno t unexpecte dsinc e bot hassay s ar e measurin g th e sam e functiona l endpoint , AR-ligan dbinding . • Th e transactivatio n assay , whic hha s alread y bee n conducte do n glyphosate , provide s dat ao f th e sam e natur e an dqualit y a s th e competitiv e bindin g assay . Thi s findin g o f

n o androge n bindin g potentia l i s furthe r supporte db y astructura l activit y relationshi p analysi s tha t

indicate s tha t

glyphosat e doe s no t

hav e androgeni c potential . Additionally , comprehensiv e i nviv o dat aobtaine dfro mchroni c studie s wit hglyphosat e demonstrat e n o effect s o n endpoint s tha t ar e sensitiv e an dspecifi c t o compound s tha t hav e androgeni c o r

anti-androgeni c activity . A detaile drevie w o f th e i n viv o dat atha t

confirm s th e i n vitr o dat atha t

show s tha t

glyphosat e i s no t

androgeni c o r

anti-androgeni c i s extensivel y reviewe di n th e section s o n th e Hershberger , mal e puberta l an dfis hscreenin g assay s presente d late r i nthi s document . Th e dat agenerate db y Kojim ae t al . (2004) , whic hdemonstrate s tha t glyphosat e i s no t

a n androge n recepto r

agonis t o r antagonist , i s substantiall y equivalen t t o th e Androge n Recepto r Bindin g Assa y (OPPT S 890.1150 ) define di n th e EDS PTie r I tes t batter y an dn o additiona l i n vitr o screenin g i s neede do r shoul db e require dt o evaluat e glyphosate' s potentia l fo r androge n recepto r agonis man dantagonism . 2 3 Aromatas e Assa y (OPPT S 890,1200 ) Th e Tie r 1 i n vitr o aromatas e assa y (OPPT S 890.1200 ) o f th e EDS Puse s recombinan t huma n aromatas e (CYP19 ) an dcytochrom e P45 0 reductas e t o detec t inhibitio no f

aromatas e activity . Aromatas e catalyze s th e conversio n o f androge n t o estrogen . I n thi s Tie r 1 i n vitr o assay , aromatas e activit y i s evaluate dusin g th e tritiate dandrostenedion e 'water-release ' method . Briefly , radioactiv e substrat e ( 3 H-androstenedione ) an dNADP H ar e adde dt o microsome s containin g th e aromatas e an dreductas e complex . Tritiated-wate r ( 3 HiO ) i s release ddurin g th e conversio n o f

androstenedion e t o estron e ove r a1 5 minut e incubatio n perio dan di s quantifie da s adirec t measuremen t

o f aromatas e activit y pe r

uni t reactio n time . Althoug ha n i n vitr o stud y designe dan dconducte da s describe di n OPPT S 890.120 0 i s no t availabl e t o evaluat e th e potentia l inhibitio no f aromatas e activit y b y glyphosate , considerabl e dat ai s availabl e fro mth e ra t subchroni c an dchroni c toxicit y studie s o f glyphosat e t o mak e th e sam e evaluation . Informatio n o f particula r

significanc e an drelevanc e t o evaluat e th e potentia l o f glyphosat e t o inhibi t

aromatas e activit y i s obtaine dfro mth e ra t multigenerationa l study . Inhibitio no f

aromatas e activit y woul dproduc e adiagnosti c patter n i n anumbe r o f sensitiv e an d specifi c endpoint s tha t

ar e evaluate di n th e ra t

multigenerationa l study . Wit hfemales , aromatas e inhibitio nca n resul t

i n increase dag e a t

vagina l openin g (VO ) an dincrease dbod y weigh t a t vagina l opening , alon g wit hasignifican t decreas e i n uterin e weight s and/o r ovaria n weight s (O'Conno r

e t al. , 2002 ; Mart y e t

al. , 1999) . I n mal e rats , inhibitio no f aromatas e activit y ca n 

### 2 5increas e th e tim e o f pre-putia l separatio n(PPS) , increas e prostat e weigh t an dincreas e semina l vesicl e weight s (O'Conno r e t al. , 2002) . Th e ra t multigenerationa l stud y i s abl e t o effectivel y an dunequivocall y identif y pattern s o f aromatas e inhibitio nan dha s bee nshow nt o b e mor e sensitiv e tha nth e puberta l assay s fo r thi s effect . Man y o f

th e sam e endpoint s tha t

indicat e aromatas e inhibitio ni n th e puberta l assa y ar e als o evaluate dfo r

i nth e ra t multigenerationa l study . Fo r example , fenarimo l wa s negativ e fo r aromatas e inhibitio nactivit y i nth e femal e puberta l study , ye t i namultigenerationa l assay i t produce dth e expecte deffect s o n parturitio nassociate dwit hlowere destroge nlevel s afte r aromatas e inhibitio n(EPA , 2007) . I nrecen t ra t multigenerationa l studies , ther e wa s n o evidenc e o f potentia l aromatas e inhibitio n b y glyphosate . N o advers e effec t o f glyphosat e wa s observe do n endpoint s indicativ e o f aromatas e inhibitio n(Moxon , 2000 ; Dhins ae t al. , 2007) . Evaluatin g th e ke y diagnosti c endpoint s i n th e femal e puberta l assa y tha t indicat e aromatas e inhibition , ther e wa s n o impac t o n ag e a t vagina l openin g (VO ) o r bod y weigh t

a t vagina l opening , an dn o decreas e i n uterin e weight s o r ovaria nweight s i n th e F l paren t animal s a t

th e highes t dose s teste d(HDT ) o f

10,00 0 pp m(Moxon , 2000 ) an d15,00 0 pp m(Dhins ae t al. , 2007) . Additionally , ther e wa s n o significan t effec t o f glyphosat e o n estrou s cyclicity , pre-coita l interval , duratio no f

gestation , numbe r

o f primordia l follicles , corpor alutea , se x distribution , anogenita l distanc e o r

histopatholog y o f

th e femal e reproductiv e tissue s (uterin e an dovary ) i n th e multigeneratio nstudie s (Moxon , 2000 ; Dhins ae t al . 2007) . Thes e finding s ar e consisten t wit hfinding s i nearlie r

chroni c an dsubchroni c studie s o nglyphosat e (Schroede r an dHoga n 1981 ; Stou t

an dJohnson , 1987) . Dhins ae t al . (2007 ) note dirregula r estru s cyclin g i n F l female s a t bot h5,00 0 an d15,00 0 ppm , bu t considere dthi s findin g incidenta l an do f n o toxicologica l significanc e sinc e n o chang e wa s note di n preciota l interval s o r matin g performance . I nterm s o f

ke y diagnosti c endpoint s i n th e mal e puberta l assay t o detec t aromatas e inhibition , ther e wa s n o impac t

o n th e tim e o f

pre-putia l separatio n(PPS ) an dn o increas e i n prostat e an d semina l vesicl e weight s (Moxon , 2000 ; Dhins ae t al. , 2007) . A findin g o f

n o effec t o n thes e sensitiv e an dspecifi c endpoint s provide s stron g an dconsisten t evidenc e tha t

glyphosat e i s no t

a n inhibito r o f aromatas e activity . Additionally , ther e wa s n o significan t effec t o n weigh t

o r histopatholog y o f

mal e reproductiv e tissue s (testicula r an daccessory se x organs) , testicula r descent , spermcoun t

an dmotility , an dexterna l genitali ai n th e multigeneratio nstudie s (Moxon , 2000 ; Dhins ae t

al. , 2007) . Thi s findin g i s consisten t wit hfinding s i nearlie r

chroni c an d subchroni c studie s (Schroede r an dHoga n1981 ; Stou t

an dJohnson , 1987) . 2.3. 1 Conclusio n - Aromatas e Inhibitio n Althoug hi n vitr o dat ai s no t availabl e t o asses s aromatas e inhibitio npotentia l o f glyphosate , othe r scientificall y relevan t

informatio no f

aequivalen t

natur e an dunquestionabl e qualit y i s availabl e fro msevera l existin g i n viv o chroni c studies . A numbe r o f mal e an dfemal e endpoint s (pathologies ) i n th e tw o generatio nra t studie s ar e predictiv e o f

aromatas e inhibitio nan d considere dt o b e functionall y equivalen t t o th e dat adevelope di n th e i n vitr o assay i n th e Tie r 1 batter y o f th e EDSP . These endpoint s include :

• testicular/uterine/ovar y weight s an dhistopatholog y • th e size , functio nan dhistopatholog y o f se x organ s 

### 2 6• fertilit y

• anogenita l distanc e

• spermcoun t

an dmobilit y • ovaria nevaluatio nfo r follicle s an dcorp alute a

• ag e an dweigh t

a t preputia l separatio nan dvagina l openin g

• numbe r

o f implantatio nsite s an dlactatio n N o treatmen t

relate deffect s wer e observe di n thes e studie s o n an y o f thes e sensitiv e an dspecifi c endpoint s tha t

woul db e indicativ e o f

aromatas e inhibitio nan dprovide s stron g an dconsisten t evidenc e tha t

glyphosat e i s no t

a n inhibito r

o f aromatas e activity . Informatio no n th e potentia l effec t o f glyphosat e o nsteroidogenesis , whic hi s als o indicativ e o f aromatas e inhibition , i s comprehensivel y discusse di n th e nex t section . Th e scientifi c informatio npresente dher e fro m existin g standar dregulator y i n viv o studie s shoul db e considere da s unequivoca l proo f tha t glyphosat e doe s no t

inhibi t aromatas e activit y i nth e mal e o r female , and , consequently , th e requiremen t

fo r performin g th e i n vitr o aromatas e assay (OPPT S 890.1200 ) o f th e EDS Pshoul d b e waived . 2. 4 Steroidogenesi s Assa y (OPPT S 890,1550 ) Th e steroidogenesi s assa y use s th e H295 Rhuma nadrenocortica l carcinom acel l lin e t o detec t interferenc e wit hth e body' s productio no f mal e an dfemal e steroi dse x hormones . Th e endpoint s measure di n thi s assay ar e testosteron e an destradio l levels . Th e biosynthesi s o f

al l steroi dhormone s begin s wit hth e transfe r o f cholestero l acros s inne r mitochondria ! spac e fro mth e oute r mitochondria l membran e t o th e inne r mitochondria l membrane ; aproces s tha t depend s o n th e actio no f th e steroidogeni c acut e regulator y (StAR ) protei n(Stocco , 2001 ; Christenso ne t al , 2001) . I t

ha s bee npropose dtha t

movemen t

o f cholestero l t o th e mitochondria l membran e b y th e StA Rprotei ni s th e rate-limitin g ste p i n steroidogenesi s (Stocco , 2001) . I n a n investigatio no f th e potentia l effec t

o f glyphosat e o n StA R protei nfunctio nan dsteroidogenesi s i nMA-1 0 Leydi g cells , i t wa s demonstrate dtha t

glyphosat e doe s no t affec t steroidogenesi s i n MA-1 0 Leydi g cell s a t th e maximu mteste dconcentratio no f 10 0 ug/m L(-59 0 uM ) (Wals he t al. , 2000) . However , Wals he t al . (2000 ) demonstrate dtha t inhibitio no f

progesteron e productio nresulte dfro macomponen t othe r tha nglyphosat e i n th e formulation . Levin e e t

al . (2007 ) demonstrate dtha t Roundup ® brande dformulatio n steroidogenesi s reporte db y Wals he t al . (2000 ) resulte dfro mexposin g cell s i n cultur e t o unrealisticall y hig hconcentration s o f th e surfactant . Steroidogenesi s i n MA-1 0 cell s stop s a t th e biosynthesi s o f

progesteron e fro mpregnenolone . Consequently , assay s wit hMA-1 0 Leydi g cell s onl y allo w fo r

apartia l evaluatio no f

th e step s responsibl e fo r

androge nan destroge n biosynthesis . However , assay s wit hthi s cel l lin e b y Wals he t al . an dLevin e e t

al . demonstrate d tha t glyphosat e doe s no t

affec t th e initia l step s an drate-limitin g step s o f steroidogenesis . Typically , inhibitor s o f

steroidogenesi s woul dinhibi t throug hadirec t

interactio nwit hth e cytochrom e P45 0 enzyme s involve di n steroi dhormon e synthesi s (Taxvi g e t al , 2008) . Th e profil e fo r

inhibitio no f

steroidogenesi s i nviv o i s characterize db y arelativel y specifi c an d diagnosti c arra y o f

i n viv o effect s a t

th e orga nlevel . Fo r

example , inhibitio no f

steroidogenesi s i n femal e rat s woul dresul t

i n a n increas e i n ag e an dweigh t a t vagina l openin g (VO) , adecreas e i n 2 7uterin e weigh t an da nincreas e i novaria n weight . I nmal e rats , expecte dresponse s fo r inhibitio n o f steroidogenesi s i s dependen t o n whethe r

ther e i s inhibitio no f

testosteron e production , inhibitio no f

5ct-reductas e o r

aromatas e activity . Wit hinhibitio no f

testosteron e biosynthesis , th e expecte dorga n leve l profil e woul dinclud e a n increas e i n ag e a t

pre-putia l separatio n (PPS ) an ddecreas e i n testes , epididymides , prostate , an d semina l vesicle s weights . Additionally , ther e woul dtypicall y b e arelate dincreas e i n live r weigh t i nmale s an dfemale s (non-reproductiv e orga n weight) . Wit hinhibitio no f

5a-reductase , th e expecte dorga n leve l profil e woul dinclud e adecreas e i n epididymides , prostate , an dsemina l vesicle s weights . Additionally , ther e woul dtypicall y b e a relate dincreas e i nlive r

weigh t i nmale s an dfemale s (non-reproductiv e orga n weight) . Wit hinhibitio no f

aromatas e activity , th e expecte dorga n leve l profil e woul dinclud e adecreas e i nbod y weigh t

a t th e tim e o f

PPS , an dincrease s i n prostat e an dsemina l vesicle s weights . Additionally , ther e woul dtypicall y b e arelate dincreas e i nlive r weigh t i nmale s an dfemale s (non-reproductiv e orga n weight) . Th e ra t multigenerationa l stud y alon g wit hth e othe r chroni c an dsubchroni c studie s provide s th e scientificall y relevan t

informatio ntha t

i s require dt o perfor ma n assessmen t fo r potentia l effect s o f glyphosat e o n steroidogenesis . A ra t multigenerationa l stud y tha t followe dth e OPPT S 870.380 0 (August , 1998 ) an dOEC D 41 6 guidelines , provide s al l o f th e critica l endpoint s require dt o asses s th e potentia l fo r detectin g inhibitio no f

steroidogenesis . Th e ra t

multi

generationa l stud y i s abl e t o effectivel y detec t aromatas e inhibitio nan dha s bee n show n t o hav e greate r sensitivit y tha n th e puberta l assays , whic hinclude s endpoint s capabl e o f detectin g aromatas e inhibition . Fo r example , fenarimo l wa s negativ e i n th e femal e puberta l study , ye t i n a multigenerationa l assa y produce dth e expecte deffect s o n parturitio n associate dwit hlowere d estroge n level s afte r

aromatas e inhibitio n(EPA , 2007) . Overvie w o f

Rat Mult i Generatio n Stud y a s i t Applie s t o a n Assessmen t o f Steroidogenesi s Ther e wer e n o effect s i n th e multigenerationa l ra t studie s b y Moxo n (2000 ) an dDhins ae t al . (2007 ) indicativ e o f

potentia l effect s o f

steroidogenesis . A comprehensiv e revie w o f th e endpoint s tha t

woul db e impacte db y a naffec t o nsteroidogenesi s i s discusse di nth e following . Ther e wer e n o effect s o n arang e o f endpoint s indicativ e o f

a n effec t o n steroidogenesi s i n rat s treate da t u p t o th e highes t

dose s teste do f

10,00 0 pp man d15,00 0 pp mglyphosat e i n thes e tw o multigenerationa l studies . Thes e endpoint s includ e th e weigh t o f testes , caud aepididymides , epididymides , semina l vesicle s an dcoagulatin g glands , uterus , ovar y an dlive r o f th e F O an dF l paren t animal s (Moxon , 2000 ; Dhins ae t al. , 2007) . Glyphosate-relate dreduction s i n bod y weigh t wer e recorde donl y fo r

F l male s treate da t

th e HOT , bu t

ther e i s n o evidenc e tha t

thi s reductio ni s a nendocrine-mediate deffect . Ther e wer e n o effect s recorde dfollowin g treatmen t a t u p t o th e HDT' s o f

10,00 0 pp man d 15,00 0 pp mglyphosat e o n th e histopatholog y o f testis , epididymes , prostate , uterus , lef t ovary , vagina , cervix , an doviducts . N o effect s o n sper mmotilit y parameter s an dsper mmorpholog y wer e note da t

al l dose s (Moxon , 2000 ; Dhins ae t al. , 2007) . Ther e wer e n o effect s o n mal e an dfemal e reproductiv e indice s tha t include dmating , fertility , conception , gestation , lactation , ag e an dweigh t a t VO , ag e a t

PPS , testicula r

descent , ovaria n evaluatio nfo r

follicles , parturition , normalit y o f externa l genitali aan dnumbe r o f implantatio n 2 8site s followin g treatmen t a t u p t o th e HDT' s o f

10,00 0 pp man d15,00 0 pp mglyphosate . Additionally , ther e wa s n o impac t o n th e numbe r

o f sperm , sper mmotilit y parameter s an dsper m morpholog y fo r

F O an dF l male s (Moxon , 2000 ; Dhins ae t al. , 2007) . 2.3. 1 Conclusio n - Steroidogenesi s I n vitr o dat aassessin g potentia l impact s o n th e complet e steroidogeni c pathwa y ar e no t availabl e fo r glyphosate ; however , dat atha t i s functionall y equivalen t t o th e i n vitr o Steroidogenesi s assay i s provide db y endpoint s include di nra t multigenerationa l studies . Specifically , i t wa s confirme d i nth e mos t

recen t multigenerationa l studie s tha t ther e wer e n o treatmen t

relate deffect s a t

hig h dietar y exposur e level s o n endpoint s tha t woul db e impacte db y a n alteratio n o f Steroidogenesi s whic hinclude :

• testicular/uterine/ovar y weight s an dhistopatholog y • size , functio nan dhistopatholog y o f se x organ s

• fertilit y • anogenita l distanc e • sper mcoun t

an dmobilit y • ovaria n evaluatio n fo r follicle s an dcorp alute a

• anogenita l distanc e • ag e an dweigh t

a t preputia l separatio n an dvagina l openin g • numbe r

o f implantatio n site s an dlactation . Glyphosate-relate dreduction s i n bod y weigh t wer e recorde donl y i n F l male s a t th e HOT , bu t ther e i s n o evidenc e tha t

thi s reductio n i s a n endocrine-mediate deffect . Additionally , ther e wa s n o effec t o n severa l endpoint s tha t woul db e impacte db y inhibitio no f Steroidogenesi s followin g treatmen t a t u p t o th e HOT . Th e informatio n fro mthes e i n viv o studie s wit hglyphosat e provide s compellin g evidenc e tha t

glyphosat e ha s n o effec t

o n Steroidogenesis . 

## 3. 0 Mammalia n I n Viv o Assay s Glyphosat e i s on e o f

th e mos t extensivel y teste dagricultura l chemical s bein g use dtoday . Multipl e regulator y toxicolog y dat aset s exis t fo r glyphosate , whic hhav e bee n reviewe db y panel s o f internationa l experts , including , mos t recently , atoxicologica l evaluatio n i n aJoin t revie w b y th e FAO/WH O i n 2004 . I n 2009 , ECETO C publishe dGuidanc e o n Identifyin g Endocrin e Disruptin g Effects , Technica l Report 106 . I n thi s guidanc e document , glyphosat e wa s reviewe da s a n example , wit hmammalia n an dwildlif e toxicolog y studie s evaluated , o f a chemica l tha t

pose s n o endocrin e disruptio n concerns . Ther e i s substantia l i n viv o mammalia n dat asupportin g th e position s tha t glyphosat e i s no t

a n endocrin e activ e compoun dan dtha t n o furthe r anima l testin g i s necessary t o characteriz e it s lac k o f endocrin e disruptin g potential . I nOPPT S Guideline s 890.140 0 (Hershberger ) an d890.160 0 (Uterotrophic) , sectio n (d ) o f eac h guidelin e discusse s "Initia l Consideration s an dLimitations " t o b e considere dbefor e proceedin g wit heithe r

assay . Fo r

th e firs t consideration , i n vitr o evaluations , adistinc t absenc e o f

potentia l fo r estroge n bindin g an dtranscriptiona l activatio n (als o evaluate di n th e Uterotrophi c assay) , an d androge n recepto r

bindin g an d5a-reductas e inhibitio n(als o evaluate di n th e Hershberge r assay ) 2 9ar e discusse di n Section s 2. 1 an d2.2 . Othe r consideration s mentio nthat , i n bothOPPT S 890.1400 an d890.1600 , metaboli c activation/deactivation , distributio nt o targe t tissue s an d clearanc e fro mth e bod y ar e distinctl y addresse di n multipl e metabolis man dADM Estudies . Thes e ADM Estudie s includ e singl e an drepea t dos e ora l studie s i n multipl e specie s (rat , rabbi t an dmonkey) , an dsingl e dos e studie s vi amultipl e route s o f administration , an dar e summarize d i nAppendi x 2 . These studie s sho w tha t glyphosat e i s no t

readil y metabolized , i s no t substantiall y absorbe dwhe ningested , i s rapidl y excreted , an ddoe s no t accumulat e i n th e body . Glyphosat e offer s lo w systemi c exposur e whe nadministere dorally , and , similarly , ha s ver y lo w derma l absorption . Give nth e lo w potentia l fo r systemi c exposur e t o glyphosate , th e valu e o f furthe r anima l testin g i nth e EDS P progra mshoul db e criticall y evaluate dbefor e conductin g th e assays . Considerabl e valu e i s foun di nmultigenerationa l reproductio nstudie s fo r th e evaluatio no f

th e potentia l fo r

asubstanc e t o potentiall y interac t wit hth e endocrin e system . A t leas t fou r suc h studie s hav e bee nconducte dt o evaluat e glyphosate . Multigenerationa l studie s cove r arang e o f specifi c an dapica l endpoints , whic hma y b e use dt o evaluat e endocrin e activity , includin g behaviora l changes , sign s o f difficul t o r prolonge dparturition , abilit y t o becom e pregnant , duratio no f

gestation , se x rati o o f

th e offspring , feminizatio no r masculinizatio no f

offspring , numbe r o f pups , stillbirths , an dgros s patholog y an dhistopatholog y o f th e vagina , uterus , ovaries , testes , epididymis , semina l vesicles , prostate , an dan y identifie dtarge t organ s (Steven s etal. , 1997) . Th e ra t multigenerationa l stud y i s th e mos t comprehensiv e o f

th e curren t

test s fo r reproductiv e an dendocrin e toxicity , providin g a n enormou s amoun t o f informatio ntha t

i s o f th e sam e natur e a s asignifican t

portio no f

th e dat aintende dt o b e generate di n man y o f th e Tie r 1 screenin g assays . Th e revise d199 8 U.S . EP A guidelin e (OPPT S 870.3800 ) fo r amultipl e generatio n reproductiv e an ddevelopmenta l toxicit y assay include s man y developmenta l benchmark s predictiv e o f

endocrin e disruptio npotential , including : • da y o f

vagina l openin g

• mea nestru s cycl e lengt han dpatter n • adivers e se t

o f femal e orga nweight s an dhistopatholog y (ovaries , uterus , vagina , an d mammar y glands )

• matin g an dfertilit y indice s • numbe r

o f implantatio nsite s

• mal e tissu e weight s an dhistopatholog y (testes , epididymis , prostate , an dsemina l vesicles )

• mal e gamete parameter s (e.g . spermnumber , motilit y an dmorphology ) 3. 1 Uterotrophi c Assa y (OPPT S 890,1600 ) Th e Uterotrophi c assay i s a n i n viv o assay i n rat s designe dt o detec t estrogeni c activit y o f

a chemica l base dsolel y o n a n increas e i n uterin e weigh t du e t o estrogen-induce dwate r imbibition s an dhypertrophy , an di s intende dt o complemen t th e i n vitr o E RCompetitiv e Recepto r Bindin g assay (OPPT S 890.1250 ) an dth e E RTranscriptiona l Activatio nassa y (OPPT S 890.1300 ) o f th e EDSP . Ther e ar e alread y atota l o f seve ni n viv o studie s o n glyphosate , conducte da t hig hdos e 3 0level s i n tw o specie s (rat s an drabbits) , wher e uterin e an dothe r organ s an dtissue s tha t

ca n b e evaluate da s indicator s o f estrogeni c potentia l hav e no t show n an y statistica l differenc e i n weigh t o r an y othe r treatmen t relate dmacroscopi c o r

microscopi c finding s tha t woul dindicat e an y estrogeni c effect s o f

glyphosate . Thi s collectio n o f data , alon g wit hth e i n vitr o E Rbindin g an d transactivatio n assay s b y Peti t e t al. , Kojim ae t

al. , an dXi e e t

al. , discusse di n Sectio n 2.0 , tha t demonstrat e tha t

glyphosat e doe s no t

interac t wit hth e ER aan dth e ERp , an dwhic hthi s i n viv o stud y i s intende dt o complement , provide s mor e evidenc e a s t o th e lac k o f estrogeni c activit y o f glyphosat e tha n thi s on e additiona l uterotrophi c assay coul dprovide . 3.1. 1 Discussio n Th e uterotrophi c assay , whic hi s base dsolel y o n th e abilit y o f E Ragonist s t o induc e a n increas e i nuterin e weigh t

du e t o hypertroph y and/o r uterin e flui dimbibition , i s widel y accepte da s a usefu l assay fo r

identifyin g estrogeni c compound s (Clar k an dMani , 1994 ; Ree l e t

al. , 1996) . I t shoul db e noted , however , tha t th e uteru s predominantl y contain s th e ER arecepto r subtype , wit h ver y littl e ER|3 . Th e uterotrophi c assa y i s designe dt o complemen t th e i n vitr o E RCompetitiv e Recepto r Bindin g assay an dth e E RTranscriptiona l Activatio n assa y o f th e EDS PT l tes t

battery . Accordin g t o OPPT S 890.1600 , immature , o r preferabl y ovariectomize drats , ( 6 pe r group ) ar e dose dvi agavag e o r

subcutaneou s injectio n fo r 3 day s an dthe n necropsied , an dE Ragonist s ar e detecte db y a n increas e i n we t o r blotte duterin e weight . However , a s note di n th e tes t guideline , th e uterotrophi c respons e i s no t alway s o f

estrogeni c origi nan dcompound s othe r tha n agonist s o r antagonist s o f

estrogen s ca n als o provid e aresponse . Fo r example , relativel y hig hdose s o f testosteron e ca n lea dt o astimulativ e response . A s previousl y discusse di n Sectio n 2.0 , existin g studie s functionall y equivalen t t o th e i n vitr o E R bindin g an dtransactivatio n assay s o f th e EDSP , alread y demonstrat e tha t glyphosat e doe s no t interac t wit hth e ER aan dth e ER 0 receptor s (Peti t e t al . 1997 , Kojim ae t

al. , 2004 , Xi e e t

al. , 2005) . Glyphosat e ha s als o bee n show n t o no t posses s estrogeni c o r

androgeni c propertie s i n severa l i nviv o studies . Fou r multigenerationa l studie s i n rats , eac hindividually , provid e consisten t evidenc e tha t glyphosat e doe s no t

exhibi t an y estrogeni c activity . Schroeder , R.E. ; Hogan , G.K . (1981 ) A Three-Generatio n Reproductio n Stud y wit h Glyphosat e i n Rats : Projec t No . 77- 2 , Stud y No . BD N 77-41 7 (Unpublishe dstudy ; prepare db y Bio/dynamics , Inc. , submitte db y Monsant o Co. , Washington , D.C. ; CDL:245909-A) ; MRID' s 00081674,0010599 5 I nthi s three-generatio n reproductio n study , wher e 1 2 mal e an d2 4 femal e Sprague-Dawle y rat s pe r grou p receive ddietar y exposur e t o glyphosat e a t 0 , 3 , 1 0 an d3 0 mg/kg/day , th e highes t dos e teste d(HDT ) wa s considere dth e no-effec t level . Endpoint s tha t

ca n b e evaluate da s indicator s o f estrogeni c potentia l i nthi s stud y include dmatin g an dfertilit y indices , ovar y weights , microscopi c evaluation s o f uteru s an dovarie s i n FO , Fi b an dF2 b parent s an dF3 b offsprin g (contro l an dhig hdoses) , an dlitte r se x ratios , al l o f

whic hha dn o significan t findings , whic h include s n o finding s o f

an y estrogeni c effects .

3 1Reyna , M . (1990) . Tw o Generatio n Reproductio n Feedin g Stud y wit hGlyphosat e i n Sprague

Dawle y Rats : La b Projec t No : MSL-10387 , Stud y No . M L88-106 . Unpublishe dstud y prepare d b y Monsant o Agricultura l Co. ; MRJ D 4162150 1 I ncomplianc e wit hOP PGuidelin e 83-4 , amultigenerationa l stud y (Reyna , 1990 ) wa s conducte d wit hSprague-Dawle y rat s (3 0 rats/sex/group ) dose da t 2,000 , 10,00 0 an d30,00 0 part s pe r millio nglyphosat e i n th e diet . Onl y aslight , transien t bod y weigh t

chang e wa s note da t

10,00 0 pp m(approximatel y 77 7 mg/kg/da y fo r male s an d66 6 mg/kg/da y fo r females) , whic hwa s considere dt o b e th e NOAE Lfo r reproductiv e an dparenta l toxicity . Endpoint s relevan t t o th e uterotrophi c assay i nF O an dFl aparent s include dovar y weights , macroscopi c evaluatio n o f uteru s an dovary , an dmicroscopi c evaluatio n o f vagina , uteru s an dovary , al l o f whic hshowe d n o glyphosate-relate deffects . Dhinsa , N . K. , Watson , P . an dBrooks , P . N . (2007 ) Glyphosat e Technical : Dietar y Tw o Generatio n Reproductio n Stud y i n th e Rat . Unpublishe dstud y t o b e submitte dt o EP A b y Nufar mAmericas . Dhins ae t al . (2007 ) conducte damultigeneratio n reproductio n stud y i n Sprague-Dawle y Crl:C D (SD ) IG S B Rrat s (F O 2 8 rats/sex/group , F l 2 4 rats/sex/group) , whic hi s i n complianc e wit hth e curren t 200 1 OEC D Tes t

Guidelin e 416 . Th e ora l administratio n o f glyphosat e technica l t o rat s b y dietar y admixtur e a t

amaximu mdos e leve l o f 15,00 0 pp mfo r

tw o successiv e generation s resulte di n possibl e treatmen t relate dchange s a t

15,00 0 ppm . Th e effects , however , wer e considere dno t

t o represen t

a n advers e healt heffect ; therefore , th e N o Observe dAdvers e Effec t Leve l (NOAEL ) wa s considere dt o b e 15,00 0 pp mfo r adul t toxicit y fo r

bot hth e F O an dF l generations . Th e NOE Lfo r reproductiv e an ddevelopmenta l toxicit y fo r bothgeneration s an d offsprin g wa s als o considere dt o b e 15,00 0 ppm . Endpoint s included :

• da y o f

ag e whe n vagina l openin g occurre di n th e F l female s • absolut e an drelativ e weight s o f th e ovarie s an duteru s (includin g oviduct s an dcervix ) o f th e F O an dF l paren t

animal s • absolut e an drelativ e weigh t o f th e uteru s o f

th e F l an dF 2 offsprin g animal s • i nfollicl e number s fro mth e ovarie s fro mtreate dF i female s • macroscopi c o r

microscopi c finding s observe di n an y tissu e fro mth e F O o r F l animals , includin g uteru s wit hoviduct s an dcervix , ovarie s an dvagin a

• mating s i n al l treatmen t group s

• rati o o f

mal e t o femal e pup s bor n • litte r siz e • viabilit y o f

offsprin g Moxon , M . E . (2000 ) Glyphosat e Acid : Multigeneratio n Reproductio n Toxicit y Stud y i n Rats ; Report No . CTL/P/6332 . Unpublishe dstud y t o b e submitte db y Syngent aCro p Protection . 3 2Moxo n (2000 ) conducte damultigenerationa l reproductio n stud y i n Wista r derive drat s (2 6 rats/sex/group) , whic hi s complian t wit hth e curren t

199 8 U S EP A OPPT S 870.380 0 guideline . Dietar y exposur e t o glyphosat e a t 0 , 1000 , 300 0 an d1000 0 part s pe r millio n(ppm ) ha dn o advers e effec t

o nreproductiv e performance , including : • da y o f

ag e whe n vagina l openin g occurre di n th e F l female s

• unadjuste dweigh t

an dorga n t o bod y weigh t rati o o f th e ovarie s o r

uteru s (includin g cervix ) o f th e F O an dF l paren t

animal s

• numbe r

o f primordia l an dsmal l growin g follicle s o f th e lef t ovar y o f th e F l paren t female s • macroscopi c o r

microscopi c finding s observe di n an y tissu e fro mth e F O o r F l animals , includin g uterus , cervix , ovarie s an dvagin a • mating s i n al l treatmen t group s

• rati o o f

male/femal e pup s bor n • litte r siz e durin g lactatio n I nadditio n t o th e fou r

multigenerationa l studies , ther e ar e thre e othe r studie s tha t

provid e evidenc e fo r

th e lac k o f estrogeni c activity— ateratolog y stud y i n th e rat , ateratolog y stud y i n th e rabbi t an da90-da y feedin g stud y i n th e rat . I n th e tw o teratolog y studies , th e uterin e weight s wer e measure dan dshowe dn o statistica l differenc e betwee n th e contro l an dth e highes t dose d tes t group . Eac ho f

thes e studie s i s discusse dbelow . Rodwell , D.E. ; Tasker , E.J. ; Blair , A.M. ; e t al . (1980a

) Teratolog y Stud y i n Rats : IRD C No . 401-054 , Stud y No . I R79-016 . (Unpublishe dstud y includin g IRD C no . 999-021 ; prepare db y Internationa l Researc han dDevelopmen t Corp. , submitte db y Monsant o Co. , Washington , D.C. ; CDL:242516-A) ; MRI D 0004636 2 Rodwell , D.E. ; Tasker , E.J. ; Blair , M. ; e t al . (1980b ) Teratolog y Stud y i n Rabbits : IRD C No . 401-056 , Stud y No . I R79-018 . (Unpublishe dstud y unde r 524-308 ; prepare db y Internationa l Researc han dDevelopmen t Corp. , submitte db y Monsant o Co. , Washington , D.C. ; CDL:242516

- B) ; MRI D 0004636 3 Glyphosat e wa s show n t o no t b e aselectiv e developmenta l toxican t i n tw o teratolog y studies , on e i n rat s dose da t

0 , 300 , 100 0 an d350 0 mg/kg/da y (Rodwel l e t al. , 1980a ) an don e i n rabbit s dose da t 0 , 75 , 17 5 an d35 0 mg/kg/da y (Rodwel l e t al. , 1980b) . I n th e ra t

study , wit hamaterna l an ddevelopmenta l NOAE Lo f 1,00 0 mg/kg/day , areduce dgai n i n mea n materna l bod y weigh t wa s note dove r

th e treatmen t

perio da t 3,50 0 mg/kg/day , a s wel l a s a n increas e i n earl y resorptions , asligh t

increas e i n postimplantatio n los s an ddecrease s i n mea n feta l bod y weigh t an dth e mea n numbe r

o f viabl e fetuse s a t

thi s maternall y toxi c dose . I n th e rabbi t study , wit ha materna l NOAE Lo f

17 5 mg/kg/da y an ddevelopmenta l NOAE Lo f 35 0 mg/kg/day , ther e wer e n o biologicall y meaningfu l difference s i nmea n numbe r o f viabl e fetuses , earl y o r

lat e resorptions , tota l implantations , corpor alutea , o r th e feta l se x distribution . I n boththes e studies , uterin e weight s wer e measure dafte r excisio n an dn o statisticall y significan t trend s wer e note dacros s thi s wid e rang e o f doses . 3 3Stout , L. ; Johnson , C . (1987 ) 90-da y Stud y o f Glyphosat e Administere di n Fee dt o Sprague/Dawle y Rats : Proj . I D ML-86-351/EH L86128 . Unpublishe dstud y prepare db y Monsant o Agricultura l Co. ; MRI D 40559401 . Stou t an dJohnso n (1987 ) conducte da90-da y ra t feedin g stud y wit hglyphosat e a t 0 , 1000 , 500 0 an d2000 0 pp mi n th e diet . Th e hig hdos e wa s considere dth e NOAEL . Microscopi c evaluatio n o f th e ovaries , ski n (wit hmammar y gland ) an duteru s (corpu s an dcervix) , i n th e contro l an d hig hdos e grou p wer e conducted . Al l th e tissue s relevan t t o th e uterotrophi c assay , showe dn o glyphosate-relate deffects , whic hprovide s furthe r evidenc e tha t

ther e wa s n o estrogeni c activit y a s well . Lastly , ther e i s alread y availabl e astud y wher e microscopi c evaluation s o f uterin e tissue s ha d bee n conducte dfollowin g chroni c exposur e t o hig hlevel s o f glyphosate . Stout , L. ; Ruecker , F . (1990 ) Chroni c Stud y o f Glyphosat e Administere di n Fee dt o Albin o Rats : La b Projec t

Number : MSL-10495 : R.D . 1014 , Stud y No . M L87-148 . Unpublishe dstud y prepare db y Monsant o Agricultura l Co. ; MRI D 41643801 . Stou t an dRucke r

(1990 ) conducte dachroni c dietar y ra t stud y dosin g a t

0 , 2000 , 800 0 an d2000 0 pp mi n th e die t

fo r tw o years . N o glyphosat e relate deffect s wer e note da t an y dos e fo r

an y microscopi c evaluation s o f uterus , ovarie s o r

mammar y gland s i n th e 1-year interi msacrifice , 2

- yea r termina l sacrific e o r

an y unschedule ddeaths . 3.1. 2 Conclusion s - Uterotrophi c Assay Owen s an dAshb y (2002 ) conducte dathoroug hcritica l revie w o f th e uterotrophi c assay , an dth e predictiv e valu e o f

th e uterotrophi c assa y itsel f wa s determine db y compariso n wit hobservation s mad e i nth e multigenerationa l reproductiv e an ddevelopmenta l toxicit y assays . The y conclude d tha t "labelin g o r

classificatio n actio n shoul dno t b e base do n i n vitr o o r

i n viv o screen s (e.g. , transcriptiona l assay s o r

th e uterotrophi c assay) , bu t awai t th e result s o f

robust , definitiv e testin g protocol s fo r

advers e effects , suc ha s th e multigenerationa l reproductiv e an ddevelopmenta l assay , s o tha t bot hth e dat ao n mechanis man dth e occurrenc e o f a n advers e effec t

ar e available. " Mos t importantly , thi s pape r

provide dacompariso n o f n o observe deffec t

level s (NOEL ) an dlowes t observe deffec t

level s (LOEL ) fo r

uterotrophi c an dra t

multigeneratio n studies , an dconclude d hig hconcordanc e fo r

NOE C an dLOE C value s fo r estrogenicity . Ther e i s consisten t

evidenc e showin g alac k

o f estrogeni c activit y o f

glyphosat e i nfou r multigenerationa l studies , asubchroni c study , an dachroni c study , al l i n rats . Thes e equivalen t dat aprovid e stron g ground s fo r th e uterotrophi c assay bein g considere dduplicativ e an d unnecessar y t o characteriz e glyphosat e potentia l a s anon-estrogeni c compoun di n a n i n viv o assay . En dPoin t

i n Uterotrophi c assa y Functionall y Equivalen t Informatio n fro mExistin g Studie s Uterin e Weigh t • N o impac t o n uterin e weight s wit hcervi x a t th e highes t

dos e teste do f 10,00 0 pp mi n die t

(Moxon , 2000 ) o f

th e F O an dF l paren t

animals . • N o impac t

o n uterin e histopatholog y whic hi s consisten t wit hn o effec t

o n uterin e weigh t

a t 10,00 0 pp mi n die t

(Moxon , 2000) . • N o impac t

o n uterin e weight s wit hcervi x an doviduct s a t th e highes t

dos e teste do f 15,00 0 pp mi n die t

(Dhins ae t

al. , 2007 ) o f

th e F O an dF l paren t 3 4En dPoin t i n Uterotrophi c assa y Functionall y Equivalen t Informatio n fro mExistin g Studie s animals . • N o impac t

o n uterin e histopatholog y whic hi s consisten t wit hn o effec t uterin e weigh t

a t 15,00 0 pp mi ndie t

(Dhins ae t

al , 2007) . • N o impac t

o n uterin e weight s a t

th e highes t

dos e teste do f

15,00 0 pp m die t (Dhins ae t

al. , 2007 ) o f

th e F l an dF 2 offsprin g animals . o n i n 

### 3. 2 Hershberge r

Bioassa y (OPPT S 890.1400 ) Th e Hershberge r

assay (OPPT S 890.1400 ) i s widel y accepte da s ausefu l tes t tha t utilize s measurement s o f

fiv e androgen-dependen t

accessory se x tissu e weight s fro mcastratedmal e rat s followin g ora l gavag e exposur e t o th e tes t substanc e fo r

identifyin g potentia l A Ragonists , A R antagonists , an d5<x-reductas e inhibitors . Thes e tissue s ar e th e ventra l prostat e (VP) , semina l vesicl e (SV ) (plu s fluid s an dcoagulatin g glands) , levato r ani-bulbocavernosu s (LABC ) muscle , paire dCowper' s gland s (COW ) an dth e glan s peni s (GP) . O'Conno r e t al . (2002 ) evaluate danumbe r o f th e EDS PTie r

1 assays , and , i n th e evaluatio no f th e Hershberge r

assay , the y summarize dth e relativ e correlation s betwee nchange s i n weigh t (a s apercentag e o f

tota l bod y weight ) o f

th e variou s targe t

organ s o f

th e assay an dth e potentia l endocrin e activity . Thos e correlation s (fro mTabl e 2 i n O'Conno r e t al. ) ar e summarize dher e i n Tabl e 2 . A s a n example , thi s tabl e illustrate s tha t i f achemica l i s astron g agonis t o f androge n recepto r activity , yo u woul dse e a n increas e i n th e relativ e weigh t (relativ e t o tota l bod y weight ) o f th e prostate , semina l vesicles , levato r ani-bulbocavernosu s muscl e an dbulbourethra l (Cowper's ) glands , an dma y o r ma y no t se e a n increas e i n th e relativ e weigh t o f th e liver . O n th e othe r hand , awea k

antagonis t

woul dresul t

i na nincreas e i nrelativ e live r weigh t an dmayb e o r mayb e no t

adecreas e i n relativ e weight s o f th e othe r fou r tissues . Non e o f th e result s fro mth e fou r

multigenerational , on e subchroni c an don e chroni c stud y wit h glyphosat e i nrat s indicat e an y androgeni c activit y whe nevaluate dagains t th e effect s correlatio n matri x o f O'Conno r

e t al . Thes e dat aar e functionall y equivalen t t o dat aexpecte dt o b e generate d fro mth e Hershberge r

assay , a s describe dbelo w i neac hsubsectio nfo r th e endpoint s note di nOPPT S 890.1400 . Therefore , th e requiremen t t o ru nthi s assay shoul db e waived . Tabl e 2 : Correlatio n o f

Effect s o n Hershberge r

Targe t Tissue s (Adapte dfro mO'Conno r e t al. , 2002 , Tabl e 2 ) Endocrin e Activity Androge nReceptor , stron g agonis t Androge nReceptor , stron g antagonis t Androge nReceptor , weak antagonis t Antiandroge n 5a-reductas e inhibito r PorV P ( % b.wt. ) t i -/ i -/ | 4 S V ( % b.wt. ) T i 

### -l { 

### -/l/ r 1 LAB C ( % b.wt. ) 

### T -/I -/ I -/ i

- B U ( % b.wt. ) t 1 -/I 

### -/4/ T 1 Live r ( % b.wt. ) -/T -/t T T

- L H (ng/mL ) T T (ng/mL )

-

P= prostate ; V P= ventra l prostate ; S V = semina l vesicles ; LAB C = levato r anibulbocevernosu s muscles ; B U = bulbourethra l (Cowper's ) glands ; L= liver ; L H = lutenizin g hormone ; T = testosterone , b.wt . = body weigh t 3 5A mor e complet e discussio no f OSR I relate dt o th e Hershberge r assay i s presente db y tissue/endpoin t

i nth e followin g subsections . Man y o f th e finding s ar e discusse di nrelatio nt o th e effect s matri x i nTabl e 2 . 3.2. 1 Prostat e /

Ventra l Prostat e Al l si x o f th e glyphosat e feedin g studie s belo w include dweigh t measuremen t

and/o r microscopi c examinatio no f

th e prostate . N o difference s i n absolut e o r relativ e weight s o f

th e prostat e o r

an y treatment-relate deffect s regardin g th e prostat e wer e note di n an y o f thes e studies . I nth e multigenerationa l reproductio nstud y o n glyphosat e b y Moxo n(2000) , ther e wer e n o difference s note di n th e absolut e weigh t o r relativ e weigh t

o f th e prostrat e glan dbetwee nth e contro l an ddos e group s i nth e F O parent s o r F l parents . I nth e mos t

recen t multigenerationa l reproductio nstud y o n glyphosat e b y Dhins ae t al . (2007) , ther e wer e n o difference s note di n th e absolut e weigh t o r relativ e weigh t

o f th e prostrat e glan d betwee nth e contro l an ddos e group s i n th e F O parent s o r F l parents . N o treatmen t relate d histopathologica l difference s o f th e prostat e wer e note dbetwee nth e contro l an dhig hdos e mal e groups . I nth e three-generatio nreproductio nstud y o n glyphosat e b y Schroede r an dHoga n(1981) , microscopi c examinatio no f th e prostat e fro mth e F O parents , Fi b parents , F2 b parent s an dF3 b offsprin g o f

th e hig hdos e grou p note dn o treatment-relate deffect s relativ e t o th e stud y controls . Th e two-generatio nreproductio nstud y b y Reyn a(1990 ) include dmicroscopi c examinatio no f th e prostat e o f

th e F O parent s an dFl aparent s i n th e contro l an dhig hglyphosat e dos e groups . Ther e wer e n o treatment-relate deffect s note di n an y o f th e groups . Bot hth e glyphosat e subchroni c ra t feedin g stud y o f

Stou t an dJohnso n(1987 ) an dth e chroni c stud y o f Stou t an dRucke r

(1990 ) include dmicroscopi c examinatio no f th e prostat e a t

9 0 day s an da t 1 2 an d2 4 months , respectively . A s wit hth e multigenerationa l studies , ther e wer e n o treatment-relate deffect s note do n th e prostat e i n eithe r study . 3.2. 2 Semina l Vesicle s Thre e o f th e fou r multigenerationa l reproductio ntoxicit y studie s o n glyphosat e include dweigh t measurement s o r

microscopi c examinatio no f th e semina l vesicles . N o differenc e i n absolut e o r relativ e weight s o r

an y treatmen t

relate deffect s wer e note di n th e semina l vesicle s i n th e thre e studies . I nth e multigenerationa l ra t reproductio nstud y o n glyphosat e conducte db y Moxo n(2000) , ther e wa s n o differenc e i neithe r th e absolut e o r

relativ e weight s o f

th e semina l vesicle s i nF O parent s o r F l parent s fro mth e contro l an ddos e groups . Microscopi c examinatio no f th e semina l vesicle s o f

th e F O parent s an dFl aparent s fro mth e contro l an dhig hdos e group s i n th e two generatio nglyphosat e reproductio ntoxicit y stud y b y Reyn a(1990 ) als o showe dn o treatmen t relate deffect s o f

an y kind , includin g an y androgeni c effects . I nth e mos t

recen t multigenerationa l reproductio nstud y o n glyphosat e b y Dhins ae t al . (2007) , ther e wer e n o difference s note di n th e absolut e weigh t o r relativ e weigh t

o f th e semina l vesicle s betwee nth e contro l an ddos e group s i n th e F O parent s o r F l parents . N o treatmen t relate d histopathologica l difference s o f th e semina l vesicle s wer e note dbetwee nth e contro l an dhig h dos e mal e groups . 

### 3 63.2. 3 Levato r An i Bulbocavernosu s (LABC ) LAB C weigh t

wa s no t evaluate di n an y o f

th e glyphosat e studies . However , th e effect s correlatio nmatri x describe db y O'Conno r (2002 ) (Tabl e 2 ) show s that , i n anumbe r o f instance s o f androgeni c effect , adifferenc e i n th e relativ e weigh t o f th e LAB C betwee ncontrol s an dtes t group s ar e als o accompanie db y change s i n th e relativ e weight s o f th e prostat e an dsemina l vesicles . N o change s i n prostat e o r semina l vesicl e weight s wer e see ni n th e 90-da y ra t feedin g stud y o n glyphosat e conducte db y Stou t an dJohnso n(1987) . A chang e i n th e relativ e weigh t o f th e LBA C ca n als o b e accompanie db y a n increas e i n th e relativ e weigh t

o f th e live r whe ni t i s th e resul t

o f androgeni c activity . Th e multigenerationa l ra t reproductio nstud y o n glyphosat e conducte db y Moxo n(2000 ) showe dastatisticall y significan t (p<0.01 ) highe r

relativ e mea nlive r

weigh t ove r th e contro l i nth e F O males , a s wel l a s a statisticall y significan t

(p<0.05 ) lowe r

absolut e mea nlive r

weigh t compare dt o th e contro l i n th e F l males , botha t

th e hig hdos e level . Dhins ae t al . (2007 ) note da nincreas e i nlive r weight s fo r female s treate da t

th e hig hdos e o f

15,00 0 pp mfro mbothgenerations . N o suc heffec t wa s note dfo r male s treate dwit h15,00 0 pp mo r fo r animal s o f

eithe r se x treate dwit h5,00 0 pp mo r 1,50 0 ppm . Accordin g t o th e effect s correlatio nmatrix , a nincreas e i nrelativ e live r weigh t ca nb e reflectiv e o f (i ) astron g androge nrecepto r agonis t whe naccompanie db y increase dprostat e an dsemina l vesicle s weight , o r

(ii ) astron g androge nrecepto r antagonist s whe naccompanie db y decrease d prostat e an dsemina l vesicl e weights . A s indicate di n Section s 3.2. 1 an d3.2. 2 above , thes e multigenerationa l ra t

reproductio nstudie s o nglyphosat e showe dn o differenc e i neithe r th e absolut e o r

relativ e weigh t

o f eithe r th e prostat e o r

th e semina l vesicles ; therefore , ther e i s n o evidenc e tha t

glyphosat e i s eithe r

astron g androge nrecepto r agonis t o r antagonist . Als o accordin g t o th e effect s correlatio nmatrix , androgeni c activit y onl y cause s a n increas e i n relativ e live r weight , neve r

adecrease ; therefore , th e decrease drelativ e live r weigh t i n th e F l mal e indicate s tha t

th e change s ar e totall y unrelate dt o endocrin e activity . Thi s conclusio ni s reasonabl e sinc e live r

weigh t change s ar e commo ni n hig hdos e group s o f toxicologica l investigations , an dth e hig hdos e male s i nthi s stud y exhibite dcompoun drelate dreduction s i n bod y weigh t

an dfoo dconsumption , whic hwoul dexplai nth e increas e i nlive r weigh t relativ e t o tota l bod y weight . 3.2. 4 Bulbourethra l (Cowper's ) Glan d Bulbourethra l glan dweight s wer e no t measure di n an y o f

th e glyphosat e studies . However , th e effect s correlatio nmatri x o f th e Hershberge r

assay (Tabl e 2 ) note s tha t i n anumbe r

o f instances , adifferenc e i n bulbourethra l glan d% bod y weigh t betwee ncontrol s an dtes t group s ar e als o accompanie db y change s i n relativ e weight s o f th e prostate , semina l vesicles , an dliver . A s discusse di n sectio n3.2.1 , th e 90-da y ra t feedin g stud y o n glyphosat e conducte db y Stou t an d Johnso n(1987 ) di dno t

fin dan y effect s followin g microscopi c examinatio no f th e prostate . Th e two-generatio nra t

reproductio ntoxicit y stud y o n glyphosat e conducte db y Moxo n(2000) , a s discusse di n Sectio n3.2.2 , di dno t fin dan y chang e i n th e absolut e o r relativ e weigh t

o f th e semina l vesicles , an da s discusse di n Sectio n3.2.3 , an di n th e nex t section , th e increas e i n relativ e live r

weigh t i nth e F O mal e an ddecreas e i nth e relativ e live r weigh t i nth e F l male , alon g wit hth e n o effec t

finding s o n th e prostat e an dsemina l vesicles , i s stron g evidenc e tha t thes e live r change s wer e no t

relate dt o endocrin e activit y an dsimpl y du e t o othe r mechanism s i n hig h dos e tes t groups . Th e mos t

recen t multigenerationa l ra t

stud y (Dhins ae t

al. , 2007 ) note dn o differenc e i n th e absolut e o r relativ e weights , o r

an y histopathologica l differences , o f eithe r th e 

### 3 7prostat e gland s o r semina l vesicle s o f

th e hig hdos e animal s whe ncompare dt o thos e o f th e contro l groups . 3.2. 5 Glan s Peni s Weigh t I nth e O'Conno r

e t al . (2000 ) evaluatio no f

th e the npropose dEDS PTie r I screenin g approaches , dat ao n glan s peni s weigh t wer e no t availabl e fo r

evaluation . However , give nth e considerabl e substantiatio no f

n o effect s o nfertilit y an dmal e reproductiv e orga nweight s i nfou r multi

generationa l reproductio nstudies , thi s endpoin t ma y b e considere dredundant . 3.2. 6 Liver

Weigh t Measuremen t

o f live r weigh t i s a n optiona l endpoin t

i n th e Hershberge r

assay (OPPT S 890.1400) . Whil e collectio no f live r weigh t woul dprovid e insigh t

int o genera l toxicity , fo r endocrin e evaluatio npurposes , live r weigh t woul donl y b e usefu l a s supportiv e i f ther e wer e othe r dat atha t suggeste dpossibl e endocrin e activity . Th e multigenerationa l reproductiv e stud y o n glyphosat e conducte db y Schroede r an dHoga n (1981 ) showe dn o difference s i nth e absolut e o r relativ e weight s o f

th e live r i nmal e F O adults , F l adults , F 2 adult s an dF 3 offsprin g betwee nth e contro l an ddos e groups . Also , n o treatmen t relate deffect s wer e identifie di nth e microscopi c examination s o f th e liver s o f

th e parenta l FO , Fib , F2 b an dweanling s fro mF3 b animals . I n th e F 2 treate dfemales , anon-dose-related , albei t statisticall y significant , decreas e i nmea nliver/bod y weigh t rati o wa s evident ; liver/brai nweigh t ratio s fo r thes e female s showe dasimila r reduction ; however , thes e difference s fro mcontro l wer e no t statisticall y significant . Th e multigenerationa l ra t reproductio nstud y o n glyphosat e conducte db y Moxo n(2000 ) showe d astatisticall y significan t

( p < 0.01 ) highe r

relativ e mea nlive r

weigh t ove r th e contro l i n th e F O males , a s wel l a s astatisticall y significan t ( p < 0.05 ) lowe r

absolut e mea nlive r

weigh t compare d t o th e contro l i n th e F l males , bot ha t th e hig hdos e level . Dhins ae t al . (2007 ) note da n increas e i nlive r weight s fo r

female s fro mbothgeneration s treate da t th e hig hdos e o f

15,00 0 ppm . N o suc h effec t wa s note dfo r

male s treate dwit h15,00 0 pp mo r fo r animal s o f

eithe r se x treate dwit h5,00 0 o r 1,50 0 ppm . Accordin g t o th e effect s correlatio nmatrix , a nincreas e i nrelativ e live r weigh t ca n b e reflectiv e o f

(i ) astron g androge nrecepto r agonis t whe naccompanie db y increase dprostat e an dsemina l vesicle s weight , o r (ii ) astron g androge nrecepto r antagonis t

whe naccompanie db y decrease dprostat e an dsemina l vesicle s weights . A s indicate di n Section s 3.2. 1 an d3.2.2 , thi s multigenerationa l ra t

reproductio nstud y o n glyphosat e showe dn o differenc e i n eithe r th e absolut e o r

relativ e weigh t

o f eithe r th e prostat e o r

th e semina l vesicles ; therefore , ther e i s n o evidenc e tha t

glyphosat e i s eithe r

astron g androge nrecepto r agonis t o r antagonist . Als o accordin g t o th e effect s correlatio nmatrix , androgeni c activit y onl y cause s a n increas e i n relativ e live r weight , neve r

adecrease ; therefore , th e decrease drelativ e live r weigh t i n th e F l mal e indicate s tha t

th e change s ar e totall y unrelate dt o endocrin e activity . Live r weigh t change s ar e commo ni n hig hdos e group s o f toxicologica l investigation s an dth e hig hdos e male s i n thi s stud y exhibite dcompoun drelate dreduction s i n bod y weigh t an dfoo dconsumption , whic hwoul d explai nth e increas e i n live r weigh t relativ e t o tota l bod y weight . Stou t an dRucke r

(1990) , i n th e chroni c ra t

stud y o nglyphosate , foun dstatisticall y significan t increase s i nliver-to-bod y weigh t ratio s i n male s a t

1 2 months , an dabsolut e live r weigh t an dliver-to-brai nweigh t ratio s a t 2 4 month s a t th e maximu mtolerate ddos e (hig hdose) . A s mentione dearlier , live r weigh t change s ar e commo ni n hig hdos e group s o f toxicologica l investigations , especiall y a t maximu mtolerate d doses . 3 8Stou t

an dJohnso n (1987 ) foun dn o change s i neithe r absolut e o r

relativ e live r

weigh t afte r 9 0 day s o f ora l dosin g i n rat s an dmicroscopi c examinatio n o f contro l an dhig hdos e liver s note dn o treatment-relate deffects . 3.2. 7 Luteinizin g Hormon e (LH ) Measuremen t

o f luteinizin g hormon e (LH ) seru mconcentratio n (ng/mL ) i s optiona l i n th e Hershberge r

assay (OPPT S 890.1400) . Increase dL H level s provid e additiona l weigh t o f evidenc e o f

stron g androge n recepto r antagonis m(Tabl e 2 ) an dwoul db e measure donl y i f ther e wer e othe r

dat atha t suggeste dtha t

th e tes t substanc e wa s a n androge n recepto r antagonist . Give nth e volum e o f

dat atha t support s th e conclusio n tha t glyphosat e i s no t

a n androge n recepto r antagonist , thi s optiona l endpoin t provide s n o adde dvalue . 3.2. 8 Testosteron e Measuremen t

o f testosteron e seru mconcentratio n (ng/mL ) i s optiona l i n th e Hershberge r assay (OPPT S 890.1400) . Sinc e n o mal e reproductiv e effect s wer e note di n th e fou r multigenerationa l ra t studie s (Dhins ae t

al. , 2007 ; Moxon , 2000 ; Reyna , 1990 ; Schroede r an dHogan , 1981) , th e measuremen t

o f testosteron e seru mconcentration s woul dprovid e n o significan t valu e t o th e understandin g o f

th e endocrin e activit y o f

glyphosate . 3.2. 9 Othe r

endpoint s o f

relevanc e t o th e Hershberge r Assay Overall , i nsi x i nviv o studie s conducte do nglyphosate , includin g fou r multigenerationa l reproductio n studies , asubchroni c feedin g stud y an dachroni c feedin g study , n o treatment relate deffect s wer e eve r

see n o n th e numbe r

o f sper mproduced , sper mmotilit y o r sperm morphology , o r

mating , pregnanc y o r

fertilit y indices . Fou r

o f thes e si x studie s showe dn o change s i n th e absolut e o r relativ e weigh t

o f th e teste s o r

epididymides , o r

an y treatment-relate d effect s o n th e microscopi c examinatio n o f th e testes . Onl y i n th e two-generatio n reproductio n stud y conducte db y Reyn awa s ther e asligh t increas e i n th e relativ e weigh t o f th e teste s (weigh t o f teste s relativ e t o tota l bod y weight ) an donl y i n th e Fl aadul t i n th e highes t

treatmen t grou p wher e th e dosin g leve l wa s i n exces s o f th e maximu mtolerate ddos e fo r glyphosate . Thi s effect , however , wa s attribute dt o th e lowe r bod y weigh t

o f th e rats , sinc e th e absolut e weigh t o f th e teste s wa s unchange dfro mtha t o f th e contro l group . Th e finding s o f eac ho f thes e studie s ar e summarize dbelow . I nth e multigenerationa l reproductio n studie s o n glyphosat e conducte db y Moxo n (2000 ) an d Dhins ae t al . (2007) , ther e wer e n o difference s note di n th e absolut e o r relativ e mea n weight s o f th e teste s o r

epididymide s betwee n contro l an ddos e group s o f th e F O parent s o r

F l parents . N o effec t o f glyphosat e o n th e numbe r o f sperm , sper mmotilit y parameter s o r spermmorpholog y wer e note dfo r

eithe r th e F O o r F l males . Schroede r an dHoga n (1981 ) foun dn o effect s o n th e absolut e o r relativ e mea n weigh t

o f th e teste s o f rat s ove r thre e generation s o f

continua l dosin g wit hglyphosate . Microscopi c examinatio no f

th e teste s fro mth e contro l an dhig hdos e group s note dn o treatment-relate d effects . Significantl y decrease dtermina l bod y weight s wer e note dfo r bot hsexe s a t

th e highes t

exposur e leve l i n th e two-generatio n reproductio n stud y o n glyphosat e conducte db y Reyn a(1990) . Th e onl y statisticall y significan t orga n weigh t

chang e wa s asligh t

increas e i n th e mea n testes-to-bod y weigh t ratio s i n th e Fl aadul t

hig hdos e group , bu t

th e mea n absolut e teste s weigh t remaine d unchanged . Thi s findin g wa s attribute dt o thei r lowe r termina l bod y weight s a t thi s dos e level , 3 9

### whic hexceede dth e maximu mtolerate ddose . N o difference s i nth e mea nrelativ e teste s weight s

wer e note di nth e F O a t

al l dos e level s o r

i nth e Fl alo w an dmid-dos e groups . Microscopi c examinatio no f

teste s fro mth e contro l an dhig hdos e groups , an do f th e epididymide s o f

th e F O an dFl aparents , note dn o treatmen t relate deffects . Mating , pregnanc y an dfertilit y indice s wer e no t affecte db y glyphosat e i neithe r th e F O o r F l animals . Microscopi c examinatio no f th e teste s an dprostat e fro mth e contro l an dhig hdos e group s note d n o treatment-relate deffect s afte r 9 0 day s o f

ora l dosin g i nth e subchroni c stud y conducte db y Stou t an dJohnso n(1987) . I nth e chroni c feedin g stud y o nglyphosat e conducte db y Stou t an dRucke r

(1990) , microscopi c examinatio no f

teste s fro mth e contro l an dhig hdos e group s note dn o treatment-relate deffect s afte r 1 2 an d2 4 month s i nth e study . 3.2.1 0 Conclusio n - Hershberge r Assay Clea r an dconsisten t

result s fro mfou r

multigenerationa l reproductio nstudie s an dtw o repeat dos e toxicit y studie s o nglyphosat e provid e stron g scientifi c evidenc e tha t glyphosat e i s no t

a n A Ragonist , A Rantagonis t o r a5a-reductas e inhibitor , an dg o beyon dth e fiv e mal e se x glan d weigh t endpoint s assesse di nth e Hershberge r assay . Base do nthi s information , th e Hershberge r assay conducte daccordin g t o OPPT S 890.140 0 guidelin e i s considere dduplicativ e an d unnecessar y t o characteriz e th e potentia l fo r glyphosat e t o interac t

wit handroge nreceptor s o r inhibi t 5a-reductas e i nvivo . Tabl e 3 : Summar y o f

Finding s Relativ e t o th e Hershberge r Assa y fo r Glyphosat e En dPoin t

i n Hershberge r

Assa y Prostate/ventra l weigh t Semina l Vesicle s weight s LAB C weight s

Bulbourethra l (Cowper's ) Glan d

Glan s Peni s weigh t Functionall y Equivalen t Informatio n fro mExisting Studie s • N o difference s i n absolut e o r relativ e weight s o f

th e prostat e o r

an y treatmen t relate deffect s regardin g th e prostat e wer e note di nasubchroni c ra t study , achroni c ra t

study , fou r

multigenerationa l ra t

studies . • N o differenc e i n absolut e o r relativ e weight s o r

an y gros s o r

microscopi c treatmen t relate deffect s wer e note di nth e semina l vesicle s (wher e evaluated ) i nmultigenerationa l studies . • LAB C weigh t

wa s no t evaluate di n an y o f

th e glyphosat e studies . However , th e absenc e o f

fertilit y issue s an dcorroborativ e mal e reproductiv e orga nweigh t dat aprovide s asubstantia l weigh t o f evidenc e acros s multipl e studies , suggestin g n o effec t o nLAB C weights . • Bulbourethra l glan dweight s wer e no t measure di n an y o f

th e glyphosat e studies . However , th e absenc e o f fertilit y issue s an dcorroborativ e mal e reproductiv e orga nweigh t dat aprovide s asubstantia l weigh t o f evidenc e acros s multipl e studies , suggestin g n o effec t o nBulbourethra l weights . • Glan s peni s weight s wer e no t measure di n an y o f

th e glyphosat e studies . However , th e absenc e o f

fertilit y issue s an dcorroborativ e mal e reproductiv e orga nweigh t dat aprovide s asubstantia l weigh t o f evidenc e acros s multipl e studies , suggestin g n o effec t o nglan s peni s weights . 

### 3. 3 Puberta l Developmen t an dThyroi dFunctio n i n Intac t Juvenil e / Peripuberta l Femal e Rat s (OPPT S 890.1450 ) O'Conno r e t al . (2002 ) state s i nthei r

revie w o f th e Tie r I femal e puberta l assay tha t "thi s assay ha s redundan t

endpoint s wit hth e Tie r

I I multigenerationa l reproductio nstudy , whic hallow s confirmatio no r

refutatio no f

screenin g result s unde r

mor e robus t

tes t conditions. " Ther e ar e 4 0alread y fou r multigenerationa l ra t

reproductio nstudie s conducte do n glyphosate . T o requir e anothe r i n viv o stud y wher e mor e tes t animal s woul db e sacrifice dt o obtai nmor e o f th e sam e informatio nalread y availabl e woul dwast e valuabl e resource s an dtim e o f bot hth e registrant s an dth e Agency . Th e femal e puberta l assay shoul db e waive dfo r glyphosate . Th e femal e puberta l assay measure s discret e endpoint s t o ascertai neffect s o npuberta l developmen t an dthyroi dfunctio ni nth e intac t juvenile/peripuberta l femal e rat . Thes e endpoint s includ e developmenta l milestones , orga nweight s an dhistopathology , estrou s cyclicity , hormon e seru m level s an dclinica l chemistr y parameters . Al l o f thes e endpoint s ar e eithe r specificall y addresse d o r capture di ndownstrea mapica l endpoint s i nmor e comprehensiv e i nviv o studies , suc ha s multigenerationa l reproduction , developmenta l toxicity , subchroni c toxicit y an dchroni c toxicit y studies . Th e multigenerationa l reproductio nstudy , whic hi s ake y Tie r 2 EDS Passay , provide s functiona l equivalenc e t o OPPT S 890.145 0 an drender s th e femal e puberta l assay redundan t (O'Conno r

etal. , 2002) . Correlation s betwee nth e variou s endpoin t effect s o f th e femal e puberta l assay an dendocrin e activit y wer e assesse db y O'Conno r e t al . (2002) . Thos e correlation s (fro mTabl e 3 i n O'Conno r e t al. , 2002 ) ar e reproduce dher e i n Tabl e 4 . T o illustrate , i f achemica l i s astron g estroge n recepto r agonist , yo u woul dexpec t t o se e adecreas e i n th e ag e a t vagina l openin g (VO) , a decreas e i n bod y weigh t

a t vagina l opening , an dadecreas e i n th e weigh t o f th e ovaries . O n th e othe r hand , evidenc e o f

aneuroendocrin e mechanis mwoul db e a n increas e i n ag e an dbod y weigh t a t vagina l openin g alon g wit hadecreas e i n th e weigh t o f th e uteru s an dovaries . A mor e complet e discussio no f OSR I relate dt o th e femal e puberta l assay i s presente db y endpoin t i n th e followin g subsections . Man y o f th e finding s ar e discusse di n relatio nt o th e effect s matri x i n Tabl e 4 . Tabl e 4 : Correlatio no f

Effect s i n Femal e Puberta l Assay (Adapte dfro mO 'Conno r e t al. , 2002 , Tabl e 3 ) Endocrin e Activity Stron g estroge nrecepto r agonis t Weak estroge nrecepto r agonis t Neuroendocrin e mechanis m Broa dsteroi dbiosynthesi s inhibito r Highl y specifi c aromatas e inhibito r Thyroi dactiv e compound s Ag e a t V O 1 -/I 

### T T T

- B.w t

at V O 1 -/i 

### T T T i Uteru s weigh t

-

1 1 

### 4

- Ovarie s weight 

### 4

I

-

- 4 Thyroi d weigh t

-

-

-

- 

### T TS H

-



### T T 4

-

1 V O = vaginal opening ; B.w t = body weight , TS H = thyroi dstimulatin g hormone ; T 4 = thyroxin e 3.3. 1 Growth , Body Weigh t Measurement s o f

growthan dbod y weigh t

wer e capture di n fou r

glyphosat e multigenerationa l reproductio nstudie s i nrats . 

### 4 1I nth e multigenerationa l reproductio nstudie s conducte db y Moxo n(2000 ) an dDhins ae t al . (2007) , n o difference s i nfemal e bod y weigh t o r i npu p weigh t

a t birt hfo r F1 A an dF2 A litter s betwee ncontrol s an ddose dgroup s wer e noted . I nth e two-generatio nra t

reproductio nstud y o f

glyphosat e conducte db y Reyn a(1990) , reduce d bod y weight s an dsof

t stool s wer e note di n adul t animal s i n th e 30,00 0 pp m(high-dose ) group . Bod y weight s o f

pup s i n thi s high-dos e grou p decrease ddurin g th e las t wee k o f lactation . I n th e 10,00 0 pp m(mid-dose ) grou p smalle r decrease s i n bod y weigh t wer e note dan donl y o n lactatio n da y 21 . Thes e bod y weigh t effect s towar dth e en do f lactatio noccurre dwhe npup s bega nt o supplemen t

thei r mil k intak e wit htreated-food . Th e ingestio no f availabl e groun dra t

cho w begin s afte r

th e eye s o f

th e pup s ope n(generall y da y 13 ) an da s the y begi nt o explor e thei r cage d environment . Therefore , th e bod y weigh t decrease s observe di n th e mid

- an dhigh-dos e pup s o n lactatio nda y 2 1 wer e considere dt o b e primaril y du e t o th e reduce drelativ e consumptio no f treate ddie t

b y th e hig hdos e offsprin g du e t o palatabilit y issue s an di s no t considere da n endocrine-mediate deffect . I nth e three-generatio nra t reproductio nstud y o f

Schroede r

an dHoga n(1981) , n o treatment relate deffect s wer e eviden t i nadul t mortalit y data , bod y weigh t o r foo dconsumptio ndat a (growt han dres t

periods) , o r

i nin-lif e physica l observatio ndat athroughou t th e stud y fo r

th e FO , F l an dF 2 generations . 3.3. 2 Ag e at

Vagina l Openin g N o effec t o n th e da y o f

ag e whe nvagina l openin g occurre di n th e F l paren t animal s i n th e tw o multigenerationa l ra t

reproductio nstudie s (Moxon , 2000 ; Dhins ae t al. , 2007) . 3.3. 3 Bod y Weigh t

at Vagina l Openin g N o differenc e i nbod y weigh t a t vagina l openin g wa s note di ntw o multigenerationa l ra t reproductio nstud y conduce do n glyphosate . Initia l bod y weight s wer e recorde dfo r F l female s a t

selectio no n da y 29postpartu mi n th e Moxo n(2000 ) multigenerationa l ra t reproductio nstudy . Th e bod y weigh t o f eac hra t wa s the n weighe da t

weekl y interval s throughou t th e prematin g period . Mea nbod y weight s o f F l female s a t da y 2 9 postpartu mwer e comparabl e t o thos e i n th e contro l group . Measure dan dadjuste d mea nbod y weight s fo r

week s 2 an d3 o f

th e F l prematin g perio dalig nwit hth e tim e o f vagina l openin g an dwer e comparabl e betwee ncontrol s an dal l dos e groups . I nth e mos t

recen t multigenerationa l stud y b y Dhins ae t al . (2007) , al l selecte dF i offsprin g wer e observe dfo r

sexua l development . Fo r

female s th e da y o f

appearanc e o f

vagina l openin g (separatio n o f th e labia ) wa s recorded . Th e bodyweigh t fo r eac hindividua l a t

th e tim e o f

sexua l maturatio nwa s als o recorded , notin g n o effect s o nbod y weigh t o nth e da y o f

vagina l opening . 3.3. 4 Uteru s Weigh t

an dHistopatholog y A s explaine di nthi s sectio nfo r eac hindividua l study , ther e wer e n o treatment-relate deffect s o n th e uteru s foun di nth e fou r ra t multigenerationa l reproductio nan dth e chroni c toxicit y studies . N o differenc e wa s note dbetwee ncontro l an ddos e group s fo r uteru s wit hcervi x weight s (absolut e o r

% relativ e t o bod y weight ) i n F O parent s o r F l parent s i n th e Moxo n(2000 ) multigenerationa l ra t

reproductio nstudy . Similarly , n o difference s wer e note dbetwee ncontro l an ddos e group s fo r

uteru s wit hoviduct s an dcervi x weight s (absolut e o r % relativ e t o bod y 

### 4 2weight ) i nF O parent s o r F l parent s i nth e Dhins ae t al . (2007 ) multigenerationa l ra t reproductio n study . Microscopi c examinatio n o f uter i o f th e F O parents , Fi b parents , F2 b parent s an dF3 b offsprin g fro mth e contro l an dhig hdos e group s wer e conducte di n th e three-generatio n ra t reproductio n stud y o n glyphosat e (Schrode r an dHogan , 1981 ) wit hn o treatment-relate deffect s noted . N o treatmen t

relate deffect s wer e note dfollowin g microscopi c examinatio n o f contro l an dhig h dos e uter i fo r

F O parents , o r

F 1 aparent s i n th e two-generatio n ra t reproductio n stud y conducte d byReyna(1990) . Stou t an dRucke r

(1990 ) conducte dmicroscopi c examinatio n o f uter i fro mth e contro l an dhig h dos e group s i n thi s chroni c toxicit y stud y an dnote dn o treatmen t relate deffect s a t

th e 12

- an d 24-mont htim e points . 3.3. 5 Estru s Cyclicity N o effec t o f glyphosat e o n mea n cycl e lengt ho r numbe r o f cycle s i n th e 3 wee k

perio dprio r

t o matin g wa s see n i n th e multigenerationa l reproductio n stud y (Moxon , 2000) . Slightl y highe r numbe r o f cycle s fo r

F O female s dose dwit h10,00 0 pp man daslightl y lowe r mea n cycl e lengt h i nF l female s relativ e t o th e contro l group s i nthi s stud y wer e considere dmargina l an d inconsisten t

acros s generation s an dno t treatmen t related . 3.3. 6 Ovarie s Weigh t an dHistopatholog y Ther e wa s n o differenc e i n absolut e o r relativ e ovar y weight s an dn o treatmen t relate deffect s note dfollowin g microscopi c examinatio n o f th e ovarie s i nth e fou r

multigenerationa l ra t reproductio nstudie s conducte do n glyphosate . I nth e multigenerationa l studie s conducte db y Moxo n (2000 ) an dDhins ae t al . (2007) , n o difference s wer e note di n th e absolut e o r relativ e ovar y weight s betwee n contro l an ddos e group s o f th e F O o r F l parents . Schroede r an dHoga n (1981 ) note dn o difference s i n absolut e o r relativ e ovar y weight s acros s FO , Fi b an dF2 b femal e adult s an dweanling s i nF3 b i nthi s 3-generatio n ra t reproductio n study , an dmicroscopi c examinatio n o f th e ovarie s note dn o treatment-relate deffects . I nth e reproductio n stud y conducte db y Reyn a(1990) , microscopi c examinatio n o f ovarie s o f

F O an dFl aparent s note dn o treatment-relate deffect s betwee n th e contro l an dhig hdose . 3.3. 7 Thyroi dWeigh t an dHistopatholog y N o treatment-relate dthyroi dweigh t effect s wer e note di n th e multigenerationa l stud y b y Dhins ae t al . (2007) . I n th e F O femal e mid-dos e o f 5,00 0 pp mth e mea n absolut e thyroi dweigh t wa s greate r tha n th e controls , whil e th e F O mal e hig han dmid-dose s o f 15,00 0 pp man d5,00 0 pp m th e mea n absolut e thyroi dweigh t wa s lowe r

tha n th e controls . Thes e finding s wer e considere d contradictor y an dunrelate dt o treatment . I f histologica l lesion s ar e no t observe di n th e thyroi dglands , the n th e chemica l i s considere dt o b e thyroi dinactiv e (OPPT S 890.1110) . N o treatmen t relate deffect s wer e note dfollowin g microscopi c examinatio n o f th e thyroi di n thre e i n viv o studie s conducte do n glyphosate .

4 3Schroede r an dHoga n (1981 ) conducte dmicroscopi c examinatio n o f th e thyroi dan dparathyroi d o f th e FO , Fi b an dF2 b parent s an dF3 b offsprin g i n th e three-generatio n ra t reproductio n stud y an dnote dn o treatment-relate deffects . Microscopi c examinatio n o f th e thyroi dafte r

9 0 day s o f

dosin g i n th e glyphosat e subchroni c stud y (Stou t

an dJohnson , 1987 ) an di n th e hig hdos e grou p a t 1 2 an d2 4 month s i n th e glyphosat e chroni c stud y i n rat s (Stou t an dRucker , 1990 ) als o note dn o treatmen t relate deffects . 3.3. 8 Thyroi dStimulatio n Hormon e (TSH ) TS H i s secrete db y th e anterio r pituitar y gland , whic hregulate s th e endocrin e functio n o f th e thyroi dgland . A s describe di n section s 3.3.1 , 3.3.2 , 3.3.3 , an d3.3.13 , n o difference s i n growth an ddevelopmen t

o r th e absolut e an drelativ e weight s o f th e pituitar y wer e note di n ra t reproductio n studie s conducte dwit hglyphosate , no r wer e ther e an y treatment-relate deffect s note dupo n microscopi c examinatio n o f th e gland . I f

n o developmenta l delay s o r histologica l lesion s ar e observe di n th e thyroi dglands , the n th e chemica l i s considere dt o b e thyroi dinactive . Th e rational e fo r

i s tha t th e thyroi dgland s ar e unde r th e influenc e o f

TS H an dan y chemica l tha t alter s circulatin g thyroi dhormon e sufficientl y t o alte r TS H secretio n wil l resul t i n histopathologica l change s i nth e thyroi dglands . Th e effect s correlatio n matri x o f O'Conno r

e t al . (2002 ) (Tabl e 4 ) fo r

th e femal e puberta l assay note s tha t a n increas e i n TS H i s supportin g evidenc e fo r thyroi dactiv e compound s alon g wit ha decreas e i nbod y weigh t

a t vagina l openin g an dovar y weight , an da nincreas e i nthyroi dweight . Multigenerationa l reproductio n studie s and/o r subchroni c an dchroni c studie s o n glyphosat e hav e show n n o decreas e i n bod y weigh t a t vagina l openin g o r

i n ovar y weigh t

(Dhins ae t

al. , 2007 ; Moxon , 2000 ; Schroede r

an dHogan , 1981 ; Reyna , 1990 ) an dn o increas e i n thyroi dweigh t (Schroede r

an dHogan , 1981 ; Stou t

an dJohnson , 1987 ; Stou t an dRucker , 1990) . Therefore , measurement s o n th e thyroi dstimulatio n hormon e woul dno t provid e additiona l informatio no n th e potentia l endocrin e activit y o f glyphosate . Furthermore , thyroi dweigh t ma y b e considere don e o f

th e mos t sensitiv e parameter s i n respons e t o xenobioti c disturbance s (Wilso ne t

al. , 1996) , a s thyroi dhormone s ma y equilibrat e t o norma l seru mlevel s du e t o homeostasi s i n repea t

dos e studies . 3.3. 9 Thyroxin e (T<t ) Althoug halteratio n thyroi dhormon e level s ar e indicativ e o f athyroid-relate deffect , i t i s insufficien t t o determin e whic hmod e o r mechanis mo f

actio n i s relate dt o th e response . An y chemica l tha t alter s circulatin g thyroi dhormon e sufficientl y t o alte r TS H secretio n wil l resul t

i nhistopathologica l change s i nth e thyroi dglands . Jus t lik e fo r a nalteratio n i nthyroi dstimulatin g hormone , th e effect s correlatio n matri x o f O'Conno r

e t al . (2002 ) (Tabl e 4 ) fo r

th e femal e puberta l assay note s tha t adecreas e i n thyroxin e level s i s supportin g evidenc e fo r thyroi dactiv e compounds , alon g wit hadecreas e i n bod y weigh t a t vagina l openin g an dovar y weight , an da n increas e i n thyroi d weight . A s discusse di n Sectio n 3.3.7 , histopatholog y o f th e thyroi di n severa l studie s i s diagnosti c o f

n o effect s o f

glyphosat e o n th e thyroi dstructur e an dfunction . Furthermore , a s discusse di n Sectio n 3.3.8 , severa l studie s als o sho w non e o f th e othe r associate dindicator s fo r a thyroi dactiv e compound . Dat ao n thyroxin e level s would , therefore , als o provid e n o additiona l informatio no n th e endocrin e modulatin g activit y o f glyphosat e beyon dwha t

i s alread y provide d b y existin g histopatholog y data . A s discusse dabove , thyroi dweigh t ma y b e considere don e o f th e mos t sensitiv e parameter s i n respons e t o xenobioti c disturbance s (Wilso n e t al. , 1996 ) a s 4 4thyroi dhormone s ma y equilibrat e t o norma l seru mlevel s du e t o homeostasi s i n repea t dos e studies . 3.3.1 0 Kidne y Weigh t

an dHistopatholog y Thre e multigenerationa l ra t reproductio n studie s alread y conducte do n glyphosat e showe dn o differenc e i n th e absolut e o r relativ e weigh t

o f th e kidney s o f

th e contro l an dtreate dtes t subjects , a s wel l a s n o microscopi c effects . Th e multigenerationa l reproductio n stud y o n rat s conducte db y Dhins ae t al . (2007 ) note dF O female s treate dwit h15,00 0 pp mdisplaye da n increas e i n kidne y weights , bot habsolut e (pO.OOl ) an drelativ e t o termina l bodyweigh t (p<0.01 ) an dF O female s treate dwit h5,00 0 pp m showe dstatisticall y significan t increase s i nabsolut e kidne y weights . Thes e finding s wer e no t note di n th e F O an dF l male s an dF l generatio n female s an dwere , therefore , considere d unrelate dt o treatment . Th e multigenerationa l reproductio n stud y o n rat s conducte db y Moxo n (2000 ) showe dn o differenc e i nth e absolut e an drelativ e kidne y weight s o f F O an dF1 A adul t

female s betwee n th e contro l an ddos e groups , an dn o tes t relate dmicroscopi c kidne y finding s wer e note dfo r th e F O an dF l females . Schroede r an dHoga n (1981 ) sa w n o difference s i nabsolut e o r relativ e kidne y weight s o f FO , Fi b an dF2 b adul t

female s an dth e F3 b offsprin g betwee n th e contro l an ddos e group s i n th e three-generatio n ra t

reproductio n study . 3.3.1 1 Liver

Weigh t Ther e wa s n o chang e i n th e absolut e o r relativ e weigh t

o f th e live r o f th e F O an dF l female s i n th e ra t reproductio n stud y conducte do n glyphosat e b y Moxo n (2000) . N o difference s i nabsolut e o r relativ e live r

weight s o f

F O an dFi b adul t

females , an do f

F3 b offsprin g wer e note dbetwee n control s an ddos e group s i n th e three-generatio n ra t reproductio n stud y conducte do n glyphosat e b y Schroede r an dHoga n (1981) . Th e absolut e live r weight s o f th e F2 b female s wer e lowe r tha n control s i n anon-dose-respons e manner . Thi s decrease d relativ e live r

weigh t wa s attributabl e t o slightl y highe r absolut e live r

weight s i nth e contro l group . Dhins ae t al . (2007) , however , note dF O female s treate dwit h15,00 0 pp mdisplaye dstatisticall y significan t

increase s i n live r

weights , bot habsolut e an drelativ e t o termina l bodyweigh t (pO.OOl) . A n increas e i n live r weigh t wa s als o note dfo r

F l female s treate dwit h15,00 0 pp m (absolute : p<0.05 , relative : p<0.01) . 3.3.1 2 Adrenal s Weigh t Th e ra t reproductio n studie s o n glyphosat e conducte db y Moxo n (2000 ) an dDhins ae t al . (2007 ) foun dn o differenc e i n th e absolut e o r relativ e weight s o f

th e adrena l glan dan dn o tes t ite m relate dmicroscopi c finding s i nth e glan do f F O an dF l females . Schroede r an dHoga n (1981 ) foun dn o differenc e i n th e absolut e an drelativ e weight s o f th e adrena l glan dbetwee n contro l an ddos e group s o f F O an dF2 b adul t

female s an dF3 b offspring . Slightl y lowe r

absolut e an drelativ e weight s o f th e adrena l glan di n F2 b female s a t th e mid-dos e leve l wer e considere dincidental , a s i t wa s no t see n a t th e hig hdos e leve l o r

note di n th e offspring . 4 53.3.1 3 Pituitar y Weigh t

N o difference s i n th e absolut e an drelativ e weight s o f th e pituitar y wer e note di n tw o ra t reproductio n studie s conducte do n glyphosate , no r wer e ther e an y treatmen t relate deffect s note d upo nmicroscopi c examinatio n o f th e gland . I nth e multigenerationa l studie s conducte db y Moxo n (2000 ) an dDhins ae t al . (2007) , ther e wer e n o effect s o f

glyphosat e o n th e absolut e o r relativ e weight s o f

th e pituitar y o f

th e F O an dF l parents . Importantly , n o treatmen t relate dmicroscopi c finding s wer e detecte di n th e pituitary , whic hsynthesize s an dsecrete s importan t endocrin e hormones , suc ha s TSH , FSH , an dLH , o f th e F O an dF l parents . N o difference s i nabsolut e an drelativ e pituitar y weight s wer e note di nth e FO , Fi b an dF2 b adul t female s an dF3 b offsprin g relativ e t o control s i n th e three-generatio n ra t reproductio n stud y conducte db y Schroede r

an dHoga n (1981) . 3.3.1 4 Clinical Chemistry I nth e 90-day dietar y stud y i n rat s (Stou t an dJohnson , 1987) , som e smal l change s i n clinica l chemistr y value s tha t

wer e no t dose-relate dwer e noted . Afte r carefu l consideration , thes e change s i n clinica l chemistr y value s wer e considere dt o b e eithe r unrelate dt o treatmen t

o r biologicall y insignificant . Stou t an dRucke r

(1990 ) measure dclinica l chemistr y parameter s a t month s 6 , 1 2 (interi m sacrifice) , 18 , an d2 4 (stud y termination) . Ther e wer e variou s change s i n hematolog y an dseru m chemistr y parameter s noted ; however , th e change s wer e no t consistentl y note da t

multipl e tim e points , wer e withi n historica l contro l ranges , smal l i nmagnitude , and/o r di dno t occu r i nadose

relate dmanner . The y were , therefore , considere dt o b e eithe r unrelate dt o treatmen t

o r lexicologicall y insignificant . 3.3.1 5 Conclusio n - Femal e Puberta l Assay Th e lac k o f potentia l fo r

glyphosat e t o interac t

wit hth e femal e endocrin e syste mha s alread y bee ncomprehensivel y addresse di nfou r multigenerationa l ra t

reproductio n studie s conducte do n glyphosate , wit hadditiona l weight-of-evidenc e provide db y subchroni c an dchroni c toxicit y studie s o n glyphosate . Th e EDS PTie r 1 assay , "Puberta l Developmen t an dThyroi dFunctio n i n Intac t Juvenil e /

Peripuberta l Femal e Rats, " i s capabl e o f detectin g estrogenic/anti-estrogeni c activit y o f chemicals , o r

agent s tha t

alte r puberta l development , vi achange s i n steroidogenesi s o r hypothalamic-pituitar y regulatio n o f th e ovar y an dthyroi dhomeostasis . Th e ke y endpoint s evaluate di nthi s stud y ar e als o evaluate di nth e multigenerationa l reproduction , subchroni c and/o r chroni c studies . A summar y o f th e finding s fro mthes e studie s a s the y relat e t o th e endpoint s evaluate di n th e Femal e Puberta l Assa y i s presente di n th e Tabl e 5 .



### 4 6Tabl e 5 : Summar y o f Finding s Relativ e t o th e Femal e Puberta l Assay fo r Glyphosat e En dPoin t Ag e at V O Bod y Weigh t

a t V O Uteru s Estru s Cyclicit y

Ovarie s Thyroi d Kidne y Live r

Adrena l

Pituitar y Clinica l Chemistr y Observation s ( N o chang e i n ag e N o chang e i n bod y weigh t N o chang e i n absolut e o r relativ e weigh t Sligh t margina l change s i nnumbe r an d mea ncycl e lengt ha t

th e hig hdos e leve l (10,00 0 ppm) ; inconsisten t acros s generations ; considere dno t treatmen t relate d N o chang e i n absolut e o r relativ e weigh t n/ a N o chang e i n absolut e o r relativ e weigh t N o differenc e i n absolut e o r relativ e weigh t i nF O an dFi b adult s an dF3 b offspring . Sligh t decreas e i nrelativ e weigh t i nth e F2 b female s attribute dt o slightl y highe r weigh t i nth e contro l group ; non-dose

relate drespons e N o differenc e i n absolut e o r relativ e weigh t i non e study , an donl y slightl y lowe r i n mid-dos e leve l o f

another ; considere d incidenta l N o chang e i n absolut e o r relativ e weigh t Smal l change s i nsom e values ; no t considere dt o treatmen t

relate do r biologicall y significan t Microscopi c Evaluatio n n/ a n/ a n o treatmen t

relate deffect s

n o treatmen t

relate deffect s n o treatmen t

relate deffect s n o treatmen t

relate deffect s n/ a

n o treatmen t

relate deffect s

n o treatmen t

relate deffect s



### Th e result s fro mth e multigenerationa l studies , whic hdemonstrat e n o effect s o npuberta l developmen t

an dthyroi dfunctio ni nth e intac t juvenile/peripuberta l femal e rats , offe r compellin g evidenc e tha t

glyphosat e doe s no t

interac t wit hth e endocrin e system . T o requir e furthe r testin g o nanimal s vi ath e Femal e Puberta l assay t o evaluat e th e endocrin e interactio npotentia l o f glyphosat e woul dprovid e n o ne w evidenc e an db e clearl y redundant . 3, 4 Puberta l Developmen t an dThyroi dFunctio n i n Intac t Juvenil e / Peripuberta l Mal e Rat s (OPPT S 890,1500 ) Th e mal e puberta l assay measure s discret e endpoint s t o ascertai neffect s o npuberta l developmen t an dthyroi dfunctio ni nth e intac t juvenile/peripuberta l mal e rat . Thes e endpoint s includ e developmenta l milestones , orga nweight s an dhistopathology , hormon e seru mlevel s an dclinica l chemistr y parameters . Al l o f thes e endpoint s ar e eithe r

specificall y addresse do r

capture di n downstrea mapica l endpoint s i nmor e comprehensiv e i nviv o studies , suc ha s multigenerational , developmenta l toxicit y studies , subchroni c toxicit y an dchroni c toxicit y studies . Th e multigenerationa l reproductio nstudy , whic hi s ake y Tie r 2 EDS Passay , provide s functiona l equivalenc e t o OPPT S 890.150 0 an drender s th e mal e puberta l assay redundan t (O'Conno r

e t al. , 2002) .

4 7A mor e complet e discussio n o f OSR I relate dt o th e mal e puberta l assay i s presente db y endpoin t i nth e followin g subsections . Man y o f th e finding s ar e discusse di n relatio n t o th e effect s matri x i nTabl e 6 . Tabl e 6 : Correlatio n o f

Effect s i n th e Mal e Puberta l Assay (Adaptedfro mO'Conno r e t al. , 2002 , Tabl e 5 ) Endocrin e Activit y Stron g estroge n recepto r agonis t Wea k estroge n recepto r agonis t Androge n recepto r antagonis t Testosteron e biosynthesi s inhibito r 5-areductas e biosynthesi s inhibito r Aromatas e biosynthesi s inhibito r Direc t thyroi d antagonis t Indirec t thyroi d antagonis t D 2 agonis t D 2 antagonis t Neuroendocrin e mechanis m Ag e

a t PP S 

### T -/t T -/t

-

- t

- t

- T BW

t a t PP S 

### -/T

-

-

- 1 4

-

- 4 1 Teste s w t 1 -/ I -/I/ t

- -/I

- 

### 4 1

-

-

- Epidid . W t 1 -/ i 1 1 1

- 1

-

-

-

- P w t 1 -/I -/ | I 

### 4 -/ t

^ 1

- 1 

## s v w t 

### 4 4 4 4 4 -/ t

4 4

-

- T 

### T -/T 4 -/ t

- 4

-

-

-

- D H T

-

-



### 4

- 4

-

-

- Thy . W t

-

-

- 

### T t

- 4 TS H 

### 4

T

-

-

- T4



### 4 4 4

- PP S = preputialseparation ; BW t = body weight ; Epidid . = epodidymides ; P= prostate ; S V = semina l vesicles ; T = testosterone ; DH T = dihydrotestosterone ; Thy . = thyroid ; TS H = thyroi dstimulatin g hormone ; T 4 = thyroxine , D 2 = dopamin e receptor . 3.4. 1 Growth , Bod y Weigh t Ther e wa s n o advers e effec t o nbodyweigh t

chang e fo r

male s throughou t

th e treatmen t

perio di nth e most recen t multigenerationa l stud y i nrat s (Dhins ae t al. , 2007) . I nth e multigenerationa l ra t reproductio n stud y conducte db y Moxo n (2000) , reduction s i n bod y weigh t relate dt o th e tes t

substanc e wer e limite dt o F1 A male s a t da y 29postpartu mfro mth e 10,00 0 pp mdietar y dos e grou p a s aresul t o f reduce dfoo dconsumptio n du e t o reduce dfoo d palatability . However , n o effec t o n pu p weigh t

a t birt hfo r F l A an dF2 A litter s wer e noted . I nth e two-generatio n ra t reproductio n stud y o f

glyphosat e conducte db y Reyn a(1990) , reduce d bod y weight s an dsof

t stool s wer e note di n adult s i n th e 30,00 0 pp m(hig hdose ) dietar y dos e



### 4 8group , an dbod y weight s o f pup s decrease ddurin g th e las t wee k o f lactation . I n th e 10,00 0 pp m (mid-dose ) group , smalle r decrease s i n bod y weigh t wer e note dan donl y o n lactatio n da y 21 . Thes e bod y weigh t

effect s toward s th e en do f lactatio n occurre dwhe n pup s bega n t o supplemen t thei r mil k intak e wit htreate dfood . Th e ingestio n o f availabl e groun dra t

cho w begin s afte r

th e animals ' eye s ope n (generall y da y 13 ) an da s the y begi n t o explor e thei r cage denvironment . Therefore , th e bod y weigh t decrease s observe di n th e mid

- an dhigh-dos e pup s o n lactatio n da y 2 1 wer e considere dt o b e primaril y du e t o th e consumptio n o f treate ddie t

b y th e offsprin g an d no t du e t o an y endocrin e effects . I nth e three-generatio n ra t reproductio n stud y o f

Schroede r

an dHoga n (1981) , n o treatment relate deffect s wer e eviden t i nadul t mortality , bod y weigh t

an dfoo dconsumptio n dat a(growt h an dres t periods) , o r

i nth e in-lif e physica l observatio n dat athroughou t th e stud y fo r

th e FO , F l an dF 2 generations . 3.4. 2 Ag e at

Preputia l Separatio n I nDhins ae t

al . (2007) , th e mea n F l da y o f preputia l separatio n o f

th e prepuc e fro mth e glan s peni s wa s statisticall y highe r fo r th e 15,00 0 pp mhig hdos e tha n th e controls , bein g recorde da s 45. 9 day s an d43. 0 days , respectively . Thi s hig hdietar y dos e substantiall y exceed s th e EP A limi t dos e o f 1,00 0 mg/kg/da y fo r

repea t dos e studie s fo r

th e lactatin g F O female s (lactatio n day s 1-4 , 174 0 mg/kg/day ; lactatio n day s 4-7 , 237 5 mg/kg/day ; lactatio n day s 7-14 , 274 9 mg/kg/day ) an dF l pup s u p t o sexua l maturit y (pre-pairin g wee k 1 , 212 7 mg/kd/da y t o pre-pairin g wee k 5 , 127 8 mg/kg/day) . I nadditio n t o substantiall y exceedin g th e limi t dos e i nth e maturin g pups , n o concomitan t

endocrine-relate dresponse s associate dwit hincrease dag e a t PPs , suc ha s decrease d weigh t o f th e testes , epididymides , prostat e o r semina l vesicle s wer e note di n th e hig hdos e F l males , supportin g thi s findin g wa s no t endocrin e mediated . N o effec t o n th e da y o f

mea n ag e whe n preputia l separatio n wa s note di n th e F l mal e parent s i n th e multigenerationa l ra t

reproductio n studie s (Moxon , 2000 ) a t dietar y dose s u p t o 10,00 0 ppm , eve n thoug hth e dose s t o thes e youn g F l mal e rat s exceede dth e limi t dos e o f 1,00 0 mg/kg/da y (u p t o 1,80 0 mg/kg/day ) ove r th e firs t si x week s o f

th e pre-matin g period . 3.4. 3 Body Weigh t

at Preputia l Separatio n I nth e multigenerationa l ra t reproductio n stud y b y Moxo n (2000) , initia l bod y weight s wer e recorde dfo r

F l male s a t

selectio n o n da y 29postpartu man dthe n agai n a t weekl y interval s throug hth e pre-matin g period . Mea n bod y weight s o f F l male s a t

da y 2 9 pos t

partu mwer e lowe r i n compariso n t o th e contro l group . Adjuste dbod y weight s fo r week s 2 an d3 o f

th e F l pre-matin g perio dnote dastatisticall y significan t ( p < 0.05 ) lowe r

bod y weigh t

fo r th e hig hdos e males . Therefore , i t

i s assume dtha t

hig hdos e mea n mal e bod y weight s a t th e da y o f

preputia l separatio n wa s lowe r

( p < 0.05 ) tha n tha t

o f th e contro l F l males . Th e non-endocrin e mediate dincreas e i nag e a t preputia l separatio n i nF l hig hdos e male s i nDhins a e t al . (2007) , resulte di nstatisticall y highe r mea n bodyweight s a t

th e tim e o f

sexua l maturatio n fo r th e 15,00 0 pp mdos e grou p du e t o thes e rat s being , o naverage , 2. 9 day s olde r tha n th e contro l grou p a t sexua l maturing . Referrin g bac k

t o th e effect s correlatio n matri x o f O'Conno r

e t al . (2002 ) (Tabl e 6) , adecreas e i n bod y weigh t

a t preputia l separatio n i s a n indicato r o f (i ) aromatas e inhibition , whe n accompanie d b y prostat e an dsemina l vesicl e weigh t increases ; (ii ) direc t

actin g thyroi dantagonism , whe n accompanie db y teste s an depididymide s weigh t decrease s an dthyroi dweigh t decrease ; (iii ) D 2 4 9recepto r antagonism , whe n accompanie db y thyroi dweigh t decrease ; o r

(iv ) neuroendocrin e mechanism , whe n accompanie db y prostat e weigh t decrease . A n increas e bod y weigh t a t preputia l separatio n i s a n indicato r o f aweak estroge n recepto r

agonist . A s discusse dfurthe r i n th e followin g sections , n o chang e i n weigh t o f th e prostate , semina l vesicles , testes , epidiymide s an dthyroi dbetwee n th e contro l an dtreate dgroup s wa s see n i n th e thre e ra t reproductio n studie s o n glyphosat e (Dhins ae t al. , 2007 ; Moxon , 2000 ; Schroede r an dHogan , 1981 ; Reyna , 1990) ; therefore , th e weigh t

o f evidenc e strongl y suggest s tha t th e decrease dmea n bod y weigh t a t preputia l separatio n o f

th e F l male s a t

10,00 0 pp m(Moxon , 2000 ) an dth e increase dmea n bod y weigh t a t preputia l separatio n o f

th e F l male s a t

15,00 0 pp m(Dhins ae t

al. , 2007 ) wer e no t relate dt o a n endocrine-mediate dpathway . 3.4. 4 Teste s Weigh t

an dHistopatholog y N o differenc e wa s note di n th e absolut e o r relativ e weigh t

o f th e teste s o f

th e F O o r F l parent s fro mth e contro l an ddos e group s i n th e multigenerationa l ra t reproductio n studie s conducte db y Moxo n (2000 ) an dDhins ae t al. , (2007) , o r

o f th e FO , Fi b an dF2 b adul t male s an dF3 b weanling s i n th e three-generatio n rat e reproductio n stud y conducte db y Schroede r an dHoga n (1981) . Microscopi c examinatio n note dn o treatment-relate deffect s o n th e testes . I nth e two-generatio n ra t reproductio n stud y o n glyphosat e conducte db y Reyn a(1990) , a statisticall y significan t

decreas e i n termina l bod y weight s wer e note dfo r bothsexe s a t

th e highes t exposur e leve l teste d(30,00 0 ppm) , whic hexceede dth e maximu mtolerate ddose . Th e onl y statisticall y significan t orga n weigh t

change , however , wa s asligh t increas e i n mea n testes to-body-weigh t

rati o (no t

mea n absolut e weight ) o f th e Fl aadul t

i n th e hig hdos e grou p an dthi s findin g wa s attribute dt o alowe r mea n termina l bod y weigh t o f th e Fl amal e a t

thi s highes t

dose . N o difference s wer e note di n th e mea n absolut e weight s o f th e teste s o r

th e relativ e weight s o f th e F O male s a t

al l dose s o r

i n th e Fl amale s a t

th e low

- an dmid-doses . Microscopi c examinatio n o f

th e teste s fo r

F O an dFl aparent s fro mth e contro l an dhigh-dos e group s note dn o treatment-relate deffects . Mating , pregnanc y an dfertilit y indice s wer e no t affecte di n eithe r

th e F O o r F l animals . 3.4. 5 Epididymide s Weigh t an dHistopatholog y N o differenc e i n th e absolut e o r relativ e weight s o f

th e epididymide s weight s o f th e F O o r F l parent s fro mth e contro l an dtreate dgroup s wer e note di n th e ra t reproductio n studie s conducte d b y Moxo n (2000 ) an dDhins ae t al. , (2007) . Microscopi c examinatio n o f th e epididymide s o f

th e F O an dF l parent s fro mth e contro l an dhigh-dos e grou p note dn o treatment-relate deffects . Mating , pregnanc y an dfertilit y indice s wer e no t affecte di n eithe r

th e F O o r Fl aanimals . 3.4. 6 Prostat e Weigh t N o differenc e i n th e absolut e o r relativ e weigh t

o f th e prostat e glan do f

th e F O o r F l parent s wa s note dbetwee n th e contro l an dhigh-dos e group s i n th e multigenerationa l ra t reproductio n studie s conducte db y Moxo n (2000 ) an dDhins ae t al. , (2007) . I nth e three-generatio n ra t reproductio n stud y conducte db y Schroede r an dHoga n (1981) , microscopi c examinatio n o f th e prostat e glan do f

th e FO , Fi b an dF2 b parent s an dth e F3 b offsprin g fro mth e contro l an dhigh-dos e group s note dn o treatment-relate deffects .

5 03.4. 7 Semina l Vesicle s Weigh t

N o differenc e i n th e absolut e o r relativ e weigh t

o f th e semina l vesicle s o f

th e F O o r F l parent s wa s note dbetwee n th e contro l an dtreate dgroup s i n th e multigenerationa l ra t reproductio n studie s conducte db y Moxo n (2000 ) an dDhins ae t al. , (2007) . Microscopi c examinatio n o f th e semina l vesicle s o f

th e F O an dF l aparent s fro mth e contro l an d hig hdos e group s o f

th e two-generatio n ra t

reproductio n stud y conducte db y Reyn a(1990 ) note d n o treatmen t

relate deffects . 3.4. 8 Testosteron e Th e effect s correlatio n matri x fo r th e mal e puberta l assa y propose db y O'Conno r (2002 ) (Tabl e 6 ) note s tha t

fo r anumbe r

o f endocrin e effects , an y difference s i ntestosteron e level s betwee n control s an dtes t

group s i s als o reflecte di n change s i n ag e a t preputia l separatio n an dweight s o f testes , epididymides , prostat e an dsemina l vesicles . However , a s discusse dpreviousl y i nthi s Section , dat afro mth e multigenerationa l ra t reproductio n studie s conducte db y Moxo n (2000 ) an dDhins ae t

al. , (2007 ) sho w tha t

change s i n thes e endpoint s betwee n th e control s an dtreate d group s wer e no t

seen . Therefore , functionall y equivalen t informatio n fo r

thi s endpoin t

i nth e mal e puberta l assay i s provide dwit hth e curren t comprehensiv e i n viv o dat ase t o n glyphosate . 3.4. 9 Thyroi dweigh t

an dHistopatholog y Althoug hsom e thyroi dweigh t effect s wer e note di nth e multigenerationa l stud y b y Dhins ae t al . (2007) , th e finding s wer e considere dcontradictor y an dunrelate dt o treatment . Fo r th e F O female s i n th e mid-dos e treatmen t o f 5,00 0 ppm , th e mea n absolut e thyroi dweigh t wa s greate r tha n th e controls , whil e fo r th e F O mal e i nth e high

- an dmid-dose s o f 15,00 0 pp man d5,00 0 ppm , th e mea n absolut e thyroi dweigh t wa s lowe r

tha n th e controls . A s previousl y stated , thes e finding s wer e considere dcontradictor y an dunrelate dt o treatment . Furthe r evidenc e a s t o glyphosate' s lac k o f effect s o nth e thyroi di s foun di nth e finding s o f microscopi c examinatio n o f th e gland . I f

histologica l lesion s ar e no t observe di n th e thyroi d gland , the n th e chemica l i s considere dt o b e thyroi dinactiv e (OPPT S 890.1110) , an d microscopi c examinatio n o f th e thyroi di n th e three-generatio n ra t reproductio n stud y an dth e subchroni c an dchroni c toxicit y studie s o n glyphosat e note dn o treatment-relate deffects . I nth e three-generatio n ra t reproductio n stud y conducte db y Schroede r an dHoga n (1981) , microscopi c examinatio n o f th e thyroi dan dparathyroi do f th e FO , Fi b an dF2 b parent s an dF3 b offsprin g fro mcontro l an dhigh-dos e group s note dn o treatment-relate deffects . Th e sam e conclusio n o f

n o treatment-relate deffect s wa s reache dfollowin g microscopi c examinatio no f

th e thyroi do f

th e contro l an dhigh-dos e group s followin g 9 0 day s o f ora l dosin g i nth e subchroni c toxicit y stud y conducte db y Stou t an dJohnso n (1987 ) an da t 1 2 an d2 4 month s i nth e chroni c toxicit y stud y o n glyphosat e conducte db y Stou t an dRucke r

(1990) . 3.4.1 0 Thyroi dSimulatio n Hormon e (TSH ) Th e effect s correlatio n matri x o f th e mal e puberta l assa y (Tabl e 6 ) indicate s tha t fo r anumbe r

o f endocrin e effects , difference s i n TS H level s betwee n control s an dtes t group s ma y als o b e reflecte di n change s i n ag e an dweigh t a t preputia l separatio n an dweight s o f testes , epididymides , prostate , semina l vesicle s an dthyroid . However , a s discusse dpreviousl y i n thi s Section , dat afro mth e multigenerationa l ra t reproductio n studie s conducte db y Moxo n (2000 ) an dDhins ae t

al . (2007 ) sho w tha t

change s i n thes e se x orga n weight s betwee n th e control s an d

5 1treate dgroup s wer e no t seen . Therefore , functionall y equivalen t informatio n fo r

thi s endpoin t

i n th e mal e puberta l assa y i s provide dwit hth e curren t comprehensiv e i n viv o dat ase t o n glyphosate . 3.4.1 1 Thyroxin e (T4 ) Th e effect s correlatio n matri x o f th e mal e puberta l assay (Tabl e 6 ) indicate s tha t fo r anumbe r

o f endocrin e effects , adecreas e i n thyroxin e level s o f treate dgroup s compare dt o th e control s ma y als o b e reflecte di n change s i n ag e an dweigh t a t preputia l separatio n an dweight s o f teste s epididymides , prostate , semina l vesicle s an dthyroi dfunction . However , a s discusse dpreviousl y i nthi s Section , dat afro mth e multigenerationa l ra t reproductio n studie s conducte db y Moxo n (2000 ) an dDhins ae t

al . (2007 ) sho w tha t

change s i n thes e endpoint s betwee n th e control s an d treate dgroup s wer e no t

seen . Therefore , functionall y equivalen t informatio n fo r

thi s endpoin t

i n th e mal e puberta l assay i s provide dwit hth e curren t comprehensiv e i n viv o dat ase t o n glyphosate . 3.4.1 2 Kidne y Weigh t an dHistopatholog y N o effect s o n mal e kidne y weight s wer e note di n th e multigenerationa l ra t stud y b y Dhins ae t

al . (2007) . Also , i n th e three-generatio n ra t reproductio n stud y conducte db y Schroede r an dHoga n (1981 ) n o difference s i n absolut e o r relativ e weigh t

o f th e kidney s o f

th e FO , Fi b an dF2 b adul t male s an dF3 b offsprin g o f th e contro l an dtreate dgroup s wer e noted . I nth e multigenerationa l ra t reproductio n stud y conducte db y Moxo n (2000) , astatisticall y significan t

( p < 0.01 ) increas e i n th e mea n kidne y weigh t adjuste dfo r

bod y weigh t

o f th e F O mal e ove r th e control s wa s note da t th e hig hdos e onl y an dno t i n th e F l males . 3.4.1 3 Live r

Weigh t N o effect s o n mal e live r

weight s wer e note di n th e multigenerationa l ra t stud y b y Dhins ae t

al . (2007) . Als o i n th e three-generatio n ra t reproductio n stud y conducte db y Schroede r an dHoga n (1981 ) n o difference s i n th e absolut e an drelativ e live r weight s o f

th e FO , Fi b an dF2 b adul t male s an dF3 b offsprin g i n th e contro l an dtreate dgroup s wer e noted . A statisticall y significan t ( p < 0.01 ) increas e i n th e mea n live r weigh t adjuste dfo r

bod y weigh t o f th e F O mal e ove r

control s a t

th e hig hdos e leve l wa s see n i n th e multigenerationa l ra t reproductio n stud y conducte db y Moxo n (2000) , an dstatisticall y significan t ( p < 0.05 ) decreas e i nth e absolut e live r

weigh t o f th e F l mal e a t

th e hig hdos e leve l relativ e t o th e controls . I t wa s note di n th e stud y repor t

tha t th e high-dos e male s exhibite dtes t substance-relate dreduction s i n bod y weigh t

an dfoo dconsumption . These inconsisten t live r weigh t finding s wer e considere d incidenta l an dunrelate dt o endocrin e effects . 3.4.1 4 Adrenal s Weigh t N o difference s i n th e absolut e o r relativ e weight s o f

th e adrena l gland s wer e note di n thre e ra t reproductio n studie s o n glyphosate . I nth e multigenerationa l ra t reproductio n studie s conducte db y Moxo n (2000 ) an dDhins ae t al . (2007) , ther e wer e n o difference s i n th e absolut e an drelativ e weight s o f th e adrena l glan di n th e F O an dF l males . I n th e three-generatio n ra t reproductio n stud y conducte db y Schroede r an dHoga n (1981) , n o differenc e i n th e absolut e an drelativ e weigh t o f adrena l glan do f

th e FO , Fl aan dF2 b adul t male s an dF3 b offsprin g i n th e contro l an dtreate dgroup s wer e noted . 5 2N o treatment-relate dmicroscopi c finding s wer e detecte di n th e adrena l gland s o f th e F O an dF l male s i n th e Moxo n (2000 ) ra t reproductio n study . Dhins ae t al . (2007) , however , note dtha t whil e ther e wer e n o treatment-relate dhistopathologica l change s i n th e adrena l gland s o f F O generatio nanimals , treatment-relate dchange s i n th e F l generatio n mal e adrena l gland s wer e note da s lowe r

incidence s an dseveritie s o f cortica l vacuolatio n o f

th e adrena l gland s whe n compare dt o controls , bu t thes e finding s wer e no t

considere dt o b e a n advers e finding . 3.4.1 5 Pituitary Weigh t N o difference s i n th e absolut e o r relativ e weight s o f

th e pituitar y gland s an dn o treatment-relate d microscopi c finding s wer e note di n thre e ra t reproductio n studie s o n glyphosate . I nth e multigenerationa l ra t reproductio n studie s conducte db y Moxo n (2000 ) an dDhins ae t al . (2007) , ther e wer e n o difference s i n th e absolut e o r relativ e weight s o f

th e pituitar y glan do f

th e F O an dF l parent s fro mth e contro l an dtreate dgroups . Als o fo r th e F O an dF l parents , n o treatment-relate dmicroscopi c finding s wer e detectedi nth e pituitary , whic hsynthesize s an d secrete s importan t

endocrin e hormones , suc ha s TSH , FSH , an dLH . I nth e three-generatio n ra t reproductio n stud y conducte db y Schroede r an dHoga n (1981) , n o difference s i n th e absolut e an drelativ e weigh t o f th e pituitar y gland s an dn o treatment-relate d microscopi c finding s o f

th e pituitar y gland s i n th e FO , Fi b an dF2 b adul t male s an dF3 b offsprin g wer e noted . 3.4.1 6 Clinica l Chemistr y I na90-da y dietar y stud y conducte db y Stou t an dJohnso n (1987) , som e change s i n clinica l chemistr y value s wer e noted ; however , thes e change s wer e smal l and/o r no t dose-related , and , therefore , considere dt o b e unrelate dt o treatmen t o r biologicall y insignificant . I nth e chroni c toxicit y stud y conducte db y Stou t an dRucke r

(1990) , clinica l chemistr y parameter s wer e measure da t month s 6 , 1 2 (interi msacrifice) , 18 , an d2 4 (stud y termination) . Ther e wer e variou s change s i n hematolog y an dseru mchemistr y parameter s considere dt o b e eithe r unrelate dt o treatmen t

o r lexicologicall y insignificant . 3.4.1 7 Conclusio n - Mal e Puberta l Assay Th e lac k o f potentia l fo r

glyphosat e t o interac t

wit hth e mal e endocrin e systemha s alread y bee n comprehensivel y addresse di nfou r multigenerationa l ra t

reproductio n studie s conducte do n glyphosate , wit hadditiona l weight-of-evidenc e provide db y subchroni c an dchroni c studie s o n glyphosate . Th e EDS PTie r 1 assay , "Puberta l Developmen t an dThyroi dFunctio n i n Intac t Juvenil e / Peripuberta l Mal e Rats, " i s capabl e o f detectin g antithyroid , androgenic , o r antiandrogeni c (androge n recepto r o r steroid-enzyme-mediated ) activit y o f chemicals , o r

agent s tha t alte r puberta l developmen t

vi achange s i ngonadotropins , prolactin , o r hypothalami c function . Al l o f

th e endpoint s evaluate di n thi s EDS Passay ar e als o evaluate di n th e multigenerationa l reproduction , subchroni c and/o r chroni c studies , o r

ca n b e evaluate dbase do n othe r informatio n fro mthes e studies . A summar y o f th e finding s fro mthes e studie s a s the y relat e t o th e endpoint s evaluate di n th e Mal e Puberta l assay i s presente di n Tabl e 7 .

5 3Tabl e 7 : Summar y o f Finding s Relativ e t o th e Mal e Puberta l Assay fo r Glyphosat e En dPoin t Growth , Bod y Weigh t Ag e a t Preputia l Separatio n Bod y Weigh t

a t Preputia l Separatio n

Teste s Weigh t

Epididymide s Weigh t Prostat e Weigh t Semina l Vesicle s Weigh t Testosteron e Thyroi dWeigh t Thyroi dStimulatio n Hormon e (TSH ) Thyroxin e (T4 ) Kidne y Weigh t

Live r Weigh t

Adrena l Weigh t Pituitar y Weigh t Clinica l Chemistr y Observation s Reductio ni nbod y weigh t limite dt o hig hdos e grou p a s aresul t

o f reduce dfoo dconsumptio n du e t o reduce dpalatability , an dno t du e t o endocrin e effect s N o glyphosat e relate dchang e i n ag e a t preputia l separatio nfo r

dose s a t o r belo w th e limi t

dos e o f l,000mg/kg/da y Mea nbod y weigh t

decreas e a t

10,00 0 pp man d increas e a t

15,00 0 ppm . Bot hdose s exceede d th e limi t dos e o f 1,00 0 mg/kg/da y durin g sexua l maturation ; however , n o othe r relate dendocrin e effect s wer e note di n se x orga n weigh t differences , therefor e th e weigh t o f th e evidenc e i s tha t bod y weigh t

change s a t

sexua l maturatio n wer e no t relate dt o endocrin e effect s N o chang e i n absolut e o r relativ e weigh t

i n on e ra t reproductio n study , an donl y asligh t increas e i nrelativ e weigh t

(no t absolut e weight ) o f

th e 30,00 0 pp mtes t

grou p (hig hdose ) du e t o a lowe r mea ntermina l bod y weigh t N o chang e i n absolut e o r relativ e weigh t N o chang e i n absolut e o r relativ e weigh t N o chang e i n absolut e o r relativ e weigh t N o othe r relate dendocrin e effect s observe di n th e multigenerationa l reproductio n studie s n/ a N o othe r relate dendocrin e effect s observe di n th e multigenerationa l reproductio n studie s N o othe r relate dendocrin e effect s observe di n th e multigenerationa l reproductio n studie s Increas e i nrelativ e weigh t onl y i nth e F O mal e a t th e hig hdose ; n o chang e i n th e F l mal e o r i n an y adul t male s o r offsprin g i nth e three

generatio nreproductio n stud y Inconsisten t

finding s (decreas e i nabsolut e an d increas e i nrelativ e weights ) i non e reproductio n stud y wer e considere dincidenta l an dunrelate d t o endocrin e effects ; n o change s i n th e three

generatio nreproductio n stud y i n male s N o change s i n absolut e o r relativ e weigh t N o change s i n absolut e o r relativ e weigh t Smal l change s i n som e values ; no t considere dt o b e treatmen t

relate do r

biologicall y significan t Microscopi c Evaluatio n

n/ a n/ a

N o treatmen t

relate deffect s

N o treatmen t

relate deffect s N o treatmen t

relate deffect s N o treatmen t

relate deffect s n/ a N o treatmen t

relate deffect s n/ a n/ a n/ a

n/ a

N o treatmen t

relate deffect s N o treatmen t

relate deffect s n/ a Th e result s fro mth e multigenerationa l studies , whic hdemonstrat e n o effect s o n puberta l developmen t

an dthyroi dfunctio ni nth e intac t juvenile/peripuberta l mal e rats , offe r compellin g evidenc e tha t

glyphosat e doe s no t

interac t wit hth e endocrin e system . T o requir e furthe r testin g 

### 5 4

### o nanimal s vi ath e Mal e Puberta l assa y t o evaluat e th e endocrin e interactio npotentia l o f

glyphosat e woul dprovid e n o ne w evidenc e an dclearl y b e redundant . 3, 5 Summar y o f

I n Viv o OSR I fo r

Mammalia n Specie s Th e multigenerationa l ra t

reproductio nstudie s conducte db y Moxo n(2000 ) an dDhins ae t al . (2007 ) ar e currentl y propose da s Tie r 2 EDS Pguidelin e studies . These studie s clearl y negat e th e nee dfo r muc ho f th e Tie r 1 screenin g batter y evaluation . Wit hth e preponderanc e o f othe r supportin g dat ai nmultipl e repea t dos e studie s discusse dthroughou t thi s document , th e Tie r

1 screenin g battery add s littl e i f an y informatio nt o th e currentl y availabl e scientifi c bod y o f knowledg e fo r

glyphosat e wit hrespec t

t o it s potentia l t o interac t wit hth e endocrin e system . I nadditio nt o th e "other

scientificall y relevan t

information " presente dhere , th e FAO/WH O 200 4 revie w o f glyphosat e (FAO/WHO , 2005 ) evaluate dfour , 4-wee k studie s i nrats , mic e an ddogs , tw o subchroni c ra t

studies , thre e subchroni c mous e studies , on e 90-da y do g study , thre e 1-yea r do g studies , a1-yea

r chroni c toxicit y stud y i nrats , tw o 2-year chroni c toxicit y studie s i nrats , on e 2-year

mous e study , a nadditiona l 2-generatio nra t reproductio nstud y an dtw o additiona l developmenta l toxicit y studie s o f glyphosat e i nrat s an drabbits . Man y o f thes e studie s wer e als o considere di nth e 200 9 ECETO C documen t "Guidanc e o nIdentifyin g Endocrin e Disruptin g Effects , Technica l Report

106, " i nwhic hglyphosat e i s highlighte da s a nexampl e o f anon endocrin e disruptin g compoun dbase do nth e extensiv e toxicolog y dat aavailable . 

### 4. 0 Non-Mammalia n I n Viv o Assay s 4. 1 Amphibia n Metamorphosi s Assa y (AMA , OPPT S 890,1100 ) Th e amphibia n metamorphosi s scree n wa s include di n th e Tie r 1 screenin g batter y a s a generalize dvertebrat e mode l t o detec t thyroi dsystem-disruptin g compounds . Throug hth e hypothalamus-pituitary-thyroi dglan daxis , thyroi dhormon e regulate s man y processe s i n organisms , includin g development , somati c growth , metabolism , an dreproduction . Metamorphosi s i nanura namphibian s i s awel l studie dmode l fo r thyroi dhormon e functio nan d i s applicabl e an dfunctionall y equivalen t t o informatio ngenerate di nstandar dregulator y studie s wit hothe r vertebrat e classes . Extensiv e scientificall y relevan t informatio no nthyroi dstructur e an dfunctio ni s collecte di nstandar dsubchronic , chroni c mammalia nstudie s an dreproductio n studies . Thi s informatio ni s functionall y equivalen t t o dat acollecte di nth e amphibia n metamorphosi s assay . Additionally , dat ao ngrowt han ddevelopmen t offis han dbird s i nth e fis h ful l life cycl e stud y an davia nreproductio nstud y provid e additiona l relevan t scientifi c informatio nt o addres s th e requiremen t fo r performin g th e amphibia nmetamorphosi s assay . Th e amphibia n thyroi dsyste mi s regulate dan drespond s i n asimilar manne r t o perturbatio n a s tha t

o f othe r tetrapods , includin g mammal s (Fort e t al. , 2007) . Therefore , dat afro mmammalia nassay s ca n b e use dt o mak e arobus t assessmen t

o f potentia l impact s o n thyroi dstructur e an dfunction . Th e commo nendpoin t i nal l thre e Tie r

1 assay s tha t

evaluat e potentia l effect s o nth e thyroi di s thyroi dglan dhistology , whic hscreen s fo r direc t toxicit y a t

th e glan ditself . Developmenta l retardation , i nth e absenc e o f overt toxicity , i s astron g indicato r o f anti-thyroi dactivity , bu t

amphibia ndevelopmenta l stag e analysi s b y itsel f i s no t considere da diagnosti c indicato r

o f anti-thyroida l activit y (OPPT S 890.1100) . I f n o developmenta l delay s o r treatmen t relate dhistologica l lesion s ar e observe di nth e thyroi dglands , the nth e chemica l i s considere dt o b e thyroi dinactiv e (OPPT S 890.1100) . Th e rational e i s that th e thyroi dgland s ar e 5 5unde r

th e influenc e o f

TS H an dan y chemica l tha t alter s circulatin g thyroi dhormon e sufficientl y t o alte r TS H secretio n wil l resul t i nhistopathologica l change s i nth e thyroi dgland s (OPPT S 890.1100) . However , i t

shoul db e emphasize dtha t th e abilit y o f

achemica l t o caus e areductio n i nseru mT 4 b y an y mechanis mwoul ddepriv e th e fetu s i n areproductio n stud y o f essentia l hormone . Thus , an y observe dreductio n i n seru mT 4 woul db e detecte di n amammalia n reproductio nstud y a s a n impac t o n developmenta l endpoint s an dca n b e evaluate di n aweight

of-evidenc e interpretatio n usin g al l o f th e dat afro mth e repea t

dos e studie s tha t

hav e evaluate d thyroi dhistopatholog y and/o r growt han ddevelopment . Whil e afe w differen t

specie s hav e bee n evaluate di nthi s screen , th e Sout hAfrica n clawe dfro g (Xenopu s laevis ) wa s chose n a s th e tes t organism . Metamorphosi s ca n b e induce do r accelerate d b y agonist s o f

th e thyroi dsystem , wherea s anti-thyroida l agent s ca n dela y metamorphosi s (Degit z e t al . 2005 , Opit z e t

al . 2005 , 2006 ; Tietg e e t

al . 2005) . Th e protoco l currentl y i n us e include s a21-da y treatmen t o f tadpoles , wit hevaluation s occurrin g o n Da y 7 an dDa y 21 . Th e tes t i s initiate da t

developmenta l stag e 51 , whic hca n b e reache dapproximatel y 1 4 day s post fertilization . Fou r

concentration s o f

th e tes t substanc e an dadilutio nwate r contro l an dfou r replicat e tank s pe r

concentratio n ar e employe di n aflow-throug hsystem . A t tes t initiation , a tota l o f 2 0 stag e 5 1 Xenopu s laevi s tadpole s ar e place di n eac ho f th e fou r replicat e tank s pe r treatmen t group . Environmenta l condition s o f 2 2 ± 1°C , p H o f

betwee n 6.5-8.0 , an da12:1 2 lightdar k photoperio dar e employed . Fiv e tadpole s fro meac htreatmen t tan k ar e randoml y remove da t

da y 7 fo r evaluatio n o f

we t weight , whol e bod y length , hin dlim b length , an dsnout ven t length . O n Da y 21 , th e remainin g tadpole s ar e evaluate dfo r thes e sam e endpoints . Histologica l evaluatio n o f thyroi dtissu e i n asubsampl e o f fiv e tadpole s fro meac hreplicat e tan k i s als o performe di nthi s scree n i norde r t o lin k an y observe dchange s i nth e rat e o f metamorphosi s t o morphologica l change s i n th e primar y thyroi dsynthesi s organ . N o treatment-relate dthyroi dweigh t effect s wer e note di nth e multigenerationa l stud y b y Dhins ae t al . (2007) . I n th e F O femal e fro mth e mid-dos e treatmen t grou p o f 5,00 0 ppm , th e mea n absolut e thyroi dweigh t

wa s greate r

tha n th e controls , whil e th e mea n absolut e thyroi dweigh t o f th e F O male s fro mth e high

- an dmid-dos e group s o f 15,00 0 pp man d5,00 0 pp mwa s lowe r tha n th e controls . Thes e finding s wer e considere dcontradictor y an dunrelate dt o treatment . Histopatholog y i n athre e generatio n reproductio n stud y (Schroede r an dHogan , 1981), a90-da y subchroni c stud y (Stou t

an dJohnson , 1987 ) an dachroni c ra t stud y (Stou t

an dRucker , 1990 ) di d no t identify an y glyphosat e treatment-relate deffect s t o th e thyroi dglands . Glyphosat e showe dn o evidenc e o f effect s o n growthan ddevelopmen t throug ha n endocrin e mechanis mi nfou r

multigenerationa l studie s i nrat s (Schroede r an dHogan , 1981 ; Reyna , 1990 ; Moxon , 2000 ; Dhins ae t

al. , 2007 ) an dn o evidenc e o f bein g aselectiv e developmenta l toxican t i ndevelopmenta l toxicit y studie s i n rat s (Rodwel l e t al. , 1980a) an drabbit s (Rodwel l e t al. , 1980b) . N o effec t o n growt ho f

th e F O an dF l minnow s wa s observe di n th e fis hfull-lif e cycl e stud y (Figure s 4 an d5 ; EG& GBionomics , 1975) . Thi s dat aprovide s consisten t an dstron g evidenc e o f an o effec t

findin g fo r

glyphosat e o nthyroi dstructur e an dfunction . Additionally , i nfou r avia n reproductio n studies , n o effect s o n growt hwer e observe da t dietar y concentration s o f greate r tha n 100 0 pp mglyphosat e (se e summar y i n sectio n 4.2.4) . 

### 5 6Tota l Lengt h 35 - •g - 30 - E.25 - JJ15 . 

### ] s 10

- o I - 5 0 3 0 day s

6 0 day s 1 1

Glyphosat e (m g a.e./L ) Figur e 4 : Growt ho f

F O fathea dminnow s (Pimephale s promelas ) continuousl y expose dt o glyphosat e fo r

3 0 an d6 0 days . Mean s ar e show n wit hth e standar derro r o f th e mea n (EG& G Bionomics , 1975) .

Tota l Lengt h 25- | 

# | : £15 H ro c



### . 3 10

- | 5 - 0 Glyphosat e (m g a.e./L ) Tota l Weigh t 100- 1 O ) E 75 H c 50 - 5 25 - 

# X

Glyphosat e (m g a.e./L ) Figur e 5 : Growt ho f

F l fathea dminnow s (Pimephalespromelas ) continuousl y expose dt o glyphosat e fo r 3 0 days . Mean s ar e show n wit hth e standar derro r o f th e mea n (EG& GBionomics , 1975) . 4.1. 1 Conclusio n - Amphibia n Metamorphosi s Assay (AMA ) Th e amphibia n metamorphosi s assa y ha s bee n include di n th e Tie r 1 battery a s ageneralize d mode l fo r impact s o nvertebrat e thyroi dfunctio nbecaus e th e amphibia n thyroi dsyste mi s regulate dan drespond s i n asimila r manne r t o perturbatio n a s tha t

o f othe r tetrapods , includin g mammal s (Fort

e t al. , 2007) . Therefore , combine dresult s fro mexistin g glyphosat e studie s provid e sensitiv e an ddiagnosti c informatio n o nthyroi dstructure , function , growthan d developmen t

tha t i s functionall y equivalen t t o tha t generate di nth e amphibia n metamorphosi s assay . Thyroi dhistopatholog y ha s bee n evaluate di n athree-generatio n reproductio n stud y (Schroede r

an dHogan , 1981) , a90-da y subchroni c stud y (Stou t an dJohnson , 1987 ) an da chroni c ra t

stud y (Stou t

an dRucker , 1990) , whic hdi dno t identify an y glyphosat e treatment relate deffect s t o th e thyroi dglands . 

### 5 7Confirmator y informatio nwa s develope dfo r a naquati c vertebrat e i nth e fis hful l life-cycl e stud y tha t show s n o impac t

o n growt han ddevelopment , whic har e sensitiv e measuremen t endpoint s fo r impact s o nthyroi dstructur e an dfunction . Additionally , informatio no f n o measureabl e effect s o nthyroi dfunctio nca nb e evaluate dwit hdat afro mavia nreproductio nstudies . N o effect s o n growt hwer e observe di n fou r avia nreproductio nstudie s tha t teste dglyphosat e dietar y concentration s greate r

tha n100 0 ppm . Tabl e 8 : Summar y o f

Finding s Relativ e t o th e Amphibia nMetamorphosi s Assa y (AMA)fo r

Glyphosat e En dPoint s i n AM A Thyroi dglan dhistopatholog y Growt han ddevelopmen t Functionall y Equivalen t Informatio nfro mExistin g Studie s • Thre e generatio nra t

reproductio nstudy , a90-da y ra t stud y an dachroni c ra t stud y di dno t

identify an y treatment-relate dmicroscopi c finding s o f th e thyroi dglands . • Growt han ddevelopmen t i nra t multigenratio nstudie s includin g developmenta l landmark s demonstrate dn o indicatio no f treatmen t relate d effects . • N o treatmen t

relate deffect s o nF O an dF l growt han ddevelopmen t i nfis h ful l lif e cycl e stud y an dfou r avia nreproductio nstudies . Give nal l o f

thi s availabl e informatio no nglyphosate , th e amphibia nmetamorphosi s screenin g assay doe s no t

offe r an y ne w relevan t

informatio nt o th e toxicolog y dat ase t fo r Tie r 1 screenin g purposes . 4, 2 Fis hShort-ter mReproductio n Assa y (OPPT S 890,1350 ) Informatio nfro msevera l guidelin e studie s fo r glyphosat e provide s dat ao f th e sam e natur e an d qualit y a s dat adevelope db y th e short-ter mfis hreproductio nstudy . Severa l o f thes e studie s hav e bee nreviewe db y EP A i n suppor t o f th e continue dregistratio no f glyphosat e an dprovid e OSRI tha t full y addresse s th e requiremen t fo r th e fis hshort-ter mreproductio nstudy . Additionally , th e potentia l fo r

glyphosat e t o induc e vitellogeni nwa s evaluate di npeer-reviewe dpublications . Th e followin g studie s provid e scientificall y relevan t informatio nequivalen t

t o dat agenerate di nth e fis hshort-ter mreproductio nstudy : 1 . Fis hful l life-cycl e toxicit y wit hth e fathea dminno w (Pimephale s promelas ) (MRI D 00108171) . 2 . Assessin g rainbo w trou t E Ractivatio nan dvitellogeni nproductio n(Petit , 1997 ; Xi e e t al . 2005) . 3 . Fis hbioconcentratio nstud y (MRI D 41228301 ; MRI D 41228302 ) 4 . Avia nreproductio nstudie s (MRI D 00108207 ; MRI D 00111953 ) 5 . Ra t multigenerationa l reproductio nstudie s (MRI D 00081674 ; MRI D 00105995 ; MRI D 41621501 ; Moxo n2000 ; Dhins ae t al. , 2007 ) Th e fis hshort-ter mreproductio nscree n(OPPT S 890.1350 ) i s initiate dwit hmatur e mal e an d femal e fis hwit hove r

a21-da y exposur e period . Th e scree nexamine s abnormalitie s associate d wit hsurvival , reproductiv e behavior , secondar y se x characteristics , gonada l histopathology , an d fecundit y (i.e. , numbe r

o f spawns , numbe r

o f egg s pe r spawn , fertility , an ddevelopmen t o f offspring ) offis hexpose dt o tes t chemical s (Se e Tabl e 1 fo r endpoint s tha t

asses s endocrin e activit y an dacompariso nwit hth e 2-generatio nra t reproductio ntest) . Dat atha t i s functionall y equivalen t

t o th e dat agenerate di n th e fis hshort-ter mreproductio nscree nwit hth e fathea d 5 8minno w (OPPT S 890.1350 ) i s provide db y th e fis hful l life-cycl e stud y (OPPT S 850.1500) . Th e

fis hful l life-cycl e stud y i s th e highes t Tie r offis hecotoxicolog y testin g an dtypicall y performe d wit hth e fathea dminno w (Pimphalespromelas ] a s afreshwate r tes t species . I nth e fis hful l life-cycl e study , th e entir e life-cycl e i s exposed ; therefore , d e fact o th e mos t sensitiv e life-stag e t o an y toxican t (includin g endocrin e activ e compounds ) i s encompasse db y th e tes t design . However , endpoint s tha t ar e potentiall y influence db y endocrin e activity , suc ha s gonada l histopathology , ar e no t typicall y measure di nth e ful l life-cycle . A standar dfis hful l life-cycl e stud y provide s acomparabl e investigatio nt o th e tes t propose di n Tie r

2 testin g fo r

fis h i nth e EDSTP , eve nthoug hacomprehensiv e assessmen t o f th e impact s o f

glyphosat e o n reproductiv e organ s (orga nweigh t an dhistopathology ) ha s alread y bee nevaluate dusin g informatio nfro mth e ra t

multigeneratio nstudie s an dth e result s o f thes e studie s demonstrat e n o treatment-relate dimpac t

o nal l measure dreproductiv e an ddevelopmenta l endpoints . I nth e firs t

o r parenta l generatio n(Fo ) th e followin g endpoint s ar e evaluated : embry o hatchin g success , embry o time-to-hatch , surviva l a t variou s tim e point s afte r

hatch , lengt han dwe t

weigh t a s a n indicato r

o f growt ha t

variou s tim e point s afte r

hatch , an dreproductiv e succes s (fecundity ) a s measure db y numbe r

o f egg s pe r femal e pe r

day , numbe r

o f spawn s an dnumbe r

o f egg s pe r spawn . I n th e secon dgeneratio n(Fi ) th e endpoint s includ e embry o hatchin g success , embry o time-to-hatch , survival , an ddevelopmen t a s measure db y lengt han dwe t weigh t a t specifie dtim e points . I nth e glyphosat e study , fathea dminnow s (F o + FI ) wer e continuousl y expose dt o mea n measure dglyphosat e concentration s o f 0.7 , 2.8 , 7.0 , 13. 0 an d25. 7 mg/ Lfo r 25 5 days . Th e curren t guidanc e i n OPPT S 890.135 0 state s "t o tes t th e highes t

concentratio ntha t

doe s no t resul t i nincrease dmortalit y o r

sign s o f over t morbidit y compare dt o controls ; o r 1/ 3 th e derive d96-h r LC 5 0 wil l serv e a s th e highes t exposur e concentratio ni n th e 21-da y test. " I n astati c 24-hou r acut e stud y ru ni nadvanc e o f th e ful l life-cycl e study , usin g ~1. 5 g fathea dminnows , th e 24

- hou r LCs o o f glyphosat e wa s determine dt o b e 84. 4 mg/Lan dmortalit y wa s show nt o resul t fro m th e lo w p H a t

th e highes t

tes t concentrations . Therefore , th e highes t teste dconcentratio ni n th e ful l life-cycl e stud y wa s approximatel y 1/ 3 th e acut e LCs o valu e a s recommende db y OPPT S 890.1350 . Thi s conclusio ni s generall y supporte db y th e rang e o f acut e toxicit y value s fo r

othe r fis hspecie s reporte di n th e Glyphosat e RE D (1993) . Additionally , glyphosat e ha s bee nshow nt o no t bioconcentrat e i n fish , wit habioconcentratio nfacto r i n whol e fis ho f

les s tha nI X (MRI D 41228301 ; MRI D 41228302) . Th e ful l life-cycl e stud y wit hfathea dminnow s expose dt o glyphosat e bega nwit htw o group s o f thirt y fertilize degg s incubate di n eac htes t aquariu ma t

25°C . Post-hatch , fis hwer e randoml y divide dint o tw o group s o f twent y an ddistribute dt o growthchamber s unti l sexua l maturit y wa s reached . Whe nsecondar y sexua l characteristic s wer e wel l develope d(circ ada y 134 ) th e numbe r offis hi n eac htan k

wa s reduce dinitiall y t o 4 male s an d4 females , an dsubsequentl y t o 2 male s an d4 females . Thi s rati o o f 2 male s t o 4 female s i s require db y th e OPPT S 890.135 0 guideline . Whe nspawnin g began , egg s wer e remove dfro mth e undersid e o f spawnin g tile s dail y an degg s o f eac hspaw nwer e counted . Fift y egg s fro meac ho f th e firs t te n spawnin g i n eac htan k wer e the noscillate di n thei r

respectiv e tes t

water s b y mean s o f

th e eg g cu p an drocke r

ar mapparatu s unti l hatchin g wa s complete d(3- 5 day s a t 25°C) . Afte r

3 0 day s exposure , fr y group s wer e terminate dan dtota l length s determine db y th e photographi c method . Tota l we t weigh t an d percen t surviva l wer e als o determine da t thi s tim e fo r

eac hfr y group . Tota l length , we t weight , 5 9se x an dgonada l condition s wer e determine dfo r eac hadul t

fis ha t th e terminatio n o f

th e experiment . Continuou s exposur e t o concentration s o f glyphosat e a s hig ha s 25. 7 mg/ Lha dn o statisticall y significan t

effect s o n an y o f

th e parameter s studie ddurin g 25 4 day s o f continuou s exposure . Hatchabilit y o f

egg s wa s excellen t

(>94% ) i n al l concentration s o f glyphosat e an dcontrols . Percen t surviva l an dtota l lengt ho f fathea dminnow s afte r

3 0 an d6 0 day s exposur e t o concentration s o f

glyphosat e a s hig ha s 25. 7 mg/Ldi dno t diffe r significantl y fro mth e control . Surviva l o f

th e fiftee norigina l fathea dminnow s place di neac hspawnin g chambe r wa s excellen t i nal l concentration s o f

glyphosat e an dcontrol s throug hda y 134 , whe n fis hwer e thinne dt o a rati o o f 4 male s an d4 females . A t termination , tota l lengt han dwe t weigh t o f th e femal e fathea d minnow s expose dt o al l concentration s o f glyphosat e wer e simila r

t o controls . Spawnin g bega n i nman y tank s aroun dda y 11 2 o f exposur e an dha doccurre di nal l tank s b y da y 148 . Th e numbe r

o f spawnings , egg s pe r

femal e an degg s pe r

spaw n di dno t

diffe r significantl y betwee n control s an dfis hexpose dt o concentration s o f glyphosat e a s hig ha s 25. 7 mg/L . On e spaw n o f 3 3 egg s wa s recovere dfro mth e B replicat e o f 25. 7 mg/ Lbefor e th e accidenta l deatho f fis hdu e t o adilute r

malfunctio nearl y i nth e spawnin g period . Prio r t o tha t time , al l fis h appeare dhealth y an dha dreache dsexua l maturity . Percentag e o f liv e fr y hatchin g i n concentration s o f

glyphosat e a s hig ha s 25. 7 mg/ Lwa s simila r t o tha t whic hwa s observe di n th e controls . Althoug hgonada l histopatholog y wa s no t evaluate di nth e fis hful l lif e cycl e study , absenc e o f

effect s o n th e numbe r

o f spawns , egg s pe r

femal e an degg s pe r

spaw n provide s stron g evidenc e o f

n o impac t

o n gonada l structur e an dfunction . Surviva l an dtota l lengt han dwe t weigh t o f secon dgeneratio n (Fi ) fathea dminnow s wa s simila r t o control s fo r

fis hexpose d3 0 day s t o concentration s o f glyphosat e a s hig ha s 25. 7 mg/L . Base d o nth e absenc e o f

an y significan t

effec t t o fathea dminnow s chronicall y expose dt o glyphosate , th e n o observe deffec t

concentratio n (NOEC ) fo r thi s specie s wa s determine dt o b e >25. 7 mg/L , th e highes t

concentratio n tested . Figur e 6 : Glyphosat e doe s no t effec t spawnin g an deg g hatchabilit y o f fathea dminnow s (Pitnephale s promelas ) continuousl y expose dt o glyphosate .

Numbe r o f Spawning s 15- ,

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4.2. 1 Result s Summar y - Fis hFul l Lifecycl e Stud y Fathea dminnow s (F o + FI ) continuousl y expose dt o hig hconcentratio n o f glyphosat e fo r

25 5 day s demonstrate dn o treatment-relate deffect s o n th e survival , growthan deg g productio n o f firs t generatio n fis ho r

o n hatchability , surviva l an dgrowt ho f second-generatio n egg s an dfry . Observin g n o effect s o n an y o f thes e endpoint s ove r

thi s concentratio n range , provide s stron g evidenc e tha t

glyphosat e doe s no t

hav e estrogenic , anti-estrogenic , androgeni c o r anti-androgeni c activity . Furthermore , a s discusse di ndetai l i nsectio n 4.3.2 , th e structur e activit y relationshi p o f glyphosat e predict s n o estrogeni c activit y no r i s glyphosat e abl e t o transactivat e a n E R responsiv e report

gene . Also , a n absenc e o f an y detectabl e effect s o n growth , developmen t an d reproductio n demonstrate s alac k o f an effec t o n steroidogenesis , gonada l functio n an dth e hypothalamic-pituitary-gonada l (HPG ) axi s function . Glyphosat e ha s bee n show n no t t o induc e vitellogeni ni n aseparat e study , a s discusse di n sectio n 4.2.3 . Additiona l relevan t

informatio n tha t

support s th e finding s o f

th e fis hfull-lif e cycl e stud y i s reviewe di nth e followin g sections . Thi s include s informatio n fro mi nvitr o an di nviv o studie s examinin g th e potentia l fo r estrogenicit y an darevie w o f th e avia n one-generatio n studies . 4.2. 2 Structur e Activity Relationshi p Indicate s Lac k o f Estrogenicity A s previousl y discusse di n th e Introductio n sectio n o f thi s document , glyphosat e doe s no t hav e th e structur e o f

amolecul e predicte dt o hav e estrogeni c o r androgeni c activity . Althoug hSA R model s ar e no t

develope dt o th e sam e degre e fo r androgens , man y o f

th e sam e principal s tha t appl y t o evaluatin g glyphosat e fo r estrogeni c potentia l appl y t o a n assessmen t o f androgeni c potential . Base do n it s chemica l structure , glyphosat e i s predicte dt o no t hav e androgeni c activit y (Deviller s e t

al. , 2009 ; Panay e e t

al. , 2008) . Th e conclusio n tha t glyphosat e doe s no t hav e estrogeni c an dandrogeni c potentia l ha s bee n empiricall y confirme di n anumbe r o f i n vitr o assay s an di nviv o tests . A ful l revie w o f th e i nvitr o an di nviv o dat ai s provide di nth e presentatio n o f

th e OSR I fo r

eac ho f th e assay s tha t

evaluat e th e potentia l fo r estrogeni c activity . 4.2. 3 Glyphosat e doe s no t induc e productio n o f

vitellogeni n Vitellogeni ni s a n eg g yol k precurso r protei n expresse di n female s offish , amphibians , reptile s (includin g birds ) an dinsects . I n fish , live r vitellogeni n productio n i s stimulate db y estradio l an d vitellogeni nproductio n i n mal e fis hha s ahistor y o f us e t o asses s exposur e t o estrogeni c compound s (Sumpte r

an dJobling , 1995) . Productio n o f vitellogeni n i n adul t

female s i s 6 1mediate db y estrogeni c activity ; thu s vitellogeni nexpressio ni s though t t o serv e a s abiomarke r fo r chemical s likel y t o alte r

estrogeni c activit y i nfis han dothe r animals . Th e estrogeni c potenc y o f glyphosat e wa s evaluate di n viv o wit hrainbo w trou t b y measurin g vitellogeni nlevel s post-glyphosat e exposure . A s previousl y discusse di nsectio n2.1.2 , seve n day s o f exposur e t o 0.1 1 mg/Lglyphosat e ha dn o effec t o n vitellogeni nlevels , indicatin g tha t glyphosat e i s no t

estrogeni c (Xi e e t

al , 2005) . Consisten t

wit hthi s result , Peti t

e t al . (1997 ) demonstrate dtha t

glyphosat e doe s no t

bin dt o an dtransactivat e arainbo w trou t E Rrecepto r (stud y summarize di nsectio n2.1.2) . 4.2. 4 Avia n one-generatio n reproductio n assay s Additiona l supportin g informatio nt o evaluat e th e potentia l fo r endocrin e activit y o f

glyphosate , particularl y th e fis hshort

ter mreproductio nstudy , i s supporte db y fou r separat e one-generatio n avia nreproductio nstudie s tha t followe dEP A OPPT S 805.2300 , EP A Sub-divisio nE guideline ? 1- 4 an dOEC D 20 6 guideline s (se e Tabl e 1) . I n thes e studies , bird s wer e fe dthre e die t concentration s containin g glyphosat e throughou t achroni c exposur e period . Bird s ar e induce db y photoperio dmanipulatio nt o la y egg s an dth e egg s ar e collecte dove r aten-wee k

period . Egg s ar e incubate dan dhatched , an dth e youn g maintaine dfo r 1 4 days . Mortalit y o f

adults , eg g production , cracke deggs , eg g shel l thickness , viability , hatchabilit y an deffect s o n youn g bird s ar e observe ddurin g th e study . Th e endpoint s tha t ar e assesse dan devaluate di n th e avia n reproductio nstud y an dth e fis hful l life-cycl e stud y ar e summarize di nth e tabl e below . Fro mthi s compariso ni t

i s clear tha t th e avia nreproductio nstud y provide s significan t an drelevan t informatio nt o evaluat e th e potentia l fo r reproductiv e effect s i nbirds . Readin g acros s t o th e mammalia nmultigenratio nstud y provide s a n additiona l lis t o f endpoint s capabl e o f

detectin g endocrin e activit y tha t

ar e evaluated . Th e origina l reproductio nstudie s wit hnorther nbobwhit e an dmallar dduc k include dates t concentratio na t

th e highes t

nomina l leve l t o tes t

i n OEC D 20 6 o f

1,00 0 m g glyphosate/k g feed . Tw o newe r

reproductio nstudie s wit hnorther nbobwhit e an dmallar dduc k wer e conducte dwit h th e highes t

teste ddietar y concentratio no f 2,25 0 m g glyphosate/k g fee d(ECETOC , 2009) . I n al l fou r o f thes e studies , n o effect s wer e observe do n measure dendpoint s tha t include dth e number s o f egg s laid , egg s damaged/laid , egg s set , viabl e embryos , liv e 3 wee k embryos , hatchlings , 14

- da y survivors , egg s laid/female , egg s laid/female/day , 14-day survivors/femal e an deg g shel l thickness . Th e result s fro mthes e avia nreproductio nstudie s ar e consisten t wit hth e result s fro m th e fis hfull-lif e cycl e stud y an dfurthe r suppor t th e conclusio ntha t

n o evidenc e o f

potentia l endocrin e activit y exist s fo r glyphosat e eve na t

extremel y hig hconcentration s an di n long-ter m continuou s feedin g studies . 4.2. 5 Conclusio n - Fis hshort-termreproductio n assay Th e methodolog y an dinformatio ngenerate dfro mafis hful l life-cycl e stud y provide s a comprehensiv e evaluatio no f potentia l endocrin e activit y an dprovide s informatio nfunctionall y equivalen t

t o informatio nobtaine dfro mth e short-ter mfis hreproductio nassay . A summar y o f th e relevan t

existin g functionall y equivalen t dat ai s summarize di nth e followin g table .



### 6 2Tabl e 9 : Summar y o f Finding s Relativ e t o th e Fis h Short-TermReproductio n Assa y fo r Glyphosat e En dPoint s i n Fis h Short-Term Reproductio n Assa y withth e Fathea dMinnow s Functionall y Equivalen t Informatio n fro mExistin g Studie s Reproductiv e behavio r • N o effec t o n spawnin g behavio r

wa s observe di n th e glyphosat e fathea d ful l life-cycl e study . However , n o impac t o nspawnin g behavio r

i s consisten t wit hn o impac t

o nreproductio n i nth e lif e cycl e study . Secondar y sexua l characteristic s • Ther e wer e n o reporte dobservation s o f a n impac t

o n mal e an dfemal e secondar y sexua l characteristic s i nth e glyphosat e fis hful l life cycl e study . However , n o impac t o n secondar y sexua l characteristic s i s consisten t wit hn o impac t

o nreproductio n i nth e lif e cycl e study . Fecundit y (gonada l output ) N o effec t o nfecundit y wa s eviden t i nth e glyphosat e fathea dful l life

cycl e study . N o treatmen t relate deffect s wer e eviden t fo r th e numbe r

o f spawns , numbe r

o f egg s spawned , numbe r

o f egg s pe r spaw n an dpercen t hatchability . I nth e glyphosat e ra t multigenerationa l studie s fecundit y wa s unaffecte di nth e F O an dF l groups . Fertilit y (fertilizatio n success ) • N o effec t o n fertilit y wa s eviden t

i n th e glyphosat e fathea dful l life-cycl e study . N o treatmen t

relate deffect s wer e eviden t fo r numbe r o f spawns , numbe r o f egg s spawned , numbe r

o f egg s pe r spaw n an dpercen t hatchability . N o fertilit y issue s wer e observe di nmultipl e ra t reproductio nstudie s an dn o effect s wer e observe dfo r sper mcoun t

an d motilit y Gonada l histopatholog y • Gonada l histopatholog y wa s no t assesse di n th e glyphosat e fathea dful l lif e cycl e study . However , histopatholog y wa s performe do n testicular , uterine , ovar y an daccessory se x organ s i n ra t multigenerationa l studie s an ddemonstrate dn o treatmen t relate deffects . Gonadosomati c inde x Gonadosomati c inde x wa s no t assesse di nth e glyphosat e fathea dful l life cycl e stud y bu t

ther e wa s n o effec t

o ngonada l outpu t

i nth e fathea dful l lif e cycl e stud y performe dfo r glyphosate . However , absolut e an d relativ e reproductiv e orga n weight s wer e comprehensivel y assesse di n th e multigenerationa l studie s an dshowe dn o treatmen t relate dimpact . Plasm aconcentratio n o f vitellogeni n(VTG ) Plasm aconcentration s o f VT Gwhic hi s aspecifi c measur e o f estrogenicit y wer e no t

measure di nth e fathea dful l lif e cycl e study . However , glyphosat e ha s bee n show n no t t o induc e VT Gproductio n b y Xi e e t al . (2005 ) i n a n intac t

organis mno r

impac t th e reproductiv e endpoint s measure di n alife cycl e study . Additionally , th e E Rresponsiv e reporte r gen e assay performe db y Peti t e t al . (1997 ) an dsummarize di n sectio n 2.1. 1 demonstrate s tha t glyphosat e i s no t

aligan dfo r

th e rainbo w trou t estroge n receptor . Thi s resul t i s consisten t

wit hth e assay s o f

Xi e e t a l an dKojim ae t

al . whic hdemonstrat e tha t glyphosat e i s no t

a n E R ligan dan dshow s n o potentia l fo r estrogenicity . Th e fis hful l life-cycl e stud y wit hglyphosat e demonstrate dn o treatment-relate deffect s o nth e survival , growthan deg g productio n o f firs t generatio n fish , o r

o n hatchability , a s wel l a s surviva l an dgrowt ho f

second-generatio n egg s an dfry , ove r a n 8 mont hexposur e perio dt o concentration s a s hig h25. 7 mg/L . Additionally , i t ha s bee n show n i n viv o tha t glyphosat e doe s no t induc e productio n o f

vitellogeni nno r

induc e a n estroge n responsiv e reporte r gen e i s atrou t cel l line , whic hi s consisten t wit hSA Ranalysi s o f

glyphosate . Th e result s fro mth e fis hfull-lif e cycl e stud y ar e als o consisten t wit hth e result s fro msubchroni c an dchroni c test s wit h mammalia nan davia n species , whic hdemonstrat e n o change s i n endocrin e functio n an dal l reproductiv e parameters . Base do n thes e studies , sufficien t informatio n alread y exist s t o negate 

### 6 3th e nee dt o perfor mth e fis hshort-ter mreproductio n study . A n additiona l fis hreproductio n

screenin g wit hglyphosat e i s no t necessary . 

## 5. 0 Conclusio n - EDS PTie r 1 OSR I Overvie w Glyphosat e ha s undergon e acomprehensiv e batter y o f i n viv o toxicolog y an decotoxicolog y testin g tha t

cove r abroa dspectru mo f

endocrin e endpoints . Thi s testin g include s multigenerationa l reproduction , subchroni c an dchroni c test s tha t hav e robus t

experimenta l designs , follo w internationally-accepte dprotocols , hav e ahig hleve l o f replicatio n an dalon g histor y o f us e i nhazar didentificatio n an dris k assessment . Thes e i nviv o studie s comprehensivel y asses s endocrin e function s tha t ar e require dfo r

reproduction , development , an d chroni c health . I t

i s importan t

t o poin t ou t tha t i n man y o f

th e chroni c studie s n o effect s and/o r n o effect s relate dt o endocrin e activit y wer e detecte da t hig hconcentration s an da t th e highes t concentratio n tested . Additionally , th e endocrine-modulatin g potentia l o f glyphosat e ha s bee n evaluate di n avariet y o f

i n vitr o assays , negatin g th e nee dfo r furthe r i n vitr o evaluation . A novervie w o f

th e OSR I addressin g th e eleve n assay s o f th e EDS PTie r

I Screenin g follows . E Rbindin g an dtranscriptio n activation : Assay s tha t provid e dat atha t

ar e functionall y equivalen t

t o dat agenerate db y th e ERa/ p competitiv e bindin g assa y usin g ra t uterin e cytoso l an dth e ER atransactivatio n assa y hav e alread y bee n conducted . Peer-reviewe darticle s b y Kojim ae t al . (2004) , Peti t

e t al . (1997 ) an dXi e e t

al . (2005) , presen t

scientifi c informatio n fro m i nvitr o tes t

system s tha t

ar e ver y comparabl e i n methodology , sensitivit y an dspecificit y t o th e tw o Tie r 1 E Rbindin g assays . Thes e assay s b y Kojim ae t al. , Peti t e t al. , an dXi e e t

al . measure d th e sam e mechanisti c endpoint s o f ligan dbindin g t o asses s agonis man dantagonism , nuclea r E R translocation , DN A bindin g t o responsiv e E Relement s upstrea mo f th e reporte r

gen e an d transactivatio no f

th e reporte r

gene . Th e result s fro mthes e assay s consistentl y demonstrat e tha t glyphosat e i s no t

a n E Ragonis t

o r antagonist . Furthermore , astructur e activit y relationshi p (SAR ) analysi s wit hmethodolog y develope db y EPA' s Offic e o f Researc han dDevelopmen t clearl y show s tha t

glyphosat e doe s no t

hav e th e chemica l structur e o f atypica l molecul e wit h estrogeni c potential . Th e i n vitr o dat ademonstratin g tha t glyphosat e doe s no t

hav e E Rbindin g potentia l i s consisten t

wit hth e comprehensiv e i nviv o dat aavailabl e fo r glyphosate . Th e i nviv o dat ademonstrate s n o effect s o n an y endpoin t tha t i s sensitiv e an dspecifi c t o compound s tha t hav e estrogeni c o r

anti-estrogeni c activity . Base do n th e weigh t o f evidence , i t

ca n b e conclude d tha t n o additiona l Tie r

1 i nvitr o screenin g i s require dt o evaluat e th e potentia l fo r E Ragonis m an dantagonism . A Rbinding : Assay s tha t provid e dat atha t

ar e functionall y equivalen t t o dat agenerate db y th e A Rcompetitiv e bindin g assa y usin g ra t prostat e cytoso l hav e als o alread y bee n conducted . Th e peer-reviewe darticl e b y Kojim ae t al . (2004 ) present s scientifi c dat atha t demonstrate s tha t glyphosat e i s no t

a n A Ragonis t

o r antagonis t

usin g a n i n vitr o tes t

syste man dmethodolog y tha t i s functionall y equivalen t t o th e methodolog y fo r

th e A Rbindin g assay i n th e Tie r 1 screenin g battery . Kojim ae t

al . evaluate dth e sam e mechanisti c endpoin t o f ligan dbindin g t o asses s agonis man dantagonis ma s th e OPPT S A Rbindin g guideline . Again , th e SA Ranalysi s indicate s tha t

glyphosat e doe s no t

hav e th e chemica l structur e o f atypica l molecul e tha t

exhibit s androgeni c potential . Lac k o f A Rbindin g i s confirme db y th e comprehensiv e i n viv o dat a availabl e fro mchroni c studie s o n glyphosate . Th e i n viv o dat ademonstrate s n o effec t o n endpoint s tha t

ar e sensitiv e an dspecifi c t o compound s tha t hav e androgeni c o r

anti-androgeni c activity . A detaile drevie w o f th e i n viv o dat atha t

confirm s glyphosate' s i s no t androgeni c o r 6 4anti-androgeni c i s extensivel y reviewe di n th e section s fo r th e Hershberger , mal e puberta l assay an dfis hscreenin g assay . Base do n th e weigh t o f evidenc e i t

ca n b e conclude dtha t

n o additiona l i nvitr o screenin g i s require dt o evaluat e th e potentia l fo r A Ragonis man dantagonism . Aromatas e Inhibition : Althoug hi n vitr o dat ai s no t availabl e t o asses s aromatas e inhibition , OSRI o f th e sam e natur e an dqualit y i s availabl e fro mexistin g i nviv o chroni c toxicit y studie s wit hglyphosate . N o treatment-relate deffect s wer e observe di n thes e studie s o n sensitiv e an d specifi c endpoint s tha t

woul db e indicativ e o f

aromatas e inhibitio nb y glyphosate , whic h provide s stron g an dconsisten t evidenc e tha t

glyphosat e i n no t

a n inhibito r

o f aromatas e activity . Scientificall y relevan t

informatio nrelate dt o steroidogenesi s i s comprehensivel y discusse di nthi s documen t an dals o applie s t o aromatas e inhibition . Consequently , th e requiremen t fo r performin g th e i n vitr o aromatas e assay ha s bee nme t b y providin g OSR I fro mexistin g standar d regulator y i n viv o studie s tha t hav e th e abilit y t o detec t

effect s o f

inhibitio no f

aromatas e activity . Steroidogenesis : I n vitr o dat aassessin g potentia l impact s o n th e complet e steroidogeni c pathwa y ar e no t

availabl e fo r

glyphosate ; however , dat atha t i s functionall y equivalen t t o th e i n vitr o steroidogenesi s assay i s provide db y endpoint s include di n multigenerationa l ra t studies . Specifically , i t

wa s confirme di nth e mos t recen t multigenerationa l stud y tha t ther e wer e n o treatment-relate deffect s a t hig hdietar y exposur e level s o n endpoint s tha t woul db e impacte db y a n alteratio no f

steroidogenesis , e.g. , th e weigh t o f testes , caud aepididymides , epididymides , semina l vesicle s an dcoagulatin g glands , uterus , ovar y an dlive r o f th e F O an dF l paren t

animal s (Moxon , 2000 ; Dhins ae t

al. , 2007) . Additionally , ther e wer e n o treatment-relate deffect s u p t o th e highes t

dos e teste d(HOT ) o n th e histopatholog y o f lef t testis , lef t epididymis , prostate , semina l vesicle s wit hcoagulatin g gland , uterus , lef t ovary , vagina , cervix , an doviducts . Furthermore , ther e wer e n o treatment-relate deffect s u p t o th e HD T o n mal e an dfemal e reproductiv e indice s tha t

include dmating , fertility , conception , gestation , lactatio nag e an d weigh t a t VO , ag e a t

PPS , testicula r

descent , ovaria nevaluatio nfo r follicles , parturition , normalit y o f

externa l genitali aan dnumbe r o f implantatio nsites . Th e i n viv o informatio nfro m thes e studie s provide s compellin g evidenc e o f n o impac t

o f glyphosat e o n steroidogenesis . Uterotrophic : A stron g weigh t o f evidenc e ha s bee nprovide dwit hfunctionall y equivalen t informatio nsupportin g alac k o f estrogeni c activit y o f

glyphosat e i nviv o fro mfou r existin g multigenerationa l studies , an dasubchroni c an dchroni c toxicit y stud y i n rats . Th e result s o f th e i nviv o studie s ar e consisten t wit hresult s o f

i n vitr o assay s tha t

demonstrat e glyphosat e i s no t a n E Rligand . Functionall y equivalen t dat ao f th e sam e natur e an dqualit y t o dat aexpecte dt o b e generate dwit hth e Uterotrophi c assay (uterin e weight s an dhistopathology ) demonstrat e tha t glyphosat e doe s no t

posses s estrogeni c activity . Hershberger : A stron g weigh t o f evidenc e ha s bee npresente dusin g functionall y equivalen t informatio nsupportin g alac k o f androge nrecepto r

agonis man dantagonis man d5a-reductas e inhibitio no f

glyphosat e i nviv o fro mfou r multigenerationa l studies , an dasubchroni c an d chroni c toxicit y stud y i nrats . Thi s functionall y equivalen t dat afro mmultigenerationa l studies , subchroni c an dchroni c studie s i n rat s demonstrat e tha t glyphosat e i s no t

a n androge nrecepto r agonis t o r antagonis t

o r a5a-reductas e inhibitor . Femal e Pubertal : Functionall y equivalen t dat at o th e femal e puberta l endpoint s ar e describe d i nSectio n3. 3 o f

thi s document , wit hacomprehensiv e overvie w o f endpoint s addresse di n th e multigenerationa l reproductive , subchroni c an dchroni c ra t studies . Endpoint s i nth e femal e puberta l assay ar e largel y asubse t o f th e multigenerationa l studie s performe do n glyphosat e an d

6 5consequentl y provid e dat ao f th e sam e natur e an dqualit y t o dat adevelope di n th e femal e puberta l assa y o f

th e Tie r 1 batter y o f

th e Endocrin e Disrupte r

Screenin g Program . Mal e Pubertal : Lik e th e femal e puberta l assay , functionall y equivalen t dat at o th e mal e puberta l endpoint s ar e describe di n Sectio n3.4 , wit hacomprehensiv e overvie w o f endpoint s addresse di nth e multigenerationa l reproductive , subchroni c an dchroni c ra t studies . Endpoint s i nth e mal e puberta l assay ar e als o essentiall y asubse t o f endpoint s fro mth e multigenerationa l studie s performe dfo r

glyphosat e an dconsequentl y provid e dat ao f th e sam e natur e an dqualit y t o dat adevelope di n th e mal e puberta l assay require dt o b e conducte di n th e EDSP . Amphibia n Metamorphosis : Th e amphibia nmetamorphosi s assay ha s bee ninclude di n th e Tie r 1 batter y a s ageneralize dmode l fo r impact s o n vertebrat e thyroi dfunctio nbecaus e th e amphibia nthyroi dsyste mi s regulate dan drespond s i n asimila r manne r t o perturbatio na s tha t

o f othe r tetrapods , includin g mammals . Therefore , combine dresult s fro mexistin g glyphosat e studie s provid e informatio ntha t i s functionall y equivalen t t o tha t generate di nth e amphibia n metamorphosi s assay . Thyroi dhistopatholog y ha s bee nevaluate di n standar dmammalia n toxicolog y studie s an ddemonstrate s n o impac t o nthyroi dstructur e an dfunction . Supportin g informatio ni s draw nfro mth e fis hful l life-cycl e stud y tha t show s n o impac t

o ngrowt han d development , whic har e sensitiv e measuremen t endpoint s fo r

impact s o nthyroi dstructur e an d function . Give nal l o f

thi s availabl e informatio no nglyphosate , th e amphibia nmetamorphosi s screenin g assa y doe s no t

offe r an y ne w relevan t

informatio nt o th e toxicolog y dat ase t fo r Tie r 1 screenin g purposes . Fis hShort-ter mReproduction : Th e methodolog y an dinformatio ngenerate dfro mafis hful l life-cycl e stud y provide s acomprehensiv e evaluatio no f potentia l modulatio no f

endocrin e activit y i nfis han dprovide s functionall y equivalen t informatio nt o informatio nfro mth e short

ter mfis hreproductio nassay . Th e fis hful l life-cycl e stud y i s th e highes t Tie r offis h ecotoxicolog y testin g an dtypicall y performe dwit hth e fathea dminno w (Pimphalespromelas ) a s afreshwate r

tes t species . I n th e study , th e entir e life-cycl e i s exposed ; therefore , d e fact o th e mos t sensitiv e life-stag e t o an y toxican t (includin g endocrin e activ e compounds ) i s encompasse d b y th e tes t design . Although , endpoint s tha t ar e potentiall y influence db y endocrin e activity , suc ha s gonada l histopatholog y an dsteroi dhormon e production , ar e no t typicall y measure di n th e ful l life-cycle , afis hful l life-cycl e stud y provide s acomparabl e investigatio nt o th e tes t propose di nth e highe r

Tie r 2 testin g fo r

fish . Th e fis hful l life-cycl e stud y fo r glyphosat e demonstrate dn o treatment-relate deffect s o n th e survival , growt han deg g productio no f firs t generatio nfis ho r

o n hatchability , a s wel l a s surviva l an dgrowt ho f second-generatio negg s an d fr y ove r a n 8 mont hexposur e perio dt o concentration s a s hig ha s 25. 7 mg/ L. Furthermore , i t ha s bee nshow ni n viv o tha t glyphosat e doe s no t

induc e productio no f

vitellogenin , whic hi s i n agreemen t wit hSA Ranalysi s o f

glyphosate . Th e result s fro mth e fis hfull-lif e cycl e stud y ar e consisten t wit hth e result s for msubchroni c an dchroni c tes t wit hmammalia nspecies , whic h demonstrat e n o change s i nendocrin e function , endocrin e orga nhistopathology , an dal l reproductiv e parameters . Additionally , th e result s fro mavia none-generatio nreproductio n studie s demonstrat e n o effec t o n an y o f

th e measure dendpoint s a t hig htes t concentrations . Base do n thi s OSRI , ther e i s sufficien t informatio nt o mee t

th e requirement s fo r

performin g a fis hshort ter mreproductio nstudy . 3 Chroni c aquati c exposur e testin g unde r th e fis hearl y life-stag e stud y (OEC D 210 ) doe s no t requir e testin g a t concentration s tha t

excee d1 0 mg/Lo f

activ e substance . Additionally , th e highes t concentratio n teste do f

25. 7 mg/Lrepresent s approximatel y 1/3 / th e 96-h r LC5 0 valu e fo r

thi s species . 6 6

### 5. 1 Glyphosat e Review s fo r Endocrin e Activit y Severa l in-dept hreview s o nth e safet y o f glyphosat e hav e bee nconducte db y severa l regulator y agencie s an dscientifi c institution s worldwid e an dhav e conclude dtha t ther e i s n o indicatio ntha t glyphosat e ha s potentia l endocrin e activit y o r wil l resul t

i nadvers e effect s o nendocrin e system s i nhuman s o r

othe r mammals . Th e point s belo w summariz e th e conclusion s o f thes e reviews . • Th e U.S . EP A (1998 ) reviewe dth e subchroni c an dchroni c mammalia nstudie s fo r glyphosat e an dconclude dtha t ther e wa s n o evidenc e t o sugges t tha t glyphosat e produce s endocrine-modulatin g effects . • I nacomprehensiv e revie w o f th e standar dmammalia ntoxicolog y studies , William s e t al. , (2000 ) conclude dtha t

glyphosat e doe s no t

hav e th e potentia l t o produc e advers e effect s o n endocrin e system s i nhuman s o r othe r mammals . • A repor t

prepare dfo r

th e USD A fores t

servic e b y SERA conclude dtha t extensiv e testin g i n experimenta l animal s an dwildlif e provide dreasonabl y stron g evidenc e tha t glyphosat e i s no t a nendocrin e disrupte r

(SERA , 2002) . • Th e lis t

o f purporte dendocrin e disrupter s compile db y th e Institut e o f Environmen t

an d Healt h(IEH , 2005 ) list s glyphosat e a s asubstanc e wit hn o evidenc e o f potentia l endocrine

disruptin g effects . • Followin g arevie w o f

th e standar dmammalia nan dwildlif e toxicolog y studies , i t wa s conclude di nth e recen t

ECETO C guidanc e documen t o nidentifyin g endocrin e disruptin g effect s tha t

glyphosat e i s no t

a nendocrin e disrupte r

usin g th e internationall y accepte d Weybridg e definition 4 

### fo r

endocrin e disruptio n(ECETOC , 2009) . 5. 2 Summar y an dConclusion s Base do nthi s weight-of-evidenc e analysi s fro mglyphosate' s extensiv e toxicologica l database , ther e i s sufficien t

OSR I tha t

i s functionall y equivalen t

an do f th e sam e natur e an dqualit y a s tha t develope di nth e Tie r

1 screenin g batter y t o conclud e tha t glyphosat e doe s no t

hav e endocrine

modulatin g potential . Therefore , th e requirement s o f Tie r 1 testin g hav e bee nsatisfie dan d additiona l testin g unde r

th e Endocrin e Disruptio nScreenin g an dTestin g Progra mi s no t warranted . Th e endocrine-modulatin g potentia l o f glyphosat e ha s bee nevaluate di navariet y o f studie s includin g i nvitr o assay s an dstandar di nviv o toxicolog y studie s tha t ar e functionall y equivalen t t o th e dat agenerate di nth e Tie r 1 screenin g battery . Th e i nviv o studie s comprehensivel y asses s endocrin e function s tha t

ar e require dfo r

reproduction , developmen t an dchroni c health . Glyphosat e ha s demonstrate dn o estrogenic , anti-estrogenic , androgeni c an danti-androgeni c potentia l i ni nvitr o assays , an dther e wa s n o indicatio no f change s i nendocrin e functio no r endocrin e orga nhistopatholog y i nan y o f th e i nviv o studies . Additionally , structur e activit y model s predic t

tha t glyphosat e doe s no t

hav e estrogeni c o r

androgeni c activity .

4 Weybridg e (1996 ) "A n endocrin e disrupte r i s a n exogenou s substanc e tha t cause s advers e healt heffect s i n a n intac t organism , o r

it s progeny , secondar y (consequent ) t o change s i n endocrin e function . A potentia l endocrin e disrupte r i s asubstanc e tha t

possesse s propertie s tha t migh t b e expecte dt o lea dt o endocrin e disruptio ni n a n intac t organism. " 

### 6 7

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7 4

## Appendi x 1

EPA' s EDS P Tie r

I Screenin g Batter y Tie r 1 In Vitro Assay s Estroge nRecepto r

bindin g assa

y E Rtranscriptiona l activatio nassay Androge nrecepto r

bindin g assay Aromatas e assa

y

Steroidogenesi s assay A nE Rbindin g assay tha t utilize s ra t

uterin e cytoso l t o examin e th e abilit y o f

ates t chemica l t o bin dwit hestroge nreceptor s A n E Rbindin g assay tha t

use s ahuma ncel l lin e t o examin e th e abilit y o f ates t chemica l t o bin dwit hon e o f th e estroge n receptor s A n A Rbindin g assay tha t utilize s ra t

prostat e cytoso l t o examin e th e abilit y o f

ates t chemica l t o bin dwit handroge nreceptor s Aromatas e i s a n enzym e responsibl e fo r estroge nbiosynthesi s tha t convert s androgen s int o estrogens , estradiol , an destrone . Th e aromatas e assa

y use s huma nrecombinan t for mo f th e protei nan d focuse s o n thi s portio no f

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

inhibi t aromatas e activity . Th e Steroidogenesi s assay utilize s H295 Rhuma nadrenocortica l carcinom acel l lin e t o detec t interferenc e wit hth e body' s productio no f

mal e an dfemal e steroi dse x hormone s (estroge n an dtestosterone) . Tie r 1 In Viv o Assay s Uterotrophi c assa

y Hershberge r

assa y

Mal e puberta l assay (rat )

Femal e puberta l assay (rat )

Fis hshor t termreproductio nassa

y

Amphibia nmetamorphosi s assa y (frog ) Th e Uterotrophi c assa y involve s th e us e o f

femal e rat s t o scree n fo r estrogeni c effects . I nthi s i nviv o assay , uterin e weigh t change s ar e measure di novariectomise do r immatur e femal e rats . Th e Hershberge r

assa y i s designe dt o detec t

androgeni c an danti

androgeni c effects . I n thi s assay , accessory se x glan dweights , includin g severa l androgen-dependen t tissues , ar e measure di n castrate do r

immatur e mal e rat s Th e Puberta l Mal e assay involve s th e us e o f rat s t o scree nfo r androgenic , anti-androgenic , an dthyroi dactivit y i nmale s durin g sexua l maturation . Thi s assay examine s abnormalitie s associate d wit hse x organ s an dpubert y markers , a s wel l a s thyroi dtissu e Th e Puberta l Femal e assay involve s th e us e o f rat s t o scree nfo r estrogeni c an dthyroi dactivit y i nfemale s durin g sexua l maturation . Thi s assay examine s abnormalitie s associate dwit h se x organ s an dpubert y markers , a s wel l a s thyroi dtissue . Th e Fis hScreen assa

y screen s fo r

estrogeni c an dandrogeni c effects . Th e assay examine s abnormalitie s associate dwit h survival , reproductiv e behavior , secondar y se x characteristics , histopathology , an dfecundit y (i.e. , numbe r o f spawns , numbe r

o f eggs/spawn , fertility ) offis hexpose dt o tes t chemicals . Th e Amphibia nMetamorphosi s assay involve s th e us e o f tadpole s t o determin e i f

chemical s affec t

th e thyroi ddurin g metamorphosi s an dconsequentl y resul t i ndevelopmenta l effects . 7 5

### Appendi x 2

Summar y o f

Glyphosat e Adsorption , Distribution , Metabolis man dExcretio n Fro mth e Glyphosat e RE D (EPA , 1993) : Tw o metabolis mstudie s wit hrat s ar e available . I nth e firs t

study , singl e o r

repeate ddose s o f

radiolabele d14 C-glyphosat e wer e administere d orall y t o mal e an dfemal e Sprague-Dawle y rats . Followin g asingl e ora l dos e o f 14C-glyphosate , 3 0 t o 36 % o f

th e dos e wa s absorbe dan dles s tha n0.27 % o f th e dos e wa s eliminate da s COi . Ninety-seve npoin t

fiv e percen t

o f th e administere ddos e wa s excrete di n th e urin e an dfece s a s th e paren t compound , glyphosate . Amin o methy l phosphoni c aci d(AMPA ) wa s th e onl y metabolit e foun di n urin e (0.2-0.3 % o f th e administere ddose ) an dfece s (0.2-0.4 % o f th e administere ddose) . Les s tha n1.0 % o f th e absorbe ddos e remaine di n tissue s an dorgans , primaril y i n bon e tissue . Repeate ddosin g a t 1 0 mg/k g di dno t

significantl y chang e th e metabolism , distributio no r excretio no f

glyphosat e (MRID s 4076710 1 an d40767102) . I nasecon dstudy , mal e an dfemal e Sprague-Dawle y rat s receive dsingl e intraperitonea l injection s o f

radiolabele d' C-glyphosate . Th e dos e leve l o f glyphosat e use dfo r

mal e an dfemal e rat s wa s 115 0 mg/kg . Bloo dsample s wer e collecte d0.25 , 0.50 , 1 , 2 , 4 , 6 an d1 0 hour s afte r injection . Femora l bon e marro w sample s wer e collecte dfro mon e thir do f th e mal e an dfemal e rat s sacrifice da t

0.5 , 4 , o r 1 0 hour s afte r

injection . Thirt y minute s afte r injectio no f

glyphosate , th e concentratio no f

radioactivit y i n th e bon e marro w o f mal e an dfemal e rat s wa s equivalen t t o 0.0044 % an d0.0072% , respectively , o f th e administere ddose . Assumin g firs t orde r kinetics , th e decreas e i nradioactivit y i nbon e marro w occurre dwit hahalf-lif e o f 7. 6 an d4. 2 hour s fo r

male s an dfemale s respectively . Similarly , th e half-live s o f th e radioactivit y i nplasm awer e approximatel y 1 hou r

fo r bot hsexes . These finding s indicat e tha t ver y littl e glyphosat e reache s bon e marrow , tha t

i t i s rapidl y eliminate dfro mbon e marro w an dtha t i t i s eve nmor e rapidl y eliminate dfro mplasma . (MRI D 00132685) . Tabl e Al : (Ridle y an dMirly , 1988) . Distributio n o f radioactivity i n rat s dose dorall y with1 0 mg/kg o f glyphosate an dsacrifice d2 4 hour s afte r dosin g (treatmen t

grou p 1 ) Sampl e Urin e Faece s Expire dCO 2 Cag e was h Tota l recovery * Male s % o f Dos e 12. 2 68. 6 0.13 7 0.053 9 8 1 Female s % o f Dos e 11. 1 70. 4 0.15 4 0.52 6 82. 2 * Total recovery i s th e mea no f individua l anima l data .

7 6Tabl e A2 : (Ridle y an dMirly , 1988) . Distributio n o f radioactivity i n rat s dose dintravenousl y with1 0 mg/kg o f glyphosat e an dsacrifice d7 day s afte r dosin g (treatmen t

grou p 3 ) Sampl e Urin e Faece s Organs/Tissue s Residua l carcas s Tai l Gastrointestina l content s Cag e was h Tota l recovery * Male s % o f Dos e 79. 0 4.6 5 0.094 1 1.1 8 0.17 2 0.039 4 0.89 0 86. 0 Female s % o f Dos e 74. 5 8.3 0 0.052 1 1.0 4 0.16 1 0.038 8 1.3 0 85. 3 * Tota l recovery i s th e mea n o f individua l anima l data . Tabl e A3 : (Ridle y an dMirly , 1988) . Distributio n o f radioactivity i n rat s dose dorall y wit h1000 mg/kg o f glyphosat e an dsacrifice d7 day s afte r dosin g (treatmen t

grou p 4 ) Sampl e Urin e Faece s Organs/Tissue s Residua l carcas s Gastrointestina l content s Cag e was h Tota l recovery * Male s % o f Dos e 17. 8 68. 9 0.035 5 0.24 8 0.025 8 3.8 6 90. 9 Female s % o f Dos e 14. 3 69. 4 0.026 6 0.20 8 0.042 9 8.0 0 92. 1 * Tota l recovery i s th e mea n o f individua l anima l data . Tabl e A4 : (Ridle y an dMirly , 1988) . Distributio n o f radioactivity i n rat s dose dorall y wit h1 0 mg/kg o f glyphosate an dsacrifice d7 day s afte r dosin g (treatmen t

grou p 5 ) Sampl e Urin e Faece s Organs/Tissue s Residua l carcas s Gastrointestina l content s Cag e was h Tota l recovery * Male s % o f Dos e 28. 6 62. 4 0.046 0 0.39 5 0.022 6 1.3 0 92. 8 Female s % o f Dos e 22. 5 69. 4 0.019 4 0.28 6 0.014 5 1.9 6 94. 2 * Total recover

y i s th e mea n o f

individua l anima l data .

7 7Tabl e AS : (Ridle y an dMirly , 1988) . Distributio n o f radioactivity i n rat s dose dorall y fo r 1 5 day s wit h1 0 mg/kg o f glyphosat e an dsacrifice d7 day s afte r dosin g (treatmen t

grou p 6 ) Sample Urin e Faece s Organs/Tissue s Residua l carcas s Gastrointestina l content s Cag e was h Total recovery * Male s % o f Dos e 30. 9 61. 0 0.047 3 0.49 7 0.013 8 0.82 0 93. 3 Female s % o f Dos e 23. 1 70. 9 0.031 3 0.31 5 0.009 5 1.9 6 96. 3 * Tota l recovery i s th e mea n o f individua l anima l data . Ridle y (1983 ) conducte dametabolis mstud y o f th e plasm aan dbon e marro w level s o f glyphosat e followin g intraperitonea l administratio n i nth e ra t an ddetermine dtha t l4 C-glyphosat e reache dmaximu mplasm aan dbon e marro w concentration s withi n 0. 5 hour s followin g intraperitonea l injectio ni nrats . Th e eliminatio n half-lif e time s o f glyphosat e i nplasm aan dbon e marro w wer e 1 hou r

an d4.2-7. 6 hours , respectively . Colvi nan dMille r

(1973 ) evaluate dth e gros s distributio n o f 14 C

- N-phosphonomethylglycin e i n rabbit s an ddetermine dtha t 14 C-glyphosat e wa s predominantl y excrete di n th e faece s (80-97 % o f th e dose ) rabbit s ove r

aperio do f

12 0 hour s followin g ora l administratio n b y gavage . Onl y a ver y limite dfractio no f

th e radioactivit y wa s retaine di n th e tissue s (0.1-1.2 % o f th e dose) . Weste r (1991 ) evaluate dth e eliminatio n o f 14 C-GIyphosat e i n rhesu s monkey s an ddetermine d tha t 94.9 % t o 98.8 % o f

th e radioactivit y wa s excrete di n th e urin e ove r aperio do f

7 day s followin g intravenou s injectio n o f 14 C-glyphosat e - nearl y al l o f th e glyphosat e excretio n occurre dwithi n 2 4 hours . Howe , Chot t

an dMcClanaha n (1988 ) evaluate dth e metabolis mo f glyphosat e i n Sprague

Dawle y rat s a t

severa l dose s vi abot hora l an dintr avenou s route s o f administration . The y determine dtha t

th e metabolis mo f

glyphosat e i n th e ra t

followin g ora l o r

intravenou s administratio nwa s ver y minor . AMPA , th e sol e metabolite , appeare dt o b e forme dt o th e exten t o f les s tha n 0.5 % b y i n viv o metabolis mo f glyphosat e i n rat s orall y dose da t 1 0 mg/kg , eithe r

i n singl e o r multipl e doses . Colvi nan dMille r

(1973 ) evaluate dth e dynamic s o f accumulatio n an ddepletio n o f orall y ingeste d14 C labele dglyphosate . Dat acomparin g tissu e uptak e an ddepletio n ar e summarize d belo w i n table s A 6 an dA 7 respectively .

7 8Tabl e A6 : (Colvi nan dMiller , 1973) : Pea k radioactivity expresse da s glyphosat e aci deq . I n rat tissue s durin g chroni c ora l dosin g wit h 4 C-glyphosat e Tissu e Live r Kidne y Muscl e Fat Gu t Splee n Heart Brai n Gonad s Bloo d 1 ng/g o r ng/m L Mal e 0.00 4 0.01 2 0.00 2 0.00 3 0.50 6 0.00 7 0.00 2 0.00 1 0.00 1 0.00 1 Femal e

0.00 4 0.00 8 0.00 1 0.00 6 0.37 0 0.01 1 0.00 4 0.00 1 0.00 9 0.00 1 1 0 ng/ g or ng/m L Mal e 0.04 1 0.13 6 0.01 8 0.06 5 3.49 4 0.07 6 0.03 8 0.01 4 0.01 4 0.01 3 Femal e

0.03 4 0.10 9 0.01 3 0.06 0 5.28 6 0.12 2 0.03 2 0.01 3 0.18 0 0.01 3 10 0 (ig/

g or (ig/mL Mal e 0.31 9 0.91 6 0.19 5 0.71 0 37.30 5 0.69 6 0.40 4 0.17 9 0.11 4 0.09 2 Female

0.29 1 0.77 9 0.12 5 0.68 1 26.90 3 0.63 6 0.20 9 0.11 6 0.59 0 0.09 0 Th e tissu e radioactivit y wa s clearl y reversibl y bound . A s soo na s th e exogenou s suppl y o f I4 C

glyphosat e wa s removed , th e radioactivit y i n th e tissue s immediatel y bega nt o decrease . Residue s i ntissue s remainin g afte r te nday s withdrawa l o nafresh-weigh t basi s ar e show ni n Tabl e A7 . Tabl e A7 : (Colvi nan dMiller , 1973) : Concentratio no f radioactivity (expresseda s glyphosat e aci deq. ) remainin g i nrat tissue s afte r

1 0 day s withdrawa l followin g chroni c ora l dosin g wit hC-glyphosate Tissu e Live r Kidne y Muscl e Fat Gu t Splee n Hear t Brai n Gonad s Bloo d 1 (ig/g o r u,g/mL Mal e 0.00 1 0.00 1 0.00 1 0.00 2 0.00 1 0.00 1 0.00 1 0.00 1 0.00 1 0.00 1 Female

0.00 1 0.00 1 0.00 1 0.00 1 O.00 1 O.00 1 <0.001 <0.00 1 0.00 0 O.00 1 1 0 (ig/

g or u.g/m L Mal e 0.00 8 0.01 3 0.01 3 0.01 6 0.00 8 0.00 8 0.01 2 0.00 6 0.00 7 0.00 6 Femal e

0.00 8 0.01 2 0.00 9 0.01 5 0.00 8 0.00 8 0.00 9 0.00 5 0.01 0 0.00 6 10 0 (ig/

g or jig/mL Mal e 0.06 9 0.10 2 0.09 7 0.11 2 0.05 9 0.06 7 0.08 4 0.05 0 0.05 7 0.05 9 Female

0.06 5 0.09 8 0.09 0 0.12 8 0.07 6 0.06 0 0.07 8 0.04 3 0.06 2 0.04 6 Colvi nan dMille r

(1973 ) conclude dtha t

upo ncontinuou s administratio no f C-glyphosat e i n feed , 8.3-10.5 % o f

th e dail y intak e wa s excrete di n urine . Th e combine dexcretio no f radioactivit y i n urin e an dfaece s a t th e en do f

th e 14-day dosin g perio dwa s i n equilibriu mwit h th e dail y intak e o f 14 C-glyphosat e a t

th e thre e fee dconcentration s tested . Upo nwithdrawa l fro m treatmen t th e excretio no f

radioactivit y droppe drapidly , bu t plateaue dafte r

fou r day s du e t o 

### 7 9redistributio no f previousl y establishe dtissu e stores . Mos t tissue s reache dmaximu m radioactivit y level s durin g th e dosin g perio di n1 0 day s o r less . Th e radioactivit y tha t wa s presen t i n th e tissue s wa s clearl y no t irreversibl y bound . Highlight s fro mth e abov e dat aaddressin g OPPT S 890.140 0 an d890.160 0 consideration s o f ADM Ean dmetabolism , negatin g th e nee dfo r i n viv o screenin g wit hth e Hershberge r an d uterotrophi c assay s includ e th e following . • Lo w G l trac t

absorptio n o f

glyphosate , • Ver y lo w derma l absorptio n (B o Neilse n e t al. , 2009 )

• Ver y littl e systemi c metabolis m/activatio n o f glyphosate , an d

• Ver y rapi dclearanc e fro mth e bod y predominantl y vi aurine .

8 0

## Appendi x 3

Cite dStudie s Submitte dt o EP A Stud y EP A Classificatio n MRID # Rodwell , D.E. ; Tasker , E.J. ; Blair , A.M. ; e t al . (1980 ) Teratolog y Stud y i nRats : IRD C No . 401-054 . (Unpublishe dstud y includin g IRD C no . 999-021 ; receive dMa y 23 , 198 0 unde r 524-308 ; prepare db y Internationa l Researc han dDevelopmen t Corp. , submitte db y Monsant o Co. , Washington , D.C. ; CDL:242516-A ) Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio no f

Glyphosate

Reregistratio nEligibilit y Decisio n(RED ) 0004636 2

Rodwell , D.E. ; Tasker , E.J. ; Blair , M. ; e t al . (1980 ) Teratolog y Stud y i nRabbits : IRD C No . 401-056 . (Unpublishe dstud y receive dMa y 23 , 198 0 unde r 524-308 ; prepare db y Internationa l Researc han dDevelopmen t Corp. , submitte db y Monsant o Co. , Washington , D.C. ; CDL:242516-B ) Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio no f

Glyphosate

Reregistratio nEligibilit y Decisio n(RED ) 0004636 3

Schroeder , R.E. ; Hogan , O.K . (1981 ) A Three-Generatio n Reproductio nStud y wit hGlyphosat e i nRats : Projec t No . 77- 2 (Unpublishe dstud y receive dSe p 22 , 198 1 unde r 524-308 ; prepare db y Bio/dynamics , Inc. , submitte db y Monsant o Co. , Washington , D.C. ; CDL:245909-A ) Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio no f

Glyphosate

Reregistratio nEligibilit y Decisio n(RED ) 0008167 4 0010599 5

Colvin , L. ; Miller , J. ; Marvel , J . (1973 ) Fina l Repor t o nC P 6757 3 Residu e an dMetabolism : Par t 9 : Th e Gros s Distributio n C P67573-14 C (N-phosphono-methylglycine- 14 C ) i nth e Rabbit : Agricultura l Researc hRepor t No . 298 . (Unpublishe dstud y receive dNo v 12 , 197 3 unde r 4G1444 ; submitte db y Monsant o Commercia l Product s Co. , St . Louis , MO ; CDL : 093849-D ) Supplementa l 0010809 9

E G& G , Bionomic s (1975 ) Chroni c Toxicit y o f Glyphosat e t o th e Fathea dMinno w (Pimephale s promelas , Rafmesque) . (Unpublishe dstud y receive dDe c 27 , 197 8 unde r 524-308; submitte db y Monsant o Co. , Washington , DC ; CDL:097759-B ) Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio no f

Glyphosate

— Reregistratio nEligibilit y Decisio n(RED ) 0010817 1

Fin k Ran dJ Beavers . (1978 ) Fina l Report : One-generatio n Reproductio nstudy—Bobwhit e Quail : Glyphosat e Technical : Projec t No . 139-141 . (Unpublishe dstud y receive dNo v 13 , 197 8 unde r 524-308 ; prepare db y Wildlif e International , Ltd. , submitte db y Monsant o Co. , Washington , DC ; CDL:235924-B ) Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio no f

Glyphosat e Reregistratio nEligibilit y Decisio n(RED ) 0010820 7

Fin k Ran dJ Beavers . (1978 ) Fina l Report : One-generatio n Reproductio nStudy—Mallar dDuck : Glyphosat e Technical : Projec t No . 139-143 . (Unpublishe dstud y receive dNo v 13 , 197 8 unde r 524-308 ; prepare db y Wildlif e Internationa l Ltd. , submitte d b y Monsant o Co. , Washington , DC ; CDL:235924-A) . Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio no f

Glyphosat e Reregistratio nEligibilit y Decisio n(RED ) 0011195 3 Ridley , W , Dietrich , M. ; Folk , R. ; e t al . (1983 ) A Stud y o f

th e Plasm aan dBon e Marro w Level s o f Glyphosat e followin g Intraperitonea l Administratio ni nth e Rat : Stud y No . 830109 . (Unpublishe dstud y receive dNo v 15 , 198 3 unde r 524-308; submitte db y Monsant o Co. , Washington , DC ; CDL:251737-F ) Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio no f

Glyphosate

— Reregistratio nEligibilit y Decisio n(RED ) 0013268 5

Stout , L. ; Johnson , C . (1987 ) 90-da y Stud y o f Glyphosat e Administere di nFee dt o Sprague/Dawle y Rats : Proj . I D ML-86

- 351/EFI L86128 . Unpublishe dstud y prepare db y Monsant o Agricultura l Co . 26 7 p . Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio no f

Glyphosat e Reregistratio nEligibilit y Decisio n(RED ) 4055940 1

8 1Stud y EP A Classificatio n MRID #

Ridley , W. ; Mirly , K . (1988 ) Th e Metabolis mo f Glyphosat e i n Spragu e Dawle y Rats—Par t I . Excretio n an dTissu e Distributio n o f Glyphosat e an dIt s Metablite s followin g Intravenou s an dOra l Administration : Laborator y Projec t No . 8613 9 (MSL-7215) : R.D . No . 877 . Unpublishe dstud y prepare db y Monsant o Co . 58 7 p . Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio n o f

Glyphosat e Reregistratio n Eligibilit y Decisio n (RED ) 4076710 1

Howe , R. ; Chott , R. ; McClanahan , R . (1988 ) Metabolis mo f Glyphosat e i nSprague-Dawle y Rats . Par t II . Identification , Characterization , an dQuantitatio n o f Glyphosat e an dIt s Metabolite s afte r

Intravenou s an dOra l Administration : Laborator y Projec t

No . MSL-7206 : R.D . No . 877 . Unpublishe d stud y prepare db y Monsant o Co . 15 5 p . Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio n o f

Glyphosat e Reregistratio n Eligibilit y Decisio n(RED ) 4076710 2

Forbi s A . (1989) . Uptake , Depuratio n an dBioconcentratio n o f 14 C-Glyphosat e t o Bluegil l Sunfis h(Lepomi s macrochints) : Projec t I D MSL-9304 . Unpublishe dstud y prepare db y Analytica l Biochemistr y Laboratories , Inc . 42 5 p . Dat aSupportin g Guidelin e Requirement s fo r

th e Reregistratio n o f

Glyphosate

— Reregistratio n Eligibilit y Decisio n (RED ) 4122830 1

Reyna , M . (1990 ) Tw o Generatio n Reproductio n Feedin g Stud y wit hGlysophat e i n Sprague-Dawle y Rats : La b Projec t No : MSL

10387 . Unpublishe dstud y prepare db y Monsant o Agricultura l Co . 115 8 Acceptable , core-guidelin e dat a (D176984,-85 ) 4162150 1

Stout , L. ; Ruecker , F . (1990 ) Chroni c Stud y o f Glyphosat e Adminitere di n Fee dt o Albin o Rats : La b Projec t Number : MSL

10495 : R.D . 1014 . Unpublishe dstud y prepare db y Monsant o Agricultura l Co . 217 5 p . Acceptable , core-guidelin e dat a (EP A Lette r

date dAugus t

25 , 1992 ) 4164380 1

Moxon , M . E . (2000 ) Glyphosat e Acid : Multi-generatio n Reproductio n Toxicit y Stud y i n Rats ; Repor t No . CTL/P/6332 . Unpublishe dstud y prepare db y Syngenta , t o b e submitted . No t Reviewed—Stud y t o b e Submitte d t o b e assigne d Dhinsa , N . K. , Watson , P . an dBrooks , P . N . (2007 ) Glyphosat e Technical : Dietar y Tw o Generatio n Reproductio n Stud y i n th e Rat . Unpublishe dstud y prepare db y Nufar mAmericas , t o b e submitted . No t Reviewed—Stud y t o b e Submitte d t o b e assigne d

8 2MRI D 0004636 2 

# International Researc h an dDevelopment

Corporatio n MATTAWAN , MICHIGAN , U.S.A . 4907 1 TELEPHON E (616 ! 668-333 6

SPONSOR : The Monsant o Company , IH.-79-01 6 TES T ARTICLE : Technica l Glyphosat e SUBJECT : Teratolog y Stud y i n Rat s DATEO FSUBMISSION : Marc h21 , 198 0

CONTAIN S TRAD E SECRET O R OTHERWIS E CONFIDENTIAL INFORMATIO N O FMONSANT O COMPAN Y

A0 0 7 4 2 3 (c ) Monsant o Compan y 198 0

401-05 4

"credenc e throug hresearch " 8 3

### MRI D 0004636 3 

# international Researc h an dDevelopmen

t Corporatio n MATTAWAN , MICHIGAN , U.S.A . 4907 1 TELEPHON E (616 ) 668-333 6



### SPONSOR : Monsant o Compan y, IR-79-01 8 TES T ARTICLE : Technica l Glyphosat e SUBJECT : 

### Teratolog y fitudy i n Rabbits DAT EO FSUBMISSION : Februar y 29 , 198 0

COMPAN Y ° N 

### ° F



### (c ) Monsant o Compan y 198 0

AA 0 0 7 4 2 1



### 401-05 6 'credenc e throug hresearch ' 8 4MRI D 0008167 4



# Bicyaynamic s Inc . Divisio n o f Biolog yan d Safet yEvaluatio n

PROJEC T NO . 77-206 3 BD N77-41 7

ATHRE E GENERATIO NREPRODUCTIO NSTUD Y I NRAT S

WIT H GLYPHOSAT E 

## Fina l Repor t Submitte d to : Monsant o Company

St . Louis , Missour i 6316 6

' Date : Marc h31 , 198 1

AA00743 7

MOHSKNTO COMPANY 198 1 \J N IAIN S I KAU t StCKt l O k ; . fHERWIS E CONFIDENTIA L 8 5MRI D 0010599 5

ADDITIONA L INFORMATIO N SUBMITTE D I N SUPPOR TO F TH E REGISTRATIO N O F ROUNDUP®

HERBICID E EP AREG . NO . 524-30 8 ATHREE-GENERATIO NREPRODUCTIO NSTUD Y I NRAT S WIT H GLYPHOSAT E

R . D . #37 4 SPECIA L REPOR T MS L #172 4

JUL Y 31 , 198 1

COMPILE D B Y R . W . STREE T

MONSANT O AGRICULTURA L PRODUCT S COMPAN Y 80 0 NORT H LINDBERG H BOULEVAR D ST . LOUIS , MISSOUR I 6316 6 CONTAIN S TRAD E SECRE T O R OTHERWIS E CONFIDENTIA L INFORMATIO N O F MONSANT O 8 6

### MRI D 0010809 9

MONSANTO COMMERCIA

L PRODUCT S CO . AGRICULTURA

L DIVISIO

N RESEARCH DEPARTMEN

T Agricultura l Researc h Repor t No . 29 8

Fina l Repor t on 

### C P 6757 3 RESIDU

E AN D METABOLISM Par t 9 : Th

e Gros s Distribution o f N-Phosphonomethylglycine- 14 

### C

(C P 67573- 14



### C ) i n th e Rabbi t

Jo b No . 9-23-760.06-786 3

Dat e Written : June 20 , 197 3 Writte n B y 

### an d Work Done By : L . B . Colvi n

J . A . Mille r Grou p Leader : J . T - Marve l AA01667 8 

### Thi s repor t contain s confidentia l information whic h i s the propert y o f Monsant o Compan

yan d whic h shal l be disclose d onl yt o dulyau thorized persons . The recipien t i s hel d accountabl e fo r the filin g an d saf e custod

yo f thi s report , whic h mus t b e returned o n demand .

8 7MRI D 0010817 1

CHRONI C TOXICIT

Y O F GLYPHO5AT E T O TH E FATHEA D MINNO W (Pimephale s promelas , Rafinesque) .

SUBMITTE D TO : . MONSANT O COMPAN

Y ST . LOUIS , MISSOUR I

E G & G , Bionomic s Aquati c Toxicolog

yLaborator

y 79 0 Mai n Stree t Wareham , Massachusett s October , 197 5

P M01354 2 8 8

# I 'fitl t MRI D 0010820 7 

# I I I I I I I ONE-GENERATIO NREPRODUCTIO NSTUDY - BOBWHIT E QUAI L GLYPHOSAT E TECHNICA L FINA L REPOR T



# I I I 1 I I I » I Submitte d t o Monsant o Compan

y St . Louis , Missour i 6316 6 

# WILDLIF E INTERNATIONA L LTD .

^03136 8 Novembe r1 , 197 8 CONTAIN S TRAD E SECRE T O R OTHERWIS E CONFIDENTIA L INFORMATIO N O F MONSANTO COMPAN Y 8 9MRI D 0011195 3

ONE-GENERATIO N REPRODUCTIO N STUD Y- MALLAR D DUC K

6LYPHOSAT E TECHNICA L FINA L REPOR T



### Submitte dt o Monsant o Compan y St . Louis , Missour i 6316 6 

# WILDLIF E INTERNATIONA L LTD .

Septembe r25 , 197 8 

# D 9 0

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MONSANT O COMPAN Y ENVIRONMENTA L HEALT H LABORATOR Y

64 5 S . NEWSTEAD ST . LOUIS , MISSOUR I 6311 0

AStud yO f Th e Plasma An d Bon e Marro wLevel s 

### O f Glyphosat e Followin g Intraperitonea l Administratio n I n Th e Ra t StudyNumber:83010 9 DME H Projec t Number : ML-83-21 8

Submitte d to : Monsant o Agricultura l Product s Compan y(Sponsor ) Through : Richar d C . Dirks , Produc t Toxicologis t



### Autho r and Stud

yDirector : Willia mP . Ridle y 

# 24 , * • 

### Willia mP . Ridley , Studj U Directo r

Marti n W . Dietrich , Gr/u p Leader / ' 

# /<y^y/ £ Roge r M . Polk , Directo r EH L / / © MONaANT O COMPAN Y 1983 ALLRIGHT S RESERVE D

AA00456 2 

### IEH L83010 9 iPAG E1

9 1MRI D 4055940 1

STUD Y TITL E 

### 90-Da yStud yo f Glyphosat e Administere d I n Fee d T o Sprague/Dawle yRat s

DAT AREQUIREMENT S Guidelin e 82-1 , 90-Da yFeedin g Stud

y- Roden t

AUTHOR S L.D . Stou t an

d C.W . Johnso n STUD Y COMPLETE D Novembe r 30 , 198 7

PERFORMIN

G LABORATOR

Y 

### Monsant o Agricultura l Compan y Environmenta l Healt h Laboratory

64 5 S . Newstea d St . Louis , Missour i 6311 0 

### LABORATORY PROJECT I.D . ML-86-351/EHL 8612 8

R.D . No . 86 0

' A '"" A0 0 038 8 

### Pag e 1 o f 26 7

9 2MRI D 4076710 1

STUD Y TITLE Volum e 1 : Th e Metabolis mo f Glyphosat e i n Spragu e DawleyRat s

- Par t I . Excretio n and Tissu e Distributio n o f Glyphosat e an

d It s Metabolite s following Intravenou s and Ora l Administration .

DAT AREQUIREMENT S Guidelin e 85-1 : Genera l Metabolis m

AUTHOR S W.P . Ridle yan

d K . Mirl y STUD Y COMPLETE D Marc h 23 , 198 8

PERFORMIN

G LABORATOR

Y Monsant o Compan

y Environmenta l Healt h Laboratory

64 5 S . Newstea d St . Louis , Missour i 6311 0

LABORATOR

Y PROJEC T HO 8613 9 (MSL-7215 ) R.D . No . 87 7 Pag e I o f VI I Followe d B

y Page s 1 thr u 58 7 8613 9 (MSL-7215 )

9 3MRI D 4076710 2 

### STUD Y TITL E Volum e 2 : Metabolis mo f Glyphosat e i n Sprague-Dawle yRats .

Par t II . Identification , Characteriziation , an d Quantitatio n o f Glyphosat e and It s Metabolite s Afte r Intravenou s and Ora l Administratio n

DAT AREQUIREMENT S Guidelin e 85-1 : Genera l Metabolis m

AUTHOR S R.K . Howe , R.C . Chott , an d R.H . McClanaha n STUD Y COMPLETE D February , 198 8

PERFORMIN

G LABORATOR

Y Monsant o Compan

y 80 0 N . Lindbergh Blvd . St . Louis , Missour i 6316 7 LABORATOR

Y PROJEC T N

O MSL-720 6 R.D . No . 87 7 

### Pag e I o f I X Followe d B

y Page s 1 thr u 15 5 MSL-720 6

AA00776 3 I 9 4MRI D 4122830 1

STUD Y TITL E Uptake , Depuration an d Bioconcentratio

n o f 14C Glyphoaat e t o Bluegil l Sunfish (Lapomi s macrochirue ) Par t I : MSL-930 4 PAT AREQUIREMEN

T Guideline 72- 6 AUTHOR A . D . Forbi s

STUD Y COMPLETE D August , 198 9 PERFORMIN

G LABORATOR

Y Analytica l Bio-Chemistr yLaboratories , Inc . Aquati c Toxicolog

yDivision 720 0 Eas t AB C Lan e P.O . Bo x 109 7 Columbia , M

O 6520 5 LABORATOR

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AA00783 4 9 5 MSL-930 4MRI D 4162150 1

STUD Y TITLE 

### Tw o Generation Reproduction Feedin g Stud ywit h Glyphosat e i n Sprague-DawleyRat s

DAT AREQUIREMEN

T Guidelin e 83- 4

AUTHOR | ' M.S . Reyna STUD Y COMPLETE D Augus t 27 , 199 0

PERFORMIN

G LABORATOR

Y 

### Monsant o Agricultura l Compan y 80 0 N . Lindberg

h Blvd . St . Louis , Missour i 6316 7

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Y PROJEC T NO . MSL - 1038 7 VOLUM E 1 o f 1 Pag e 1 o f 115 8



# •

AA0 0 2 ( f b ~MRI D 4164380 1

STUD Y TITLE 

### Chroni c Stud

yo f Glyphosat e Administere d i n Fee d t o Albin o Rat s DAT AREQUIREMEN

T Guidelin e 83- 5

AUTHOR S L.D . Stou t an

d F.A . Ruecke r STUD Y COMPLETE D Septembe r 26 , 199 0 PERFORMIN

G LABORATOR

Y 

### Monsant o Agricultura l Compan y ; 

### 80 0 N . Lindbergh Blvd . St . Louis , Missour i 6316 7 LABORATOR

Y PROJECT NO . MSL-1049 5 R.D . NO . 101 4 VOLUM E 1 o f 1 

### Pag e I o f I X followe d b

ypage s 1 t o 217 5

AA00202 0 9 7