DuPont-2983 8



### TRAD E SECRE T Position Paper

Title 

### METHOMYL : OTHE R SCIENTIFICALLYRELEVAN TINFORMATIO N PERTAININ G T O EP ATES TORDE R EDSP-090301-6 2 Author s Kari n S . Bentley , Ph.D . Carl aThomas , Ph.D . Test Guidelin e No t Applicabl e Dat e Complete d February 18 , 201 0 Applicant/Sponso r E . I . d u Pont d e Nemour s an dCompany Wilmington , Delawar e 1989 8 U.S.A . Author s Locatio n DuPon t Cro p Protectio n Stin e Haskel l Researc hCente r 109 0 Elkto n Roa d Newark , D E 1971 1 DuPon t Projec t Identificatio n DuPont-2983 8

••••• • • ' • • • • . • • • 

### • * I •

• 

## • . . " ••• » 

## •

Pag e 1 o f 4 1DuPont-2983 8 

## STATEMENT S O F CONFIDENTIALITY Th e Followin g Statemen t Applie s T o Countrie s Othe r Tha n Th e Unite dState s o f America : Thi s report

i s th e property o f

E . I . du Pon t

d e Nemour s an dCompan y an dcontain s confidential an dtrad e secret information . Excep t

a s require db y law , thi s report shoul dno t

b e partiall y o r

full y (i ) photocopie do r

release di n an y for mt o an outsid e party withou t

th e prio r writte n consent

o f E . I . du Pon t

d e Nemour s an dCompan y o r it s affiliates , o r

(ii ) use db y aregistratio n authority t o suppor t th e registratio n o f

an y other produc t withou t th e prio r writte n consent

o f E . I . du Pon t

d e Nemour s an d Company o r

it s affiliates .
Th e Followin g Statemen t Applie s T o Th e Unite dState s o f America : 

## STATEMEN TO F N o DATACONFIDENTIALIT Y N o clai mo f

confidentiality is.mad e fo r an y informatio n containe di n thi s study o n th e basi s o f it s fallin g withi n th e scop e o f FIFRA Sectio n 10(d)(l)(A) , (B) , o r (C) . Company E . I . du Pont

d e Nemour s an dCompan y i Company Agent : U.S . Produc t

Registratio n Manager 

# '1 0 Date W e hav e submitte dthi s materia l t o th e Unite dState s Environmenta l Protectio n Agenc y specificall y unde r th e provision s containe dh i FIFR A a s amende dand , . . , hereby , consen t

t o us e an ddisclosure o f

thi s material b y EP A accordifig*lt>*f?IFR/? . *• • * Notwithstandin g th e wordin g o f ou r markin g TRAD E SECRET , thi s marldfig , b y itself , convey s n o supplemental claim s o f confidentiality under

FIFRA'Jftfiljon s o r 10(b) . I n submittin g thi s material t o EP A accordin g t o metho dan dfora& t ^ . . requirement s containe di n P RNotic e 86-5 , w e d o no t waiv e an y prot&ftflto^o r

rigfa j . . • involvin g thi s material that woul dhav e bee n claime db y th e Compan y titffi s material ha dno t bee n submitte dt o th e EPA , no r d o w e waiv e an y protectio n o r righ t pro'SklsA,. . unde r FIFR A Sectio n 10(g) . *... . •..".".* •DuPont-2983 8



### GOO D LABORATOR YPRACTIC E COMPLIANC E STATEMEN T U.S . EP A Goo dLaborator y Practic e Standard s (4 0 CF RPai l 160 ) d o no t appl y t o th e current document . Applicant/Sponso r E . I . du Pon t

d e Nemour s an dCompan y Wilmington , Delawar e 1989 8 U.S.A . Autho r Kari n S . Bentley , Ph.D . ( / Senior Regulatory Toxicologis t Dat e Applicant/Sponsor DuPont Representativ e Date-DuPont-2983 8



### SIGNATUR E METHOMYL : OTHE R SCIENTIFICALLYRELEVAN TINFORMATIO N PERTAININ G T O EP ATES TORDE R EDSP-09030 1 -6 2 Position Pape

r by : Kariri S . Bentley , Ph.D . ~j j Date (/ Senior Regulatory Toxicologis t \ \ Carl aN . Thomas , Ph.D . Date Regulatory Ecotoxicologis t Date Completed : February 18,201 0 Applicant/Sponsor : E . I . d u Pont de Nemour s an dCompany Wilmington , Delaware 1989 8 U.S.A .

### DuPont-2983 8



### TABL E O F CONTENT S Titl e Pag e 1 Statement s o f

Confidentialit y 2 Goo dLaboratory Practic e Complianc e Statemen t ' . 3 Signatur e ; 4 Table o f Content s . 5 1. 0 Summary 6 2. 0 Introductio n an dBackgroun d6 2. 1 Othe r Scientificall y Relevan t

Informatio n (OSRI ) . ' 8 2. 2 Toxicologica l Profil e o f Methomy l 9 3. 0 Summary o f

Relevan t Mammalia n Toxicolog y Studie s 1 1 3. 1 Discussio n o f

studie s supportin g registratio n 1 1 3. 2 Discussio n o f

availabl e informatio n i n th e ope n literatur e 2 3 3.2. 1 Summar y o f

th e Potentia l t o Modulat e Estroge n o r Androge n Recepto r Functio n 2 3 3.2. 2 Summar y o f

th e Potentia l t o Modulat e Placenta l Aromatas e Activit y I n Vitr o 2 5 3.2. 3 Summary o f

I n Viv o Studies : ..........2 6 3. 3 Availabl e Informatio n fro mth e EP A ToxCast TM Projec t 2 8 4. 0 Summary o f

Relevan t Ecotoxicolog y Studie s : 2 8 4. 1 Discussio n o f

studie s supportin g registratio n 2 8 4. 2 Discussio n o f

availabl e informatio n i n th e ope n literatur e 3 2 5. 0 DuPon t

Positio n an dConclusion s 3 3 5. 1 Potentia l fo r

Methomy l t o Interac t

wit hth e Endocrin e Syste m3 3 5. 2 Functionall y Equivalen t Informatio n Fulfillin g th e EDS PTie r IBatter y 3 5 5. 3 Overal l Conclusio n 3 7 6. 0 Reference s 3 7 

### TABLE S Tabl e 1 Cite dMethomy l Toxicit y Studie s Supportin g Registratio n 4 0 Tabl e 2 Endocrin e Tissue s Evaluate di n Mammalia n Toxicolog y Studie s

Conducte dwit hMethomy l ; . • 4 1

### DuPont-2983 8



### METHOMYL : OTHE R SCIENTIFICALLYRELEVAN TINFORMATIO N PERTAININ G T O EP ATES TORDE R EDSP-09030 1 -6 2 1. 0 SUMMAR Y

Thi s documen t

i s i n respons e t o EP A Tes t Orde r EDSP-090301-6 2 requirin g DuPon t t o tes t th e pesticid e activ e ingredien t Methomy l (CAS #16752-77-5 ) i n th e Tie r 1 screenin g battery o f

th e Endocrin e Disruptio n Screenin g Progra m(EDSP) . DuPon t i s supplyin g scientificall y relevan t informatio n whic hfulfill s th e purpos e o f th e Tie r 1 EDS Pbattery - t o asses s th e potentia l t o interac t wit hestrogen , androge n an dthyroi d >hormon e processes . Methomy l ha s bee n evaluate di nalarg e numbe r o f mammalia n ' toxicolog y an decotoxicolog y studie s supportin g th e registratio n o f thi s produc t

whic h hav e assesse dpotentia l subchronic , chronic , reproductiv e o r developmenta l toxicit y i n rats , mice , dogs , rabbits , fish , o r birds . Severa l studie s fro mth e ope n literatur e hav e reporte dresult s for

the testin g of

methomy l in estroge n andandroge n transactivatio n assays , an dmethomy l ha s bee n include dfo r testin g i n th e man y endocrine-relate di n vitr o test s i n th e EP A ToxCast TM project . Th e result s overwhelmingl y demonstrat e th e lac k o f interactio n b y methomy l wit hestrogen , androgen , o r

thyroid . Therefore , furthe r assessmen t

o f methomy l i n th e Tie r

1 EDS P screenin g assay s i s no t

warranted . 

## 2. 0 INTRODUCTIONAN

D BACKGROUN D I n Augus t 1996 , Congres s passe dth e Foo dQuality Protectio n Ac t tha t require dth e U S Environmenta l Protectio n Agenc y (EPA ) "t o develo p ascreenin g progra musin g appropriat e validate dtes t

system s an dothe r

scientificall y relevan t

informatio n t o determin e whethe r

certai n substance s ma y hav e a n effec t i n human s tha t

i s similar t o an effec t produce db y anaturall y occurrin g estrogen , o r suc hothe r

effect s a s [EPA ] ma y designate" . EP A subsequentl y initiate dit s Endocrin e Disrupte r Screenin g Programi n 199 8 base do n th e recommendation s o f th e Agency' s Endocrin e Disruptio n Screenin g an dTestin g Advisory Committe e (EDSTAC) , whic hwa s compose do f

stakeholder s representin g industry , governmenta l agencies , researc h scientists , an denvironmenta l an dpubli c healt hgroups . A two-tiere dapproac htha t addresse s potentia l huma n an decologica l effect s an dexamine deffect s o n estrogen- , androgen- , an dthyroi dhormone-relate dprocesse s wa s recommended . Th e purpos e o f Tie r 1 "screening " i s t o identif

y substance s tha t

hav e th e "potential " t o interac t wit hestrogen , androgen , o r thyroi dhormon e system s usin g abatter y o f assays . Th e purpos e o f

Tie r 2 "tests " i s t o identif

y an destablis h"dose-respons e relationships " fo r an y advers e effect s tha t

migh t resul t fro mth e interaction s identifie di n th e Tie r 1 assays . Ove r

th e nex t year s th e Agenc y move dforwar dt o develo p an dvalidat e a numbe r o f Tie r I assays . I n Octobe r

2009 , EP A announce dth e availabilit y o f th e fina l Tie r 1 screenin g battery an dth e relate dtes t guidelines . Th e fina l Tie r I endocrin e screenin g battery i s compose do f th e followin g assays :DuPont-2983 8 I nvitr o assays : • • estroge n recepto r

bindin g - a n i n vitr o mode l whic hassesse s th e abilit y o f a substanc e t o interac t

wit hth e estroge n receptor s i n ra t uterin e cytoso l • androge n recepto r

bindin g - a n i n vitr o mode l whic hassesse s th e abilit y o f a substanc e to interac t

wit hthe androge n receptor s in rat prostat e cytoso l • estroge n transcriptiona l activatio n (HeLa990 3 cells ) - a n assa y tha t assesse s th e bindin g o f

achemica l t o th e estroge n recepto r an dth e subsequen t

downstrea m transcriptio n o f

areporte r gen e produc t • aromatas e - a n i n vitr o model t o identif y substance s tha t

ma y inhibi t

th e catalyti c activity o f th e enzym e ( C YP19 ) responsibl e fo r th e conversio n o f

androgen s t o estrogen s • steroidogenesi s (H295 Rcells ) - a n i n vitr o mode l intende dt o identif y substance s tha t interfer e wit hsteroi dhormon e synthesi s resultin g i n testosteron e an d estradiol/estron e productio n I n viv o mammalia n assays :

. • ra t uterotrophi c - a n i n viv o mode l t o asses s th e abilit y o f asubstanc e t o elici t uterin e weigh t

change s consisten t

withagonist s o r

antagonist s o f

natura l estrogen . • ra t Hershberge r

- a n i n viv o mode l t o asses s th e abilit y o f asubstanc e t o elici t mal e reproductiv e organ weigh t change s consisten t

wit handroge n agonist s an d antagonist s an d5a-reductas e inhibitor s • femal e ra t

pubertal - a n assay t o identif y substance s tha t

interac t wit hth e endocrin e syste mb y assessin g endpoint s associate dwit hpuberta l developmen t an dthyroi dfunctio n i n femal e animal s • mal e ra t

puberta l - a n assay t o identif y substance s tha t

interac t wit hth e endocrin e syste mb y assessin g endpoint s associate dwit hpuberta l developmen t an dthyroi d functio n i nmal e animal s • . In viv o wildlif

e assays : • Amphibia n metamorphosi s - ageneralize dvertebrat e mode l t o identif y substance s whic hinterfer e wit hth e norma l functio n o f th e hypothalamic

pituitary-thyroi d(HPT ) axi s • Fis hshort-termreproductio n i n fathea dminno w - ashort-termassay measurin g reproductiv e fitnes s an dbiochemica l endpoint s tha t reflec t effect s associate dwit h th e disturbanc e o f

th e hypothalamus-pituitary-gonada l (HPG ) axi s I n Apri l 2009 , EP A publishe dafina l lis t o f substance s t o b e initiall y teste di n th e Tie r 1 screenin g battery (Federa l Registe r 74 ; 17579) . Beginnin g Octobe r 2009 , EP A ha s issue dtes t

order s t o registrant s o f

th e liste dsubstance s whic hmandat e testin g unde r th e EDSP . A tes t

orde r fo r Methomy l ha s bee n receive db y DuPont , Order/DC I • EDSP-090301-62 , issue do n Novembe r 12 , 2009 .

### DuPont-2983 8

2. 1 Othe r

Scientifically Relevan t

Informatio n (OSRI

) EP A ha s provide dregistrant s a n opportunity t o eithe r submi t o r cit e existin g dat athat ma y fulfil l i n part

o r completel y th e requirement s o f th e Tie r 1 EDS Pscreenin g battery (EPA' s Approac hfo r Considerin g OSRI unde r

th e EDSP , Marc h26 , 2009) . EP A ha s provide dth e followin g understandin g o f wha t constitute s OSRI : "Other scientificall y relevan t

informatio n " i s informatio n tha t inform s th e determinatio n a s t o whethe r th e substanc e may hav e an effec t tha t i s similar t o an effec t produce db y asubstanc e tha t interact s wit hth e estrogen , androgen , and/o r thyroi dhormona l system s (e.g. , informatio n tha t identifie s substance s a s havin g th e potentia l t o interac t wit hestrogen , androgen , and/o r thyroi d system(s) ; informatio n demonstratin g whethe r substance s hav e a n effec t

o n th e functionin g o f

th e endocrin e system) . OSR I may eithe r

b e functionall y equivalen t t o informatio n obtaine dfro mth e Tie r 1 assay s —tha t

is , dat afro massay s tha t perfor mth e sam e functio n a s EDS P Tie r 1 assay s —o r

may includ e dat atha t provid e informatio n on apotentia l consequenc e or effec t tha t coul dbe due to effect s o n th e estrogen , androgen , o r thyroi dsystems . EPA furthe r

state s tha t

the followin g type s of

studie s may contai n informatio n that th e Agenc y woul dlikel y regar da s scientificall y relevan t information : studie s tha t correspon dt o assay s i n Tie r 1 an d2 ; metabolis mstudies ; o r othe r vertebrat e o r invertebrat e toxicit y studies , including , bu t no t limited.to , developmenta l toxicit y tests , carcinogenicit y tests , toxicogenomi c data , an dreproductiv e toxicit y tests . EP A goe s o n t o indicat e tha t

othe r type s o f studie s may als o b e considere dscientificall y relevant . Fo r

instance , testin g conducte dunde r 4 0 CF RPart

15 8 dat arequirement s t o suppor t pesticid e registratio n ma y b e considered . Judgment s b y EP A abou t whethe r th e OSRI satisfie s al l o r

part o f th e Tie r 1 Tes t Orde r wil l base do n th e "weight

o f th e evidence " fro mal l relevan t informatio n availabl e takin g int o accoun t th e datase t

a s a whol e fo r th e subjec t

chemical . , ; O n Octobe r

2 , 200 9 th e Whit e Hous e Offic e o f Managemen t

an dBudge t

(OMB ) approve dth e pendin g Informatio n Collectio n Reques t (ICR ) require dfo r

th e implementatio n o f

th e initia l Tie r

1 screenin g unde r

th e EDSP . I n approvin g th e initiatio n o f

thi s program , OM B urge dEP A t o "promote an dencourage " tes t orde r recipient s t o submi t

"functionall y equivalent " dat ai n lie u o f "performin g al l o r

som e o f th e Tie r 1 assays " an dt o accept

existin g FIFR A tes t

guidelin e dat a"t o th e greates t exten t possible" . A s part o f th e 4 0 CF RPart

15 8 dat arequirement s fo r registratio n unde r

FIFR A Sectio n 3 , pesticid e activ e ingredient s ar e require dt o b e evaluate di n abroa drang e o f mammalia n toxicolog y studie s includin g test s fo r acute , subchronic , an dchroni c toxicity , mutagenicity , acut e an dsubchroni c neurotoxicity , reproductiv e an d developmenta l toxicity , an dcarcinogenicit y an di n avariet y o f wildlif e studie s includin g test s fo r

toxicity i n fis han deffect s o n avia n reproduction . Man y o f thes e studie s provid e amean s fo r assessin g th e potentia l o f

asubstanc e t o alte r

endocrin e

### DuPont-2983 8 functio n (Steven s e t

al , 1997 ; ECETOC , 1996) . O f particular valu e i n th e assessmen t

o f advers e endocrine-mediate deffect s i n th e intac t organis mar e th e followin g type s o f

regulatory studies :

• Subchroni c toxicity studie s i n rats , mice , an ddoe s - Thes e studie s ar e capabl e o f detectin g hormonall y mediate dmacroscopi c an dmicroscopi c change s i n morpholog y and/o r siz e o f th e pituitary , thyroid , adrenals , uterus , ovary , mammar y gland , testes , epididymis , an dothe r

se x accessory organ s a s aresul t o f repeate dtreatment . Both DBS

- an dDDT-induce dincrease s i n uterin e weigh t wer e observe dh i thi s typ e o f study (Buri n e t

al , 1993) . • Prenata l developmenta l toxicity studie s i n rat s an drabbit s - Thes e studie s examin e hormonall y induce deffect s sucha s abortion , resorption , o r premature delivery , abnormalitie s i n reproductiv e development , an dmasculinizatio n o r feminizatio n o f

femal e an dmal e fetuses , respectively . Substance s suc ha s th e phytoestroge n coumestero l an dth e anti-androge n cyproteron e acetat e wer e detecte d i n thi s typ e o f

stud y (van Ravenzwaay , 1992) . • Multigeneratio n reproductio n stud y i n rat s - Th e multigeneratio n reproductio n study i s recommende db y EDSTA C (1998 ) a s th e definitiv e Tie r 2 stud y fo r

th e assessmen t

o f advers e effect s o f

hormonall y activ e substance s i n th e intac t animal . Therefore , thi s stud y play s apivota l rol e i n th e determinatio n o f advers e endocrin e effect s relativ e t o reproduction , growth , an ddevelopment . These studie s ar e capabl e o f detectin g hormonall y induce deffect s coverin g every phas e o f developmen t

an d reproductio n includin g behavior , maintenanc e o f pregnancy , gestatio n length , difficul t o r prolonge dparturition , fertility , se x rati o o f offspring , feminizatio n o r masculinizatio n o f

offspring , numbe r

o f pups , stil l births , an dgros s an dmicroscopi c patholog y o f

th e vagina , uterus , ovaries , testes , epididymis , semina l vesicles , prostate , etc . Procymidone-induce dhypospadia s an dvinclozolin-induce dfeminizatio n o f mal e pup s wer e identifie di n astud y o f thi s typ e (van Ravenzwaay , 1992) . • Combine dlifetim e chroni c toxicity/oncogenicity studie s i n rat s an dmic e an d chroni c toxicity studie s i n dos s - Thes e studie s includ e extensiv e histopatholog y o f primar y endocrin e tissue s an dsecondary accessory organ s includin g pituitary , thyroid , adrenals , ovary , uterus , vagina , mammar y gland , testes , epididymis , prostate , an dsemina l vesicles . Weak hormonall y mediate deffect s whic hresul t i n tumor s o f endocrin e tissues , suc ha s testicular tumor s (Hseu he t

al. , 1977 ) o r

mammar y glan d tumor s (Grugg s e t

a/. , 1983) , ar e ofte ndetecte di n thes e studies . 2. 2 Toxicologica

l Profil e o f

Methomy l Th e mammalia n toxicity o f methomy l i s wel l characterize dbase do n th e result s o f a n extensive'batter y o f

toxicolog y studie s conducte di n rats , mice , dogs , rabbit s an d Guine apig s an dwel l a s th e result s o f anumbe r o f i n vitr o assays . Th e primar y tbxicologica l effec t

o f thi s substanc e i n mammalia n specie s i s th e inhibitio n o f acetylcholinesters e i n th e nervou s system , whic hi s rapidl y reversible . A s aresul t methomy l demonstrate s hig hacut e toxicit y b y th e oral , ocular , an dinhalatio n routes . Th e primar y symptom s ar e thos e associate dwit hcholiriesteras e inhibition : N o cumulativ e effect s o n th e nervou s syste moccu r wit hrepeate dexposure .

### DuPont-2983 8 Hematolog y change s occurre donl y afte r chroni c exposur e an dwer e generall y mil d andregenerative . Methomy l doe s not induc e tumor s in rat s or

mic e andis classifie d b y EP A a s Grou p E(no t

likel y t o b e carcinogeni c t o humans) . Methomy l i s no t mutageni c an dha s produce dnegativ e result s i n abattery genotoxicity studies . N o increase dsensitivity o f

offsprin g wa s note di n multigeneratio n reproductio n an d developmenta l toxicity studie s a s offsprin g NOAEL s ar e equivalen t t o o r greate r tha n thos e fo r parenta l animals . Th e principl e effect s i n thes e studie s wer e mortality , clinica l sign s o f

cholinesteras e inhibition , bod y weigh t reductions , an dreduce d number s o f

pup s du e t o systemi c toxicity . Methomy l i s considere dneithe r a reproductiv e no r

developmenta l toxicant . Throughou t th e mammalia n toxicolog y database , ther e ha s bee n n o evidenc e suggestiv e o f an advers e effec t

o n an y componen t

o f th e endocrin e system . Methomy l ha s bee n extensivel y teste di n acut e an dchroni c studie s wit hanumbe r o f specie s o f ecologica l importanc e (birds , fish , aquati c an dterrestria l invertebrates , non-targe t plants) . Th e ecotoxicit y o f methomy l i s therefor e wel l characterized . Th e primar y toxicologica l effec t of methomy l in vertebrat e andinvertebrat e anima l specie s i s th e inhibitio n o f

acetylcholinesteras e i n th e nervou s system . Thi s effec t i s rapidl y reversible . A s aresult , methomy l demonstrate s hig hacut e toxicity . Th e mai n effect s i n chroni c exposur e wer e reduce dsurviva l an dgrowthi n fish , an dreduce d bod y weigh t

i n birds . Thes e effect s ar e indicativ e o f th e inheren t

toxicity o f

th e carbamat e insecticide . Th e existin g dat aavailabl e fo r methomy l fro mtoxicolog y an d ecotoxicolog y database s provid e compellin g evidenc e tha t methomy l i s no t

expecte d t o hav e a n advers e effec t

o n an y componen t

o f th e endocrin e system . Summarie s o f

relevan t existin g 4 0 CF RPart

15 8 toxicolog y an decotoxicolog y studie s tha t

support th e curren t

FIFR A Sectio n 3 registratio n o f methomy l an d provid e informatio n o n th e potentia l o f thi s produc t

t o interac t

withth e endocrin e syste mar e provide di n Section s 3 an d4 o f thi s document . Thes e studie s includ e subchroni c an dchroni c toxicit y studie s i n rats , mice , an ddog s an dreproductio n an d developmenta l studie s i n rat s an drabbits . Als o provide dar e summarie s o f studie s whic hhav e bee n conducte dt o evaluat e an y potentia l developmen t an dreproductiv e effect s o f methomy l i n bird s an dfis hspecies . Tabl e 1 list s fo r eac hstudy th e MRI D numbers , report

numbers , titles , whethe r

th e stud y wa s reviewe db y EPA , an dth e Agency' s classificatio n a s t o acceptability o f eac hstudy . I n addition , a s requeste db y EPA , har dcopie s o f

th e fron t pag e o f eac hreport

that ha s bee n previousl y submitte d t o EP A ar e bein g supplie dunde r separat e cover . Finally , informatio n availabl e fro m th e ope n literatur e tha t

i s relevan t

t o th e potentia l interactio n o f methomy l wit hth e endocrin e syste mi s als o summarized . Har dcopie s o f thes e paper s ar e bein g provide d unde r separat e cove r

a s acourtes y t o EP A fo r

th e Agency' s interna l us e wit hth e understandin g that

DuPon t doe s no t hav e authorit y t o gran t

EP A permissio n t o distribut e or

pos t thes e thir dparty studie s on the EPA website .

1 0DuPont-2983 8



### 3. 0 SUMMAR YO F RELEVAN TMAMMALIA N TOXICOLOG YSTUDIE S 3. 1 Discussio n of

studie s supportin g registratio n Paynter , O.E . (1966) ; Thre e mont hdietar y administratio n - rat s insecticid e 1179 . DuPon t Repor t No . MRO-848 , Hazleto n Repor t No . 201-151 , MRID 00007190 , satisfie s Guidelin e 82-1 . Material s an dMethods : I n a13-week

feedin g study , Methomy l (purit y 100% ) wa s administere dt o mal e an d femal e Crl:CD®B Rrat s (10 . rats/sex/group ) at concentration s o f

0 , 10 , 50 , an d 25 0 ppm . A fifthgrou p o f mal e an dfemal e rat s wa s fe d12 5 pp mfo r 6 week s an d 500 ppmfor

the remainde r

of the study . In thi s grou p only , 5 animals/se x wer e sacrifice da t

2 month s an dth e remainde r wer e sacrifice da t

stud y termination . Parameter s evaluate dinclude dbod y weight,'bod y weigh t gain , foo dconsumption , clinica l signs , haematology , urinalysis , plasm aan dre dbloo dcel l cholinesteras e activity , orga n weights (includin g adrenals , pituitary , an dtestes) , an dgros s an d microscopi c patholog y (includin g adrenals , testes , epididymis , ovary , uterus , pituitary , an dthyroid) . Findings : Genera l observations : N o tes t substance-relate dclinica l sign s o f toxicit y wer e note dat an y dos e level , wit hth e exceptio n o f transien t tremor s observe di n on e femal e rat fro mth e 25 0 pp mgrou p at 1 3 weeks . Tes t

substance-relate dmortalit y di d no t occu r durin g th e cours e o f

thi s study . Growthwa s suppresse di n th e 25 0 pp m male s an ddurin g th e sevenththroug hth e thirteenthweek s i n th e 50 0 pp mmale s an d female s (significantl y differen t fro mcontro l value s i n th e 50 0 pp mmale s only) . Foo d consumptio n fo r

th e 50 0 pp mmale s wa s lowe r bu t no t significantl y differen t

from'th e controls . Foo dconsumptio n fo r th e 25 0 pp mmale s an d50 0 pp mmale s an dfemale s wa s slightl y lowe r

compare dwit hcontrols . Haematology , clinica l chemistry , cholinesteras e activity , urinalysis : Mino r haematologica l change s wer e noted . Compare dwit hcontrols , significantl y lowe r , haemoglobi n value s wer e observe di n th e 25 0 pp mmale s a t 2 months . A t

3 months , re dbloo dcel l count s fo r

th e 25 0 pp mfemale s wer e significantl y lowe r bu t stil l withi n norma l limits . N o significan t difference s wer e foun di n cholinesteras e activity o r urinalysi s Values . Gros s pathology , orga n weights , histopathology : N o tes t substance-relate dgros s morphologica l change s wer e observe da t necropsy . Significantl y decrease dlive r weight s an dincrease dbrain/bod y weigh t ratio s wer e observe dfo r

th e 125/50 0 pp m an d25 0 pp mmale s sacrifice dafte r 13-weeks . Thes e difference s fro mcontro l value s wer e relate dt o lowe r

bod y weight s o f

thes e animal s whe n compare dt o controls . Microscopi c examinatio n o f tissue s fro mth e 25 0 pp mrat s reveale dmoderat e erythroi dhyperplasi ai n th e bon e marro w o f al l mal e rat s i n thi s group . Ther e wer e n o tes t substanc e relate deffect s suggestiv e o f a n advers e effec t

o n th e endocrin e syste m


### 1 1DuPont-2983 8 Conclusion : Th e NOAE Li n th e 90-da y feedin g stud y i n rat s wa s 5 0 pp m (approximatel y 3. 6 mg/k g bw/day ) fo r male s base do n decrease dbod y weight/foo d consumptio n at

concentration s > 25 0 pp man da n increase dincidenc e o f erythroi d hyperplasi ai n th e bon e marro w o f male s a t 25 0 ppm . Th e NOAE Lfo r female s wa s 5 0 pp m(approximatel y 4. 1 mg/k g bw/day ) base do n th e lowe r RB C an dhaemoglobi n a t 25 0 pp man dbod y weight/foo dconsumptio n effect s a t 50 0 ppm . Ther e wa s n o evidenc e o f

adirec t advers e effec t

o n th e endocrin e syste mi n thi s study . Cox , R.H . (1979a) ; A subacut e (9 0 day ) evaluatio n ofNudri n i n th e Fischer-34 4 ra t fo r Shell Oi l Company . DuPon t Repor t No . 52 B 101-630-78 , MRID none . Material s an dMethods : I n a90-da y feedin g study , Methomyl , dissolve di n a n acetone/co moi l mixtur e (2 % w/w) , wa s administere dt o mal e Fische r rat s (2 0 rats/sex/group ) a t concentration s o f 0 , 100 , 200 , 40 0 an d60 0 ppm . Additionally , on e grou p o f 2 0 male s wa s fe d 2 0 pp mfro mweek s 1- 9 o f th e stud y followe db y 100 0 pp mfro mweek s 10-1 3 o f th e stud y an danothe r

grou p o f 2 0 male s wa s fe d5 0 pp mfro mweek s 1- 4 followe db y 80 0 pp mfro mweek s 5-13 . Femal e Fische r rat s (2 0 rats/sex/group ) wer e administere dconcentration s o f 0 , 135 , 270 , 540 , an d81 0 ppm . Additionally , on e grou p o f 2 0 female s wa s fe d2 7 pp mfro mweek s 1- 9 followe db y 150 0 pp mfro m week s 10-1 3 an danothe r

grou p o f 2 0 female s wa s fe d6 8 pp mfro mwee k 1- 4 followe db y 108 0 ppm'fro mweek s 5-13 . Parameter s evaluate dinclude dbod y weight , foo dconsumption , clinical signs , haematology , clinica l chemistry , urinalysis , plasm aan dRB C (re dbloo dcell ) cholinesteras e activity , orga n weights (includin g adrenals , teste s an depididymis , ovary an dfallopia n tubes , uterus) , an dgros s an d microscopi c patholog y (includin g adrenal s pituitary , testes , epididymis , ovary , fallopia n tube , uterus , cervix , an dthyroid) . Findings : Genera l observations : Tes t substance-relate dmortalit y di dno t occu r durin g th e cours e o f thi s study . Tes t

substance-relate dtransien t bod y weigh t

difference s wer e note dthroughou t

th e stud y i n male s an dfemale s administere d100 0 pp man dhigher ; however , ther e wer e n o difference s note db y th e en do f th e study . Transien t significan t decrease s i n foo dconsumptio n wer e als o note dthroughou t th e study . Haematology , clinica l chemistry , cholinesteras e activity , urinalysis : Clinica l patholog y change s indicativ e o f asystemi c effec t

include ddecrease s i n haemoglobi n at dose s o f 40 0 pp man d50/80 0 fo r

male s an d54 0 pp mo r

greate r i n females . RB C count s wer e decrease di n 60 0 pp mmale s an d81 0 pp mfemales . Reticulocyte s wer e significantl y increase di n 600 , 50/800 , an d20/100 0 pp mmale s an di n female s a t 20 0 pp man dgreater . Decrease dglucos e level s wer e note dfo r mal e rat s give n 400 , 600 , an d50/80 0 pp mfo r

6 week s bu t

no t afte r 1 3 weeks . Glucos e wa s als o decrease d i n female s rat s receivin g 54 0 pp mo r greater . Ure awa s increase di n 27/150 0 pp m female s at wee k 13 . N o distinc t

o r apparen t tes t substance-relate ddifference s wer e observe di n th e result s o f

th e urinalysi s conducte ddurin g wee k 7 o r a t termination . Decrease s i n plasm acholinesteras e activit y wer e note dfo r female s receivin g 

### 1 2DuPont-2983 8 81 0 pp mo r

greate r bu t similar change s wer e no t

observe di n th e mal e rats . I n contrast , ther e wa s a n apparen t treatmen t

relate ddecreas e i n RB C cholinesteras e activit y fro mth e 50/80 0 pp mmale s a t termination , bu t

similar change s wer e no t observe di n th e femal e rats . Gros s pathology , orga n weights , histopathology : Absolut e and/o r relativ e splee n weight s wer e increase di n male s receivin g 60 0 pp mo r greate r an di n female s receivin g 54 0 pp mo r

greater . Relativ e weight s o f th e liver s an dkidney s wer e increase dfo r

female s receivin g at

an dabov e 54 0 pp man d81 0 ppm , respectively . No.distinc t

o r consisten t

tes t substance-relate dmicroscopi c change s wer e note di n tissu e section s evaluated . Ther e wer e n o tes t substanc e relate deffect s suggestiv e o f a n advers e effec t

o n th e endocrin e system . Conclusion : Th e NOAE Li n male s wa s 20 0 pp m(13. 6 mg/k g bw/day ) base do n haematolog y change s at

40 0 pp man dgreater . Th e NOAE Li n female s wa s 13 5 pp m (10. 0 mg/k g bw/day ) base do n haematolog y change s at 27 0 pp man dgreater . Ther e wa s n o evidenc e o f

adirec t advers e effec t

o n th e endocrin e syste mi n thi s study . Cox , R.H . (1979b) ; A subacut e (9 0 day ) evaluatio n ofNudri n i n th e B6C3F 1 mous e fo r Shell Oi l Company . DuPon t Report No . 52 E 004-630-79 . MRID none . Material s an dMethods : In a90-day feedin g study , Methomy l was administere dto mal e andfemal e Crl:CD®B Rmic e at

dietar y concentration s o f

0 , 75 , 150 , 300 , 480 , 600 , an d96 0 pp m (20/sex/grou p fo r

control s an d10/sex/grou p fo r tes t substanc e group s excep t

th e 60 0 pp mgrou p whic hha d1 1 male s an d9 females) . Parameter s evaluate dinclude d bod y weight , foo dconsumption , clinica l signs , clinica l pathology , organ weight s (includin g adrenals , testes , ovarie s wit hfallopia n tubes , anduterus) , andgros s and microscopi c patholog y (includin g adrenals , testes , prostate , ovaries , fallopia n tube , an duterus) . Findings : Genera l observations : Tes t substance-relate dmortalit y di dno t occu r durin g th e cours e o f thi s study . There wer e n o consisten t tes t substance-relate dclinica l sign s note do r effect s o n bod y weight s o r foo dconsumption . Haematology : Haematologi c difference s indicativ e o f asystemi c effec t

include d decrease dlevel s o f

haemoglobi n i n male s at

an dabov e 30 0 pp mwit happaren t compensatio n at

60 0 an d96 0 ppm , a s indicate db y increase s i n erythrocyte . count s an dhaematocri t

accompanie db y decrease dreticulocyt e counts . Female s at 15 0 pp m an dhighe r

ha ddecrease derythrocyt e count s wit hincrease s i n reticulocyt e count s at " an dabov e 48 0 pp mindicativ e o f th e initiatio n o f

compensation . Haematocri t wa s als o significantl y decrease dfo r th e 15 0 pp man d60 0 pp mfemales . Ther e wer e n o tes t substanc e relate deffect s o n clinica l chemistry o r urinalysi s parameters . Gros s pathology , orga n weights , histopathology : Mal e mic e receivin g 48 0 pp m an dabov e ha dincrease dabsolut e an drelativ e live r weights . Othe r

orga n weigh t difference s wer e considere ddu e t o mino r difference s i n fina l bod y weight s an dno t tes t substance-related . Ther e wer e n o consisten t tes t substance-relate dgros s o r 

### 1 3DuPont-2983 8 microscopi c patholog y finding s noted . Ther e wer e n o tes t substanc e relate deffect s suggestiv e o f

an advers e effec t

o n th e endocrin e system . Conclusion : Th e NOAE Li n mal e mic e wa s 15 0 pp m(approximatel y 26. 6 nag/k g bw/day ) base do n haematolog y change s a t 30 0 pp man dabove . Th e NOAE Li n -femal e mic e wa s 7 5 pp m(approximatel y 15. 6 mg/k g bw/day ) base do n haematolog y change s at

15 0 pp man dabove . Ther e wa s n o evidenc e o f adirec t advers e effec t

o n th e endocrin e syste mi n thi s study . Sherman , H . (1967) ; Three-mont hfeedin g stud y o n dog s wit hS-methy l N-[(methylcarbamoyl)oxy ] thioacetimidat e [Lannat e Methomy l insecticide ; INX-1179] . DuPon t

Repor t No . HLR168-67 , MRID 00009010 , doe s no t satisf y Guidelin e 82-1 , bu t

sufficien t dat afro machroni c stud y i s available . Material s an dMethods : In a90-da y feedin g study , Methomy l (purit y 97.5% ) was administere dto mal e and femal e beagl e dog s ( 4 dogs/sex/group ) at concentration s o f

0 , 50 , 100 , an d40 0 pp m i n th e diet . Parameter s evaluate dinclude dbod y weight , foo dconsumption , clinica l signs , haematology , clinica l chemistry , orga n weight s (adrenal , pituitary , testes , prostate , an dthyroid), , an dgros s an dmicroscopi c patholog y (includin g adrenal , pituitary , testes , epididymis , prostate , ovary , uterus , fallopia n tube , mammar y gland , an dthyroid) . Findings : Genera l observations : N o tes t substance-relate dclinica l sign s wer e note ddurin g th e cours e o f th e study . Tes t

substance-relate dmortalit y di dno t occur . Ther e wer e n o tes t substance-relate deffect s o n bod y weight s o r foo dconsumption . Haematology , clinica l chemistry , urinalysis : N o tes t substance-relate deffect s o n haematology , urinalysis , o r clinica l chemistr y parameter s wer e noted . Gros s pathology , orga n weights , histopathology : N o tes t substance-relate dgros s lesion s wer e observe dat

necropsy . N o tes t

substance-relate dchange s i n mea n orga n weight s o r

orga n weight s relativ e t o fina l bod y weigh t wer e apparen t

a t an y dietary , concentration . N o gros s o r microscopi c lesion s wer e note dtha t coul db e attribute dt o administratio n o f

th e tes t substance . Ther e wer e n o tes t substanc e relate deffect s suggestiv e o f

a n advers e effec t

o n th e endocrin e system . Conclusion : Th e NOAE Li n th e 90-da y feedin g stud y i n dog s wa s 40 0 pp mfo r male s an dfemale s (14. 7 an d12. 5 mg/k g bw/day , respectively) , th e highes t dos e tested . Ther e wa s n o evidenc e o f adirec t advers e effec t

o n th e endocrin e syste mi n thi s study .



### 1 4DuPont-2983 8 Kaplan , A.M . (1981) ; Long-termfeedin g stud y i n rat s withS-methy l N-((methylcarbamoyl)oxy ) thioacetimidate ) (Methomyl ; INX-1179) . DuPon t Repor t No . HL R235-81 , MR1 D 00078361 , satisfie s Guidelin e 83-5 , 83- 1 a , 83-2a . • • 

### \ Material s an dMethods ; I n a2-yea r

chroni c toxicit y an dcarcinogenicit y feedin g study , Methomy l (purit y 99% ) wa s administere dt o mal e an dfemal e Chr:CD ® rat s (8 0 rats/sex/grou p plu s a n additiona l 2 0 rats/sex/group designate dfo r erythrocyt e an dbrai n cholinesteras e determinations ) at

concentration s o f

0 , 50 , 100 , an d40 0 ppm . Te n rat s pe r grou p wer e sacrifice dafte r

approximatel y 1 yea r

o n study , an dal l survivin g rat s wer e sacrifice dafte r

approximatel y 2 year s o n study . Parameter s evaluate dinclude dbod y weight , bod y weigh t

gain , foo dconsumption , foo defficiency , clinica l signs , clinica l pathology , organ weights (includin g adrenal , pituitary , an dtestes) , gros s an d microscopi c patholog y (includin g adrenal , pituitary , testes , epididymis , prostate , ovary , uterus , mammary , andthyroid) , anderythrocyt e andbrai n cholinesteras e activity . . Findings : Genera l observations : N o tes t substance-relate dclinica l sign s o f toxicity wer e note ddurin g th e study . Ther e wa s n o tes t substance-relate deffec t

o n mortality . Mal e an dfemal e rat s fe d40 0 pp mha dsignificantl y lowe r bod y weigh t

gai n durin g th e firs t year o f th e study . Male s i n th e 40 0 pp mgrou p ha dasimila r reductio n i n rat e o f weigh t gai n fo r tes t day s 0-560 . Ther e wer e n o tes t substanc e relate deffect s o n foo d consumptio n o r

foo defficiency . Haematology , clinica l chemistry , urinalysis : Female s i n th e 40 0 pp mgrou p ha d significantl y lowe r

erythrocyt e counts , haemoglobi n values , an dhaematocri t whe n compare dt o controls . A similar tren doccurre di n erythrocyt e count s an d haemoglobi n value s i n mal e rat s fe d40 0 ppm ; however , th e change s wer e no t statisticall y significant . Ther e wer e n o tes t substance-relate dclinica l chemistr y o r urinalysi s finding s noted . \ Erythrocyt e cholinesteras e activity : Erythrocyt e cholinesteras e activit y wa s slightl y lowe r

(no t statisticall y significant ) fo r th e 50 , 100 , an d40 0 pp mmale s durin g th e 0.25 , 0.5 , an d1-mont

hevaluations ; however , ados e response-relationshi p wa s no t eviden t an dther e wer e n o effect s i n females . There wer e n o effect s o n brai n cholinesteras e activit y a t

eithe r 1 o r 2 years . Gros s pathology , orga n weights , histopathology : N o tes t substance-relate dgros s lesion s wer e observe da t

necropsy . Ther e wer e n o histopatholog y finding s noted . Ther e wer e n o tes t

substance-relate dabsolut e orga n weigh t change s i n eithe r

se x at an y concentratio n no r

wer e ther e an y relativ e orga n weigh t finding s i n mal e rats . Femal e rat s at

an dabov e 10 0 pp mha dsignificantl y increase drelativ e liver weights ; however , thi s di dno t

correlat e wit han y histopathologica l changes . Female s fe d 40 0 pp mals o ha dincrease drelativ e splee n weight s whic hma y hav e bee n correlated ' wit hrea r footpa ddermatitis , acommo n naturall y occurrin g lesio n i n wir e cage-reare d agein g rats . Ther e wa s n o increas e i n neoplasti c lesion s i n eithe r male s o r female s a t 

### 1 5DuPont-2983 8

an y dietar y concentration . Ther e wer e n o tes t substanc e relate deffect s suggestiv e o f a n advers e effec t

o n th e endocrin e system . Conclusion : Th e NOAE Li n th e 2-year feedin g stud y i n rat s wa s 10 0 pp mfo r male s an dfemale s (4. 8 mg/k g bw/da y fo r male s an d6. 3 mg/k g bw/da y fo r females) . Thi s NOAE Lwa s base do n decrease dweigh t gai n an dhaematolog y change s at 40 0 ppm . Unde r th e condition s o f

thi s study , Methomy l wa s no t acarcinogen . Ther e wa s n o evidenc e o f

adirec t advers e effec t

o n th e endocrin e syste mi n thi s study . Snyder , F . G . (1981) ; 104-wee k chronic toxicity an dcarcinogenicity stud y i n mic e Methomyl ; H- l 1,135 . DuPon t Repor t No . HL O 253-81 , Hazelto n Report No . 201

- 510, MRID 00078423 , satisfie s Guidelin e 83-2b . ' Material s an dMethods : I n a104-week

carcinogenicit y feedin g study , Methomy l (purity 99% ) wa s administere dt o mal e an dfemal e CD®- 1 mic e (approximatel y 8 0 mice/sex/group ) a t concentration s o f

0 ppm , 5 0 ppm , 10 0 pp m(reduce dt o 7 5 at wee k 39 ) an d80 0 pp m (reduce dt o 40 0 at

wee k 2 8 an dfurthe r

reduce dt o 20 0 at

wee k 39) . Parameter s evaluate dinclude dbod y weight , bod y weigh t gain , foo dconsumption , foo d efficiency , clinica l signs , haematology , organ weight s (includin g adrenal , pituitary , an dtesti s wit hepididymis) , an dgros s an dmicroscopi c patholog y (includin g adrenal , pituitary , testes , epididymis , prostate , ovary , uterus , mammary , an dthyroid) . Findings : Genera l observations : N o tes t substance-relate dclinica l sign s wer e note ddurin g th e study . Th e incidenc e o f

gros s o r palpabl e masse s wa s similar fo r al l groups . Decrease dsurviva l occurre di n 10 0 pp mfemale s an d80 0 pp mmale s an dfemales . Du e t o thi s result , th e hig hdos e grou p wa s reduce dt o 40 0 pp ma t wee k 2 8 an d subsequentl y reduce dt o 20 0 pp mat wee k 39 . Th e 10 0 pp mgrou p wa s reduce dt o 7 5 pp mals o at

wee k 39 . Surviva l continue dt o b e lowe r i n 7 5 an d20 0 pp mmale s an dfemale s an dwa s lowe r i n al l treate dmale s at

wee k 104 . Ther e wer e n o tes t substance-relate deffect s o n bod y weight s o r bod y weigh t

gains . Ther e wer e n o tes t substance-relate deffect s o n foo dconsumptio n o r foo defficiency . Haematology : Significantl y lowe r haematocri t

wa s note di n 10 0 an d80 0 pp mmale s at wee k 13 . Significantl y lowe r

haemoglobi n leve l an dre dcel l coun t wer e note di n 80 0 pp mmale s at

wee k 26 . Decrease s i n haemoglobi n level s wer e note di n th e 10 0 an d80 0 pp mfemale s at

week s 1 3 an d2 6 (significantl y lowe r i n 10 0 pp mfemale s at wee k 13) . Decrease s i n re dcel l count s wer e als o note di n 10 0 an d80 0 pp mfemale s at wee k 26 : Thes e change s ar e indicativ e o f ates t substance-relate deffec t

o n re dcel l mass . Thi s tren dwa s no t

note dafte r

wee k 26 : Gros s pathology , orga n weights , histopathology : Ther e wer e n o tes t substance

relate dgros s o r

histopathologica l finding s noted . Th e incidenc e o f neoplasti c lesion s wa s simila r

amon g al l groups . Th e orga n weigh t dat awa s unremarkabl e excep t fo r a significantl y highe r

absolut e an dslightl y elevate drelativ e adrena l weigh t i n 80 0 pp m males . However , ther e wa s n o correspondin g chang e i n th e relativ e adrena l weight . 

### 1 6DuPont-2983 8 Moreover , th e increase dabsolut e adrena l weigh t wa s mos t likel y th e resul t

o f a n unusuall y hig hadrena l weigh t i n asingl e anima l (0.01 4 gm) . Th e remainin g adrena l weight s i n thi s dos e grou p range dfro m0.00 5 t o 0.01 0 gm , whic hwa s similar t o th e rang e o f adrena l weight s observe di n th e contro l .group (0.00 4 t o O.'Olgm) . Therefore , th e increase dabsolut e adrena l weigh t i n th e 40 0 pp mmal e mic e wa s no t considere d t o b e tes t substance-related . Ther e wer e n o tes t substanc e relate deffect s suggestiv e o f a n advers e effec t

o n th e endocrin e system . Conclusion : Th e NOAE Li n th e 104-wee k feedin g stud y i n mic e wa s 5 0 pp mfo r bothmale s andfemale s (8. 7 mg/k g bw/da y for male s and10. 6 mg/k g bw/da y for females) . Thi s NOAE Lwa s base do n increase dmortalit y i n 7 5 pp man dgreate r male s an dfemales . Unde r th e condition s o f

thi s study , Methomy l wa s no t a carcinogen . Ther e wa s n o evidenc e o f adirec t advers e effec t

o n th e endocrin e syste m i n thi s study . Busey , W.M . (1968) ; Two-yea r dietar y administratio n —Dogs : Lannat e Methomy l insecticide (S-methyl-N-((methylcarbamoyl)oxy ) thioacetimidate) . DuPon t Repor t No . Hazleto n 201-165 MRID 00007091 , satisfie s Guidelin e 83- 1 b . Material s an dMethods : I n a2-year

feedin g study , Methomy l (purity 90-100% ) wa s administere dt o mal e an d femal e beagl e dog s ( 4 dogs/sex/group ) at concentration s o f

0 , 50 , 100 , 400 , an d 100 0 ppm . Parameter s evaluate dinclude dbod y weight , foo dconsumption , clinica l signs , haematology , clinica l chemistry , urinalysis , orga n weight s (includin g adrenals , testes , an dthyroid) , an dgros s an dmicroscopi c patholog y (includin g adrenals , pituitary , testes , ovaries , uteru s an dthyroid) . Findings : Genera l observations : Ther e wer e n o tes t substance-relate dclinica l sign s note da t o r belo w 40 0 pp mi n eithe r

sex . Tw o 100 0 pp mmale s exhibite dtes t substance

relate deffect s includin g tremors , salivation , incoordination , an dcirclin g movements . Ther e wa s als o a n increase dincidenc e o f diarrhoe ai n 1 mal e administere d100 0 pp m whe n compare dt o controls . On e 100 0 pp mfemal e wa s foun ddea ddurin g wee k 9 . Thi s do g wa s replace do n stud y an dth e replacemen t do g als o die dafte r

1 8 days o n test . Deatho f

th e replacemen t

do g wa s precede db y convulsiv e seizure s an dcoma . Th e death s o f

thes e 2 female s wer e considere dtes t substance-related . Ther e wer e n o tes t substance-relate deffect s o n bod y weight s o r foo dconsumption . Haematology , clinica l chemistry , urinalysis : Haematologica l studie s reveale d sligh t t o moderat e anaemi ai n fou r 100 0 pp mdog s a t

th e 3-monthinterva l an d persisten t tes t substance-dependen t

anaemi ai n on e o f

th e 100 0 pp mmales . Temporary tes t

substanc e withdrawa l durin g 1 0 consecutiv e week s resulte di n improvemen t

o f th e condition . Ther e wer e n o tes t substance-relate dclinica l chemistr y o r

urinalysi s finding s noted . Plasm acholinesteras e activit y wa s slightl y lowe r tha n th e contro l valu e i n on e mal e do g an don e femal e do g administere d 

### 1000 ppm . 1 7DuPont-2983 8 Gros s pathology , orga n weights , histopathology : On e 1000 pp mmal e exhibite da n enlarge dsplee n an dliver . Th e enlarge dlive r contribute dt o ahighe r

mea n absolute ari drelativ e live r

fo r th e 100 0 pp mmales . Th e splee n weigh t wa s s o larg e i t

wa s exclude dfro mth e calculatio n o f th e mean . Kidney/bod y weigh t ratio s fo r thre e 1000 pp mmale s an don e 40 0 pp mmal e wer e slightl y t o moderatel y increase d compare dt o controls . Tes t substanc e relate dhistopathologica l alteration s wer e see n i n th e spleen s an dkidney s o f th e 40 0 an d100 0 pp mmal e ari dfemal e group s an di n th e liver s an dbon e marro w o f th e 100 0 pp mmal e an dfemal e groups . Th e change s see n i n th e kidney s wer e characterise db y a n increas e i n pigmentatio n an dsligh t swellin g o f

th e epithelia l cell s o f

th e proxima l convolute dtubules . Pigmen t depositio n wa s see n i n th e 40 0 an dlOO O pp mmale s bu t onl y a t 100 0 pp mi n females . Increase dpigmen t

an dextramedullary hematopoiesi s wer e observe di n th e spleen s o f 100 0 pp manimals , an dpigmen t wa s observe di n th e spleen s o f 40 0 pp manimals . Minima l t o sligh t

bil e duc t proliferatio n i n th e live r

an dslightl y increase derythroi d an dmyeloidcell s i n th e bon e marro w wer e observe di n 100 0 pp manimals . Ther e wer e n o tes t

substanc e relate deffect s suggestiv e o f a n advers e effec t

o n th e endocrin e system . . Conclusion : Th e NOAE Li n th e 2-year feedin g stud y i n dog s wa s 10 0 pp mfo r male s an dfemale s (3. 0 mg/k g bw/day) . Thi s NOAE Lwa s base do n histopatholog y change s i n th e kidne y an dsplee n at 40 0 pp man dabove . Ther e wa s n o evidenc e o f a direc t advers e effec t

o n th e endocrin e syste mi n thi s study . Lu , C.C . (1983) ; Nudri n 2 generatio n reproductio n stud y i n rats . DuPon t Repor t No . WRCRIR-27 5 (HL O 519-95) , MRID 4325070 1 an d43769401, , satisfie s Guidelin e 83-3a . Material s an dMethods : I n atwo-generatio n reproductio n study , Methomy l wa s administere di n th e die t t o mal e an dfemal e CD ® rat s at concentration s o f

0 , 75 , 600 , an d120 0 ppm . Eac hdos e grou p consiste do f

1 3 mal e an d2 6 femal e rat s fo r th e F O generation . Du e t o lo w conceptio n rate s fo r

F O rats , additiona l rat s wer e selecte dfo r th e F I generatio n a t

wee k 1 2 t o increas e eac hgrou p t o 2 0 male s an d4 0 females . Th e 120 0 pp mdos e grou p ha d 3 8 female s instea do f

4 0 du e t o ashortag e o f

animal s at

th e tim e o f

selection . Th e F O rat s wer e bre dwithi n thei r treatmen t group s t o produc e F I litter s afte r 10 0 day s o n test . Th e F I rat s wer e bre dwithi n thei r respectiv e treatmen t

group s t o produc e F 2 litter s afte r

12 0 day s o n test . Parameter s evaluate dinclude dbod y weight , foo d consumption , clinica l signs , clinica l pathology , bloo dcholinesteras e activity , reproductiv e indices , spermcounts , litte r an dpu p parameters , orga n weight s (includin g ovary , uterus , an dteste s i n F O an dF I adult s an di n F I an dF 2 pups ; adrenal s i n F I adult s an dF I an dF 2 pups) , an dgros s an dmicroscopi c patholog y (includin g adrenal , uterus , an dteste s i n F O adults ; adrenals , pituitary , testes , prostate , ovary , uterus , mammary , an dthyroi di n F I adult s an dF I an dF 2 pups) .



### 1 8DuPont-2983 8 Findings : F n an dF i Parenta l Rat s Genera l observations : Withth e exceptio n o f alopeci aobserve da t

an dabov e 60 0 ppm , ther e wer e n o tes t substance-relate dclinica l sign s observe di n eithe r generatio n throughou t

th e duratio n o f

th e study . Tes t

substance-relate dmortality di d no t occu r durin g th e cours e o f

thi s study . Bod y weight s wer e reduce di n parenta l F O an dF I male s an dfemale s i n th e 60 0 an d120 0 pp mgroups . Bod y weight s i n th e F I parenta l female s i n th e 7 5 pp mgrou p wer e slightl y lowe r (3-9 % lowe r

compare d t o controls) . Althoug hth e bod y weight s o f 7 5 pp mfemale s wer e significantl y lowe r compare dt o control durin g prematin g an dgestation , th e averag e differenc e fro m control durin g thes e period s wa s 6.5% . Thi s sligh t differenc e wa s no t

considere d lexicologicall y significant . Bod y weight s i n th e 7 5 pp mF ] parenta l male s wer e reduce dat

sporadi c intervals . Thes e bod y weigh t reduction s i n th e 7 5 pp m F I parenta l male s an dfemale s reflec t th e reduce dstartin g weights i n thes e groups , sinc e thei r rat e o f growthwa s comparabl e t o th e contro l group . Foo dconsumptio n fo r rat s i n th e 7 5 pp mF O parental s wa s sporadicall y increased . Foo dconsumptio n i n th e 60 0 an d120 0 pp mF O parental s tende dt o b e reduce dwit hsporadi c statisticall y significan t difference s fro mcontrols . • Sporadi c reduction s i nfoo dconsumptio n i nth e 7 5 pp mF I parental s als o occurred . Foo dconsumptio n in'th e F I 60 0 an d120 0 pp m parenta l animal s wa s significantl y reduced . Reproductiv e parameters : N o tes t substance-relate deffect s o n reproductio n wer e detecte di n analyse s o f

mea n spermcounts , gestationa l length , lactation , an dfertilit y indice s i n an y o f

th e dos e group s i n bothgenerations . Ther e wer e n o apparen t tes t substanc e relate deffect s o n endocrin e syste mfunctio n relativ e t o reproductiv e performance , foeta l development , sexua l maturation , an dgenera l growthan d development . Gros s pathology , orga n weights , histopathology , i n parenta l animal s an dpups : N o tes t substance-relate danatomica l change s wer e note dgrossl y o r microscopicall y i n tissue s o f

parenta l animal s an dselecte dpup s i n an y dos e grou p fro meithe r generation . I n th e F O parents , th e followin g orga n weight s wer e significantl y differen t fro mcontro l du e t o bod y weigh t reductio n i n th e 60 0 an d120 0 pp mgroups : absolut e uterus , spleen , an dbrai n weights ; relativ e spleen , brain , an dteste s weights . I n th e F I parents , th e followin g orga n weight s wer e significantl y differen t fro mcontro l du e t o reductio n i n bod y weigh t o f 60 0 an d1200 pp mrats : absolut e brain , heart , liver , adrenal , kidney , testes , ovary , uterus ; relativ e spleen , brain , heart , kidney , testes . I n th e F I an dF apups , th e followin g organ weight s wer e significantl y differen t fro m' contro l du e t o reductio n i n bod y weigh t o f 60 0 an d120 0 pp mpups : absolut e heart , spleen , adrenal , kidney , testes ; relativ e testes , liver , kidney , spleen , adrena l weights . Ther e wer e n o histopathologica l change s i n an y o f thes e tissues . Occasiona l spuriou s orga n weigh t

difference s wer e als o note di n 7 5 pp man dF I an dF arat s fo r whic hther e wer e n o histopathologica l changes . Ther e wer e n o tes t substanc e relate deffect s o n orga n weigh t

o r histopathologica l change s suggestiv e o f a n advers e effec t

o n th e endocrin e system .



### . 1 9DuPont-2983 8 Bloo dcholinesteras e activity , clinica l patholog y i n parenta l animal s an dpups : N o tes t substance-relate deffec t

o n bloo dcholinesteras e fo r th e F O rat s wa s observe d i n an y group . Significan t

reduction s i n mea n value s o f re dbloo dcel l count , haemoglobin , an dhaematocri t wer e observe donl y i n th e F O females;i n th e 60 0 an d 120 0 pp mgroup s whe n compare dwit hcontrols . Othe r bloo dparameter s wer e no t affected . F i andF i litte r

an dpu p dat a 

### \ Genera l observations : Durin g lactation , pu p weight s i n th e 60 0 an d120 0 pp m group s wer e significantl y reduce din bothgenerations . In the FI generatio n only , bod y weights fo r

th e 7 5 pp mgrou p wer e approximatel y 9 % lowe r than control s at birthan dremaine dlowe r

b y th e sam e proportio n throughou t th e lactatio n period . Thi s effec t

becam e statisticall y significan t b y da y 4 . However , th e lowe r weigh t o f th e 7 5 ppniF ] pup s i s no t

considere dtes t

substance-relate dfo r

th e followin g reasons : 1 ) There wa s n o effec t

o n bod y weigh t

at 7 5 pp mi n th e F ^ pup s at

th e sam e concentratio n whos e parent s wer e expose dfo r alonge r perio do f time . 2 ) Th e litte r

. siz e o f th e 7 5 pp mF I pup s wa s th e larges t i n eithe r generation . Base do n greate r nutritiona l an dphysiologica l demands , pu p siz e an dweigh t ar e ofte n reduce dan dca n remai n reduce dthroughou t lactation . T o illustrat e thi s point , th e bod y weigh t o f th e 7 5 pp mF I pup s ar e compare dt o thos e o f th e F2,controls , whic har e close r i n litte r siz e tha n th e F I controls . Whe n th e 7 5 pp mF I mea n pu p weight s ar e examine d relativ e t o th e F I controls , i t i s apparen t

tha t ther e ar e n o tes t

substance-relate dbod y weigh t effect s i n th e 7 5 pp mF I pups , whe n compare dwit hacontro l litte r o f similar size . Litte r siz e an dpu p survival : A tes t substance-relate ddecreas e i n th e litte r siz e an d th e numbe r

o f liv e pups,wa s observe dfo r 120 0 pp mF I pup s an d60 0 an d120 0 pp m p 2 pups . A spuriou s decreas e i n litte r siz e an dliv e pup s i n th e 7 5 pp mF 2 generatio n wa s observe ddu e t o on e femal e tha t delivere dasingl e dea dpup . A large r p 2 contro l litte r (large r tha n th e F I generation ) contribute dt o th e statistica l significanc e o f th e 7 5 pp mF 2 litte r

siz e o n day .1 . Becaus e th e smalle r litter siz e wa s primaril y du e t o a n even t i n asingl e litter , i t

i s considere dt o b e neithe r biologicall y significan t

o r treatmen t related . Pu p surviva l wa s reduce dfo r bothF I an d¥ 2 generation s i n th e 120 0 pp mgrou p only . Reduce dpu p surviva l occurre dpredominantl y durin g th e firs t fou r day s of lactation . Ther e wer e no tes t substance-relate dchange s on the proportio n o f

mal e an dfemal e pup s i n eithe r th e F I o r F 2 generation . Conclusion : Ther e wa s n o evidenc e o f adirec t advers e effec t

o n th e endocrin e syste mi n thi s study . Th e NOAEL s i n thi s stud y ar e th e following : Parenta l toxicity : 7 5 pp m(4. 6 mg/k g bw/da y fo r F O males , 4. 8 mg/k g bw/da y fo r

F O females , 6. 7 mg/k g bw/da y fo r F I males , 6. 3 mg/k g bw/da y fo r

F I females) , base do n decrease d bod y weigh t

i n F O / F I male s an dfemale s a t

60 0 pp m an dabove . Reproductio n an d120 0 pp mbase do n n o effect s i n th e F O / F ] generatio n fertility : (th e highes t concentratio n tested) .
2 0DuPont-2983 8 Pu p growthan d7 5 pp mbase do n decrease dpu p weigh t i n F ] / ? 2 pup s development : durin g lactatio n at 60 0 pp man dabove . Culik , R. , Rogers , A.S . (1978) ; Oral teratogeni c stud y i n rat s wit hLannat e (INX-1179) . DuPon t

Repor t No . HLR498

- 78 , MRID 00008621 , satisfie s Guidelin e 

### 8 3- 3 a . Material s an dMethods ; In adevelopmenta l toxicit y study , Methomy l (purity 99% ) was administere din die t to mate dChr-C D femal e rat s (25/dos e group ) o n gestatio n days 6-15 . Dose s wer e 0 , 50 , 100 , o r 40 0 ppm . Thi s wa s equivalen t t o 0 , 4.9 , 9.4 , an d33. 9 mg/kgbw/day , respectively . Parameter s evaluate di n dam s wer e bod y weight , foo dconsumption , survival , clinica l signs , reproductiv e outcome s (includin g corpor alute ai n eac hovary , implantatio n site s i n eac hhorn , numbe r an dlocatio n o f

foetuses) , an dgros s pathology . Parameter s evaluate di n foetuse s wer e bod y weight , incidence s o f dea d foetuse s and/o r

foeta l resorptions , crown-rump lengtho f eac hliv e foetus , an d incidence s o f

external , visceral , an dskeletal malformation s an dvariations . Findings : Materna l effect s Genera l Observations : N o tes t substance-relate dclinica l sign s o f toxicit y o r mortalit y wer e observe di n an y o f th e tes t group s durin g th e study . Bod y weights wer e significantl y lowe r

tha n control s i n th e 40 0 pp mdos e group . Rat s administere d 40 0 pp mha dlowe r

foo dconsumptio n compare dt o controls . Reproductiv e outcomes : Parameter s measurin g th e outcom e o f pregnanc y an d foeta l development , includin g th e numbe r o f litter s wit hpartia l an dtota l resorptions , an dth e bod y measurement s o f th e foetuse s wer e no t

differen t fro mthos e o f

th e concurren t

controls . Ther e wer e n o apparen t test-substanc e relate deffect s o n endocrin e functio n pertainin g t o maintenanc e o f pregnanc y o r

foeta l development . Gros s pathology : Tes t

substance-relate dan dadvers e gros s morphologica l change s wer e no t observed . Th e uteru s an dovarie s wer e give n agros s patholog y examination . , Foetal effect s Smal l subcutaneou s haematoma s an dpetechia l haemorrhage s o n variou s part s o f th e bod y wer e foun di n foetuse s fro mlitter s o f al l groups , includin g controls . On e run t wa s foun di n eac ho f

th e thre e tes t

groups . Mos t

anomalie s an dmalformation s foun d i n th e soft-tissu e sectionin g wer e isolate doccurrence s wit htw o exceptions : apparen t hydroriephrosi s (foun di n al l group s a t approximatel y th e sam e rate ) an dpeliosi s (observe di n al l bu t

40 0 pp mfoetuse s at

arat e similar

t o controls ) o f

th e liver . N o tes t substance-relate dmalformatio n o r abnormalitie s o f

th e skeleta l syste mwer e noted .



### 2 1DuPont-2983 8 Conclusion : Th e NOAE Li s 10 0 pp m(9. 4 mg/k g bw/day ) fo r materna l effects . Th e materna l NOAE Li s base do n bod y weigh t reductio n a t

40 0 ppm . Th e foeta l NOAE L i s 40 0 pp mbase do n n o effect s at th e highes t

dos e tested . Ther e wa s n o evidenc e o f a direc t advers e effec t

o n th e endocrin e syste mi n thi s study . Feussner , E.L. , Hoberman , A.M. , Christian , M.S . (1983);,Embryo-fetal toxicity an d teratogenicity stud y ofMethomy l i n th e rabbit . DuPon t Report No . HL O 331-83 , MRID 00131257 , satisfie s Guidelin e 83-3b . Material s an dMethods : I n adevelopmental toxicity study , Methomy l (purit y 98.7% ) wa s administere db y ora l gavag e t o time-mate dDLLNZ Wfemal e rabbit s (20/dos e group ) o n gestatio n day s 7-19 . Gavag e dose s i n revers e osmosi s processe ddeionise dwate r wer e 0 , 2, ' 6 , an d 1 6 mg/k g bw/day . Th e dos e volum e wa s 5 mL/k g bw . Parameter s evaluate di n dam s wer e bod y weight , bod y weigh t gain , foo dconsumption , survival , clinica l signs , reproductiv e outcome s (includin g corpor alutea , numbe r an dplacemen t

o f implantations) , an dgros s pathology . Uterin e weigh t wa s measure dwit han dwithou t th e product s o f

conception . Parameter s evaluate di n foetuse s wer e bod y weight , incidence s o f

dea dfoetuse s and/o r

foeta l resorptions , an dincidence s o f external , viscera l (including-interna l confirmatio n o f sex) , an dskeletal , malformation s an d variations . . ; Findings : Materna l effect s Genera l Observations : Tes t substanc e relate dcentra l nervou s syste mstimulatio n wa s observe di n th e 1 6 mg/k g bw/da y rabbits . Clinica l sign s o f toxicit y include d tremors , hyperactiviry , bod y jerks , exces s salivation , aggressiv e behaviour , hyperpnea , convulsions , ataxia , impaire do r los t rightin g reflex , ski n lesions , dyspnea , hypersensitivity , pin-poin t pupils , an dvocalisation . Th e incidenc e o f tes t substance

relate danorexi awa s significantl y decrease dfo r rabbit s fe d1 6 mg/k g bw/day . Thi s observatio n wa s considere dt o b e areboun di n foo dconsumptio n afte r th e tes t substanc e wa s n o longe r

administered . Seve n o f

th e rabbit s i n thi s dos e grou p died . Mortality occurre di n on e rabbi t fe d6 mg/k g bw/da y whic hwa s no t considere dt o b e tes t substance-relate dsinc e th e deathoccurre d8 day s followin g th e cessatio n o f dosing . A smal l tes t

substance-relate dincreas e i n materna l bod y weigh t gai n occurre dbetwee n day s 0 an d2 9 o f gestation ; howeve r

onl y th e gai n o f

th e 1 6 mg/k g bw/da y dos e grou p differe dsignificantl y fro mcontrols . Thi s differenc e wa s du e t o th e decrease dincidenc e o f anorexi ao f

th e dam s i n th e 1 6 mg/k g bw/da y grou p durin g th e latte r

portio n o f

gestation . Reproductiv e outcomes : A tes t substance-relate dincreas e i n averag e foeta l bod y weigh t occurred ; th e differenc e fro mth e contro l valu e wa s statisticall y significan t only'fo r th e 6 an d1 6 mg/k g bw/day.dos e groups . Thi s increas e i n foeta l bod y weigh t wa s no t considere dt o b e a n advers e effec t sinc e i t wa s no t observe dt o b e du e t o th e presenc e o f

oedema . Methomy l di dno t adversel y affec t

th e incidenc e o f

pregnanc y o r th e averag e numbe r

o f corpor alutea , implantations , resorptions , an dliv e foetuse s 

### 2 2DuPont-2983 8 pe r dam . Ther e wer e n o apparen t test-substanc e relate deffect s o n endocrin e functio n pertainin g t o maintenanc e o f pregnanc y o r

foeta l development . Foetal effect s Administratio n o f

2 , 6 , an d1 6 mg/k g bw/da y di dno t resul t i n advers e effect s o n embryo/foeta l viabilit y o r foeta l se x ratio . Similarly , n o gros s external , sof t tissu e o r skeleta l malformations , developmenta l variation s o r variation s du e t o retarde d developmen t

observe dfo r

foetuse s wer e attribute dt o tes t substanc e administration . Conclusion : NOAEL : 6 mg/k g bw/da y fo r materna l effect s base do n decrease dbod y weight , mortality , an dclinica l sign s o f cholinesteras e inhibitio n at 1 6 mg/k g bw/day . Th e foeta l NOAE Lwa s 1 6 mg/k g bw/da y base dn o effect s at th e highes t

dos e tested . There wa s n o evidenc e o f adirec t advers e effec t

o n th e endocrin e systemi n thi s study . 3. 2 Discussio n o f

availabl e informatio n i n th e ope n literature 3.2. 1 Summary o f

th e Potentia l t o Modulat e Estroge n o r Androge n Recepto r

Functio n I n aserie s o f

publications , researcher s use di n vitr o assay s t o evaluat e th e abilit y o f methomyl , an daclos e structura l analogu e oxamyl , t o modulat e activity o f th e estroge n (ER) , androge n (AR) , o r progesteron e (PR ) receptors . I n th e report

b y Klotz , Arnol dan dMcLachla n (1997) , methomy l an doxamy l wer e evaluate dfor

their abilit y to modulat e ERandPRactivit y usin g transactivatio n assay s (MCF- 7 huma n breas t

cance r cells , Ishikaw ahuma n endometria l cance r cells , an d T47 D huma n breas t

cance r cells ) an dwhole-cel l competitiv e bindin g assays . I n th e transactivatio n assays , methomy l andoxamy l wer e teste dat aconcentratio n of 10 0 n M an dth e response s compare dt o 17p-estradio l (50 0 p M i n MCF- 7 cells ; 1 n M i n Ishikaw acells ) o r

progesteron e ( 1 n M i n T47 D cells) . I n th e whole-cel l competitiv e bindin g assays , methomy l an doxamy l wer e teste da t concentration s of . 10 0 nM , 1 uM , an d1 0 jj, M an dth e abilit y t o inhibi t bindin g o f

17p-estradio l (50 0 p M i n MCF- 7 cell s fo r

E Rbindin g assay ) o r

th e P Ragonis t

R502 0 ( 1 n M i n T47 D cell s fo r P Rbindin g assay ) wa s measured . The y repor t tha t bot hmethomy l an doxamy l weakl y activate dER

- an dPR-responsiv e reporte r gene s an ddecrease destradiol

- o r progesterone-induce dreporte r gen e activity i n breas t

an dendometria l cance r

cells . Th e author s als o report

tha t a s aclass , th e carbamate s teste ddemonstrate dlimite d capacit y t o displac e E Ro r P Rrecepto r

ligand s fro mth e E Ro r

P Ri n whol e cel l assays . The y sugges t

tha t th e carbamate s evaluate dac t a s partia l steroi dhormon e recepto r agonists/antagonist s i n bindin g assay s throug hamechanis mseparat e fro m ligan dbinding . Nishihar aan dcoworker s (2000 ) evaluate dmor e tha n 50 0 chemicals , includin g methomyl , i n ayeas t

two-hybri dtransactivatio n assay syste mwit hth e estroge n recepto r ER aan dth e coactivato r

TIF2 . Methomy l wa s teste du p t o amaximu m concentratio n o f

2x10~ 4 M , an dth e respons e wa s compare dt o th e activit y o f 17(3-estradiol . Methomy l showedn o activit y unde r th e condition s o f

th e study . I n th e report

b y Anderse n an dcoworker s (2002) , methomy l wa s evaluate dfo r it s abilit y t o modulat e E Ran dP Rrecepto r activit y usin g transactivatio n assay s 

### 2 3

### DuPont-2983 8 [MCF7 cells , an dChines e Hamste r Ovar y (CHO ) Cells] , an dit s abilit y t o induc e cel l proliferatio n i n MCF- 7 huma n breas t cancer cells , amarke r

o f estrogeni c activity . I n th e transactivatio n assays , methomy l wa s teste dove r aconcentratio n rang e o f

10" 8 

### t o 5xlO" 5 

### M an dth e response s wer e compare dt o th e activit y o f 17(3-estradio l (1 0 n M i n MCF- 7 cells ) o r

th e P Ragonis t

R188 1 (0. 1 n M i n CH O cells) . I n th e cel l proliferatio n assays , methomy l wa s teste dover aconcentratio n rang e o f

10" 8 

### t o 5xlO" 5 

### M i n MCF- 7 cell s an dth e response s wer e compare dt o th e activit y o f 17(3-estradio l (concentratio n rang e o f

10~ 1 2

### t o 12" 8 

### M) . Fo r

th e transactivatio n assay s an dth e cel l proliferatio n assay , methomy l wa s als o teste dafte r co-administratio n with 17p-estradio l (0.0 1 n M i n th e cel l proliferatio n assay i n MCF- 7 cells ; 1 0 n M fo r th e E Rtransactivatio n assay i n MCF- 7 cells ) o r R188 1 (0. 1 n M fo r

th e transactivatio n assay i n CH O cells) . Unde r th e condition s o f

th e study , methomy l showe dn o significan t activit y i n an y o f

th e E Ro r A Rtransactivatio n assay s o r th e cel l proliferatio n assay . I n th e report

b y Kojim aan dcoworker s (2004) , methomy l an doxamy l wer e evaluate d fo r it s abilit y t o modulat e recepto r activit y usin g transactivatio n assay s fo r huma n ERa , huma n ERp , o r

huma n A Ri n CH O cells . I n al l o f th e assays , methomy l an d .oxamy l wer e teste dove r

aconcentratio n rang e u p t o 10" 5 

### M an dth e response s wer e compare dt o th e activit y o f 17p-estradio l o r

dihydrotestosteron e (DHT ) fo r th e E Ro r A Rassays , respectively . Relativ e luciferas e activity wa s calculated'relativ e t o th e positiv e control s (17p-estradio l fo r ER aan dERP ; DH T fo r

AR) . I n addition , fo r measuremen t

o f antagonis t

activity , assay s wer e evaluate dwit hth e tes t materia l co-administere dwit hnp-estradio l (10' " M fo r ERa ; 10" 1 0 

### M fo r

ERp ) o r DH T (10" 1 0 

### M ) fo r

th e E Ran dA Rtransactivatio n assays , respectively . Under th e condition s o f

th e study , methomy l an doxamy l showedn o significan t activit y i n an y o f E Ro r A Rtransactivatio n assays . I n th e repor t

b y Nendz aan dWenze l (2006) , methomy l an doxamy l wer e evaluate d fo r thei r abilit y t o modulat e E Ractivit y usin g aYeas t Estroge n Scree n (YES) . I n th e assay , methomy l an doxamy l wer e teste dove r aconcentratio n rang e o f

10 0 mg/ Lt o 5 0 (ig/mL , an dth e response s wer e compare dt o th e activit y o f 17p-estradio l (concentratio n 3,00 0 ng/ml t o 1. 5 ng/ml) . Unde r th e condition s o f

th e study , methomy l an doxamy l showe dn o significan t activit y i n th e YE S assay . I n th e report

b y Jia o an dcoworker s (2008) , atransgeni c yeas t screenin g syste musin g fis hE Rwa s establishe dan duse dt o evaluat e anumbe r o f xenobiotic s includin g methomyl . Methomy l wa s teste dat concentration s o f

10" 9 

### , 10" 7 

### , an d10" 5 

### M and.th e response s wer e compare dt o th e activit y o f 17p-estradio l o r

dihydrotestosteron e (DHT ) fo r th e E Ro r A Rassays , respectively . I n addition , fo r measuremen t

o f antagonis t activity , assay s wer e evaluate dwit hth e tes t materia l co-administere dwit h 17p-estradio l (concentratio n no t stated) . Unde r

th e condition s o f

th e study , methomy l showe dn o activit y t o th e fis hE Ri n an y o f th e transactivatio n assay s tha t wer e performed . Together , th e dat afro mthes e report s sugges t tha t methomyl , an dit s clos e structura l analogu e oxamyl , d o no t

interac t withth e E Ro r

AR . Th e dat afro mfiv e o f th e si x report s wer e al l negative . I n th e report b y Klot z an dcoworkers 1 

### , th e author s clai m methomy l an doxamy l activate dth e E Ran dPR . However , the y als o report that 'al l 2 4DuPont-2983 8

o f th e carbamate s teste dweakl y activate dER

- o r PR-responsiv e reporte r

gene s an d decrease destradiol

- o r

progesterone-induce dreporte r gen e activity i n breas t

an d endometria l cance r

cells . Th e author s als o report tha t th e carbamate s teste d demonstrate dlimite dcapacit y t o displac e E Ro r P Rrecepto r

ligand s fro mth e E Ro r P Ri n whol e cel l assays . The y sugges t tha t th e carbamate s ac t

a s partia l steroi d hormon e recepto r

agonists/antagonist s i n bindin g assay s throug hamechanis m separat e fro mligan dbinding . However , severa l factor s i n thes e studie s sugges t tha t th e observe deffect s wer e non-specifi c an dunrelate dt o tru e receptor-mediate d effects . Thes e response s di dno t occu r i n adose-dependen t

manner , aserin e proteas e inhibito r (phenylmethy l sulfony l fluoride ) demonstrate dvery simila r activity , an d finally , th e compound s appeare dt o affec t bothth e E Ran dP Rsystem s t o a n equal extent . Thes e ar e importan t point s i n tha t

whil e ther e i s "cross-talk " betwee n recepto r system s i t i s rar e tha t

acompound , endogenou s o r exogenous , wil l equall y affec t mor e tha n on e recepto r

system . I n fact , man y compound s evaluate di n thi s stud y ha d similar potencies , suggestin g tha t ther e ar e non-specifi c factor s involved . Thi s stud y provide s n o soli devidenc e tha t methomy l o r

oxamy l ar e steroi dhormon e recepto r agonist/antagonist , an dth e author s themselve s stat e tha t th e reporte dactivit y wa s no t recepto r mediated . Th e weigh t

o f evidenc e suggest s tha t

neithe r methomy l no r oxamy l interac t

wit hth e E Ro r

AR . Tw o recen t

paper s describ e effort s b y Roncaglionre ? al. , (2008 ) an dCeli k e t al , (2008 ) t o develo p structura l activity model s fo r interactio n wit hth e huma n estroge n receptor . Roncaglio n e t

al. , correlate dth e result s o f a900-chemica l databas e fo r huma n ER arecepto r

bindin g an dreporte r

gen e assay s wit hvariet y o f parameter s tha t characterize drecepto r

binding . The sourc e of

activit y dat ain the ERbindin g assay s wa s the , Japanes e MET I databas e (OEC D EDTA , 2002) , whic hi s note db y th e author s t o b e on e o f

th e larges t

collection s o f

dat afo r ER . Methomy l i s indicate da s bein g inactiv e fo r

E Ractivit y accordin g t o thi s database . I n th e pape r b y Celi k e t al , acomparativ e dockin g stud y o f wel l know n huma n ER aligand s an danumbe r o f selecte dorgani c compound s wa s conducte dt o asses s th e potentia l o f thes e substance s t o interac t wit hth e ligan dbindin g domai n o f th e receptor . Methomy l wa s amon g . thre e pesticide s examine dtha t gav e lo w affinit y score s suggestin g tha t i t ma y bin d ver y poorl y o r

no t a t al l t o th e huma n ERa . 3.2. 2 Summar y o f

th e Potentia l t o Modulat e Placenta ! Aromatas e Activit y I n Vitro I n th e repor t

b y Anderse n an dcoworker s (2002) , methomy l wa s evaluate dfo r it s abilit y t o modulat e CYP1 9 aromatas e activity i n huma n placenta l microsome s usin g th e classi c tritiate dwate r

releas e method . Methomy l wa s teste da t asingl e concentratio n (5 0 uM ) an dth e respons e wa s compare dt o th e activit y o f 4-androsten

4-ol-3,17-dion e ( 1 jaM) . Whe n teste dat asingl e concentratio n (5 0 urn ) unde r th e condition s o f

th e assay , methomy l induce daromatas e activit y t o approximatel y 120 % o f control . Whil e thes e dat asugges t tha t methomy l ma y hav e th e potentia l t o modulat e aromatas e activity , th e significanc e o f th e findin g i s unclear

give n th e result s wer e no t

repeate di n thi s study , onl y asingl e tes t concentratio n o f

methomy l wa s evaluated , an dn o finding s suggestiv e o f a n advers e effec t

o n aromatas e ha s bee n observe di n th e man y anima l studie s conducte dwit hthi s product . 

### 2 5DuPont-2983 8

3.2: 3 Summary o f

In Viv o Studies : Th e purpose o f

th e researc hconducte db y Porte r an dcoworker s (1993 ) wa s t o determin e th e effect s o f

aldicarb , methomyl , and/o r metribuzin , alon e o r

i n combination , o n plasm ahormon e (thyroxin e an dsomatotropin ) concentration s i n Sprague-Dawle y rats . Thyroxin e (T4 ) i s athyroi dhormon e tha t participates i n th e regulatio n o f

metabolis man dfoo dintake , a s wel l a s differentiatio n an dmaturatio n o f numerou s cel l types . Somatotropin , als o know n a s growthhormone , i s secrete db y th e pituitar y glan dan dregulate s soffran dbon y tissu e growth , a s wel l a s othe r metaboli c processes . Thes e hormone s wer e selecte da s indicator s o f norma l bod y function . I n thi s study , 6 Sprague-Dawle y rats/sex/grou p wer e treate dwithaldicarb ( 1 o r 1 0 ppb) , metribuzi n (100 0 o r 10,00 0 ppb) , and/o r

methomy l (10 0 o r

100 0 ppb ) i n th e drinkin g water

fo r 6 o r 1 6 weeks . Bloo dwa s draw n fro mfemal e rat s afte r 2 an d6 week s o f

exposur e an dfro mmal e rate s afte r 7 , 13 , an d1 6 week s o f

exposure . Plasm awa s analyze dfo r

T4 , Tj , uptake , an dsomatotropin . Afte r 2 week s o f

exposur e femal e rat s ha dplasm aT 4 concentration s tha t wer e significantl y increase db y eithe r metribuzi n o r

th e combinatio n o f

th e thre e chemical s a t th e hig hdos e levels . T 4 concentration s wer e significantl y increase db y metribuzi n afte r 6 week s o f

exposure . Methomy l alon e ha dn o effec t o n T 4 plasm a concentrations . Ther e wer e n o significan t effect s o n somatotropi n at 2 o r 6 week s o f exposur e b y compound s individuall y o r i n combination . Mal e rat s ha dplasm aT 4 concentration s tha t

wer e significantl y increase db y metribuzi n afte r 7 , 13 , an d 1 6 week s o f

exposure . Th e combinatio n o f th e thre e chemical s resulte di n aslight , thoug hstatisticall y significant , increas e i n somatotropi n level s afte r 1 3 week s o f exposure . Althoug hno t

statisticall y significant , ther e wer e sligh t decrease s i n somatotropi n level s afte r

7 week s o f

exposur e t o aldicarb a s wel l a s followin g exposur e t o acombinatio n o f th e 3 chemical s fo r

1 6 weeks . Th e author s conclude dtha t thes e substances , eithe r

individuall y o r

a s amixture , ar e responsibl e fo r

learnin g impairmen t

an dothe r neurologica l effects , change s i n immun e function , andendocrin e changes . The author s als o sugges t tha t the finding s support th e concep t

o f th e interconnectednes s o f th e nervous , endocrine , an dimmun e system s an drais e th e likelihoo do f impact s o n al l thre e system s i f on e i s show n t o b e affected . However , w e disagre e wit hth e conclusion s o f Porte r e t al , (1993 ) an dbeliev e tha t ther e ar e severa l seriou s flaw s i n bot hth e experimenta l desig n an dth e conclusion s tha t hav e bee n draw n fro mthi s study . These includ e th e following : Individua l anima l hormon e level s ar e know n t o var y greatly . No t onl y wer e sampl e size s o f 6 rats/sex/grou p to o smal l t o detec t areproducibl e effec t

bu t actua l hormon e level s wer e no t

reported . Therefore , i t

i s difficul t

t o evaluat e th e biologica l significanc e o f

th e reporte dresults . Rat s wer e anesthetize ddurin g th e collectio n o f blood , an dth e anesthesi ama y hav e confounde dan y tru e compound-relate deffect . : Th e tim e o f

da y fo r bloo dcollectio n wa s no t

specifie dno r

wa s i t state da s bein g don e consistentl y (i.e. , th e pape r di dno t stat e tha t al l bloo dwa s collecte da t aspecifi c tim e o f day , suc ha s betwee n 7- 8 a.m.) . Sinc e hormon e level s fluctuat e throughou t th e day

_

### DuPont-2983 8

an dar e als o affecte db y foo dintake , acarefull y controlle d.bloo dcollectio n i s essentia l t o accuratel y asses s alteration s i n hormon e levels . Measuremen t

o f thyroxin e alon e i s no t

adequat e t o determin e a n advers e effec t o n th e thyroi dglan dsinc e thyroi dhormone s i n normal , health y animal s fluctuat e t o maintai n hormona l homeostasis . Actua l intak e o f

eac hcompoun dwa s no t

provided . Pathology , bod y weights , an dclinica l sign s wer e no t show n s o a s t o corroborat e th e hormona l measurements . Th e author s depende dheavil y o n statistica l analysi s t o infe r biologica l significance , withou t scientifi c proo f

tha t th e suggeste dbiologica l effect s actuall y occurred . N o evidenc e wa s provide dtha t eithe r th e nervous , endocrine , o r immun e system s wer e affecte di n th e study . Althoug hi t

wa s suggeste dtha t

th e combinatio n o f

compound s ma y hav e ha dawea k effec t o n somatotropi n concentrations , th e biologica l significanc e o f th e reporte d change s ar e questionabl e sinc e ther e wa s a n inconsistenc y i n th e state dresponse s i n mal e rat s (level s wer e slightl y increase da t 13.week s an dslightl y decrease da t 1 6 weeks) . A s discusse di n th e previou s sectio n o f thi s paper , traditiona l toxicolog y studie s conducte dwithmethomy l hav e give n n o indicatio n tha t th e thyroi di s atarge t

organ fo r methomy l toxicit y o r

tha t th e leve l o f

somatotropi n i s affected . Therefore , th e stud y b y Porte r

an dcoworker s (1993 ) provide s n o evidenc e tha t methomy l i s a n endocrin e disrupter ; i n fact , i t suggest s th e opposit e sinc e th e reporte dthyroi deffects . appear t o b e relate dt o th e treatmen t wit hmetribuzin. . I n astud y conducte db y El-Medan y an dcoworker s (1993) , th e author s reporte da decreas e i n seru mtestosteron e an da n elevatio n o f FSH , LH , an dprolacti n level s i n mal e rat s orall y dose dwith1 7 m g methomyl/k g bod y weight/da y fo r 2 months . Th e author s reporte dtha t

pathologica l evaluatio n o f th e teste s reveale ddegenerativ e change s i n th e seminiferou s epitheliu mwit hsloughe dcell s i n th e tubul e lumen , containin g nucle i tha t

wer e pyknoti c an dlackin g i n nuclear an dcytoplasmi c membranes . Additionally , histochemica l analysi s revealedincrease s i n variou s testicular enzyme s (alkalin e phosphatase , aci dphosphatase , an dalph aesterase) . Whil e thi s dat awoul dappear t o indicat e tha t

th e teste s ha dbee n affected , th e LD 5 

### o : 

### fo r methomy l i s 3 2 mg/k g bod y weight . Thi s fac t alon e lead s on e t o questio n th e stud y b y El-Madan y an dcoworkers . I n thei r paper , the y stat e tha t

adosag e o f

1 7 - mg/k g bod y weigh t

i s 1/3 0 th e LDs o an dcit e Haye s (1975) . However , Haye s (1975 ) i s aboo k entitle d"Toxicolog y o f

Pesticides " and , whil e methomy l doe s appear i n an appendi x withnumerou s othe r compounds , methomy l no r it s LDs o ar e foun dwithi n th e text . El-Medan y an dcoworker s stat e i n thei r discussio n that

Sale man d coworker s (1988 ) reporte dtha t chroni c exposur e t o methomy l resulte di n decrease d libido , ejaculat e volume , spermviability , an dincrease dabnormal spermi n mal e rats . However , th e cite dwork

i s actuall y astud y o f

th e effect s o f

dimethoat e (a n organophosphate ) an ddeltamethri n ( apyrethroid ) o n seme n characteristic s i n mal e rabbits . Muc ho f

thi s stud y appear s t o hav e bee n subsequentl y republishe di n apape r 2 7DuPont-2983 8 b y Mahgou b an dEl-Medan y (2001 ) a s th e stud y design , dos e level , an dth e abov e dat aappear

t o b e th e sam e a s tha t

reporte dearlier . Thi s mor e recen t publicatio n include s arecovery grou p i n whic htreatmen t wa s withdraw n fo r

on e month . Th e author s report

tha t th e hormona l an dtesticula r change s wer e stil l presen t following , thi s recovery period . Th e discrepancie s i n th e cite dliterature , th e presume dus e o f th e LDs o dos e leve l fo r

a2 monthstudy , an dth e absenc e o f similar effect s o n th e teste s an dsper mnumbe r

i n standar dmammalia n toxicolog y studie s (summarize dabove ) rais e question s regardin g th e validit y o f th e result s reporte db y El-Medan y e t al . 3. 3 Availabl e Informatio n fro mth e EPA ToxCasf TM Project EP A ha s initiate daprogra mt o scree n alarg e numbe r o f environmenta l chemical s i n a battery o f i n vitro , high-throughpu t

screens . I n th e firs t

phas e o f thi s project , 30 9 substance s hav e bee n selecte dfo r testing , mos t

o f whic har e food-us e pesticide s fo r whic hextensiv e anima l toxicolog y testin g result s ar e availabl e (Judso n e t al. , 2009) . Thes e substance s ar e bein g evaluate dwit hnin e i n vitr o assay s technologie s coverin g 46 7 cell-fre e o r

cel l base dassays . Th e tota l numbe r o f endpoint s measure d fo r eac hchemica l i s 624 . ToxCas t ha s profile dman y o f

th e EDS P chemical s selecte d fo r initia l testin g i n th e Tie r

I endocrin e battery , includin g methomyl , usin g assay s whic hcharacteriz e recepto r binding , activation , inhibition , an dtarge t gen e regulatio n tha t ar e relevan t

t o estrogen , androgen , an dthyroi dan dothe r endocrine-relate d processes . Include dar e si x assay s relate dt o estrogen , fou r assay s relate dt o androgen , fou r assay s relate dt o thyroid , an d1 assa y relate dt o aromatas e a s wel l a s 7 0 othe r nuclear receptor s an dxenobioti c metabolizin g enzyme s tha t hav e potentia l fo r endocrin e signalin g (Kavloc k e t al. , 2009) . Th e result s fo r th e 30 9 chemicals*i n Phas e I hav e bee n presente db y Rei f e t al. , (2009) ; base do n th e pictoria l presentatio n o f th e results , methomy l appear s t o hav e produce dn o "hits " fo r an y o f th e endocrine

relate di n vitr o endpoints . 

### 4. 0 SUMMAR YO F RELEVAN TECOTOXICOLOG YSTUDIE S 4. 1 Discussio n o f

studie s supportin g registratio n Beavers , J.B. , Hawrot , R. , Lynn , S.P. , Jaber , M . (199la) . A one-generatio n reproductio n stud y -withth e mallar d(Ana s platyrhynchos);H- l 7 , 940 : DuPon t Repor t No. : HL O 336-91 , MRID 41898601 , U.S . EPA 71-4(1988) . Material s an dMethods : Fou r treatmen t group s wer e randoml y distribute dwit h1 6 pair s o f bird s withon e mal e an don e femal e mallar d(Ana s platyrhynchos } pe r pen . Eac htreatmen t

grou p receive d methomy l (98.4 % purity ) i n fee dat concentration s o f

0 , 50 , 150 , o r

50 0 pp mfo r 1 8 weeks . Bird s i n th e 5 0 pp mtreatmen t grou p receive dapproximatel y 150-pp m methomy l fo r

th e firs t fou r week s o f th e study , du e t o a n erro r

i n premi x preparation , bu t i t wa s determine dtha t

thi s ha dn o effec t

o n th e study . Reproductiv e parameter s wer e measure dbeginnin g a t th e onse t o f eg g laying . Adul t

bird s wer e observe dfo r abnorma l behaviour , mortality , sign s o f toxicity , an dchange s i n bod y weigh t an d foo dintake . 

### 2 8DuPont-2983 8 Findings : Genera l observations : Ther e wer e n o mortalities , overt sign s o f toxicit y o r treatmen t relate deffect s observe dupo n foo dconsumption . Th e 500-pp mmethomy l tes t rat e showe dapparen t

treatmen t relate ddecrease s i n bod y weigh t i n hen s an da statisticall y insignifican t

decreas e i n percent

o f viabl e embryos . Th e weigh t differenc e i n drake s wa s slight , an dexcep t fo r th e firs t tw o week s o f

th e study , th e chang e i n bod y weigh t

wa s comparabl e t o th e control group . Thus , th e statistica l differenc e i s no t

deeme dbiologicall y relevant . I t wa s no t determine di f

th e sligh t decreas e in embry o viabilit y was biologicall y relate dor treatmen t related . No tes t substance-relate dgros s lesion s wer e observe da t necropsy . Reproductiv e parameters : Ther e wer e n o statisticall y significan t o r apparen t treatmen t relate deffect s upo n reproductiv e parameter s at tes t concentration s 50 , 150 , an d500-pp mmethomy l whe n compare dt o contro l value s Conclusion : Th e NOE C fo r mallard s expose dt o methomy l i n th e die t fo r 1 8 week s wa s 15 0 ppm , base do n effect s upo n bod y weigh t o f hen s at 50 0 ppm . Beavers , J.B. , Hawrot , R. , Jaber , M. , Lynn , S.P. (199Ib) . A one-generatio n reproductio n stud y wit hth e norther n bobwhit e (Colinu s viginianus) ; H-17 , 940 : DuPon t Repor t No. : HL O 337-91 , MRID 41898602 , U.S . EP A 71-4 (1988

) . Material s an dMethods : Sixtee n pair s (male/female ) o f youn g adul t

norther n bobwhit e (Colinu s virginianus ) receive dmethomy l (98.4 % purity ) i n th e fee dat concentration s o f

0 , 50 , 150 , an d 50 0 pp mfo r

2 0 weeks . Reproductiv e parameter s wer e measure dbeginnin g a t th e onse t o f eg g laying . Adul t

bird s wer e observe dfo r

abnorma l behaviour , mortality , sign s o f toxicity , an dchange s i n bod y weigh t an dfoo dintake . Al l bird s tha t die d wer e necropsie dafte r

death . Thos e bird s tha t

survive dunti l stud y terminatio n wer e als o necropsied . Findings : Genera l observations : N o treatment-relate dmortalities , over t sign s o f toxicit y o r treatmen t relate deffect s wer e observed . A t th e 500-pp mmethomy l tes t group , ther e wa s asligh t

reductio n i n bod y weigh t o f th e cocks . However , thi s differenc e wa s influence db y on e bird , whic hwa s note da s feathe r pecke dan dpenworn . Ther e wer e n o treatmen t

relate dbod y weigh t

effect s fo r

th e hens . N o tes t

substance-related-gros s lesion s wer e observe dat

necropsy . x Reproductiv e parameters : Ther e wer e n o apparen t treatmen t relate deffect s o n reproductiv e parameter s fo r 5 0 o r 15 0 ppm-methomy l groups . A t th e 50 0 ppm-methomy l concentratio n group , ther e wa s areductio n i n th e tota l numbe r o f egg s lai dpe r

he n tha t wa s determine dno t

t o b e statisticall y significant . Subsequently , ther e wa s areductio n i n th e numbe r o f offspring . N o othe r

tes t substance-relate deffect s were 1 observe do n reproductiv e parameters .



### 2 9DuPont-2983 8 Conclusion : Th e NOE C fo r northern bobwhit e quai l expose dt o methomy l i n th e die t fo r 2 0 week s wa s 15 0 ppm , base do n th e effec t upo n eg g productio n (total numbe r o f egg s lai dpe r

hen ) at 50 0 pp m(th e highes t

leve l tested) . Driscoll , R.R. , Muska,C.F . (1982) . Earl y Lif e Stage Toxicit

y o f Methomy l to Fathea d Minnow . DuPon t

Report No. : HLR-528-82 , MRID 00131255 . Material s an dMethods : Th e effect s o f

methomy l (>99 % purity ) o n embryo s an dlarva e o f fathea dminnow s (Pimephale s promelas ) wer e evaluate di n a28-da y post-hatc hcontinuou s exposur e study . Treatment s consisted?o f adilutio n wate r

control an dnomina l concentration s o f 31 , 62 , 125 , 250 , an d500-jj.g/ Lmethomyl . Fift y embryo s wer e impartiall y distribute dt o eac ho f

1 2 embry o cups . Dea dembryo s an dfungal-infecte dembryo s wer e counte dan dremoved . T o initiat e th e 28-da y larva l exposure , 2 0 larva e wer e impartiall y selecte dfro meac hembry o cu p an dtransferre dt o th e respectiv e tes t aquaria . Observation s o f

larva l mortality , behaviour , an dappearanc e wer e mad e daily. Th e numbe r

o f liv e fry i n eac haquariu mwa s estimate dan drecorde donc e pe r week . A t th e en do f

th e post-hatc hexposur e period , th e numbe r o f survivin g larva e wa s recorded . Standar dbod y lengthan dwe t bod y weigh t

o f eac hsurvivin g fis hwa s measured . Findings : Mean measure dconcentration s o f methomy l wer e 27 , 57 , 117 , 243 , an d491-u g as/ L methomyl . Percen t

embry o hatc ha t

al l th e concentration s teste dwa s no t significantl y differen t fro mth e controls . Th e 28-day larva l surviva l wa s th e mos t sensitiv e indicato r of methomy l toxicit y in thi s study . The lowes t tes t concentratio n resultin g i n asignifican t

reductio n i n surviva l o f

fathea dminno w wa s 11 7 u.g/L . A t th e lowe r tes t concentrations , fis hsurvival wa s no t statisticall y differen t

fro mcontro l responses . Larva l growth , a s measure db y standar dlengthan dwe t bod y weight , wa s significantl y reduce dat

24 3 jag/L . Conclusion : O n th e basi s o f reduce dpercen t

surviva l o f

larvae , th e MAT C fo r

th e fathea dminno w (Pimephale s promelas ) wa s estimate dt o b e greate r tha n 5 7 jig/

Lbu t les s tha n 117-ja g methomyl/L . Rhodes , J.E . (1991) . Earl y life-stag e toxicity o f IN-X1179-394 to th e fathea dminno w (Pimephale s promelas ) underflow-throug hconditions . DuPon t Repor t No . HL O 702-91 . MRID 46015305 , U S EPA , 1986 . HazardEvaluatio n Division , Standard Evaluatio n Procedure . Fish Earl y Life Stag e Test . Material s an dMethods ; Th e effect s o f

methomy l (98.4 % purity ) t o egg s an dfry o f th e fathea dminnow , Pimephale s promelas , wa s determine di n aflow-through , earl y life-stag e 35-day toxicit y test . Treatment s consiste do f adilutio n wate r

contro l an dnomina l
3 0DuPont-2983 8 concentration s o f

19 , 38 , 75 , 150 , an d300-^g/Lmethomyl . Tw o replicate s containin g 2 5 egg s eac hwer e expose dt o eac htreatmen t concentratio n an dcontro l i n incubatio n cups . Al l incubatio n cup s wer e observe ddaily . Dea degg s an dfungal

infecte degg s wer e removed . Th e 29-da y post-hatc hgrowthperio dbegan o n stud y Da y 6 , whe n 95 % hatc hwa s attained . Observation s o f abnorma l behaviour , physica l changes , an dmortalit y wer e recorde ddaily . A t th e en do f

th e study , standar dlength an dblotte dwe t

weigh t wer e measured . Findings : Mea n measure dconcentration s o f methomy l wer e 18.0 , 39.3 , 73.0 , 145 , an d 261-fi g as/

L methomy l an drange dfro m8 7 t o 103 % o f nomina l concentrations . Eg g hatchabilit y an dfry surviva l wer e no t significantl y reduce da t

an y tes t concentration . Th e n o observe deffec t

concentratio n (NOEC ) wa s 73.0-u g methomyl/L . Th e lowes t observe deffec t

concentratio n (LOEC ) wa s 145-u g methomyl/L , an dth e maximu m acceptabl e toxican t

concentratio n (MATC ) wa s 104-ja g methomyl/L . Al l chemica l an dphysica l parameter s fo r th e 35-day stud y wer e withi n acceptabl e ranges . Th e followin g endpoint s wer e determine dfo r th e methomy l tes t

concentration s an d contro l (i n parenthesis) : Mea n percen t surviva l range dfro m8 6 t o 94 % (92 % fo r wate r control) ; Mea n % eg g hatche drange dfro m8 8 t o 96 % (94 % fo r wate r control) ; mea n standar dlengthrange dfro m18.5 6 t o 21.1 7 m m(21.1 5 m mfo r wate r control) ; mean we t weigh t range dfro m0.11 2 t o 0.14 8 g (0.14 6 g fo r wate r control) . Conclusion : Th e 35-da y NOE C fo r th e fathea dminno w (Pimephalespromelas } wa s 73.0-ji g methomyl/L , th e LOE C wa s 145-ji g methomyl/L , base do n mean , measure d concentration s o f

methomyl . Th e MAT C wa s 104-u g methomyl/L . Strawn , T. , Rhodes , J.E. , Leak , T . (1993) . Ful l life-cycl e toxicit y o f DPX-X1179-394 (methomyl ) t o th e fathea dminno w (Pimephale s promelas ) underflow-throug h conditions . DuPon t

Report No . HL O 47-93 , MRID 43072101 , U.S . EPA 72- 5 (1988) . Material s an dMethods ; . Th e toxicit y o f

methomy l (98.4 % purity ) t o th e fathea dminno w (Pimephalespromelas} , wa s determine di n aflow-throug hful l life-cycl e toxicit y study . Newl y fertilise degg s wer e use dfo r th e stud y withexposur e continuin g fo r 19 3 days . Treatment s consiste do f adilutio n wate r

control , an dnomina l concentration s o f

19.0 , 38.0 , 75.0 , 150 , an d300-u g methomyl/L . Fou r replicates , wit h50 egg s each , wer e randoml y set-u p for the control andthe five tes t concentrations . Egg s wer e place di n incubatio n cup s an dallowe dt o hatch . A t Day 7 , th e fr y wer e thinne dt o 2 5 pe r replicat e an drelease dint o thei r respectiv e chambers . Measurement s fo r

lengt han dblotte dwe t

weight s wer e recorded . Observation s o f abnorma l behaviour , .abnorma l physica l change , an dmortalit y wer e recorde ddail y fo r bot hth e parenta l grou p an dfirs t generatio n offsprin g exposures .



### 3 1DuPont-2983 8 Findings : Mean measure dexposur e concentration s o f methomy l wer e 18.0 , 40.7 , 76.0 , 142 , an d 280-u g methofnyl/L . Thes e value s range dfro m93. 3 t o 107 % o f th e targe t nomina l tes t concentrations . Parenta l generatio n eg g hatchabilit y an dsurviva l wer e significantl y reduce da t

th e 442.an d280-u £methomyl/L . A t 28 , 56 , an d8 4 days post-hatch , parenta l generatio n growth , in term s of length , was significantl y reduce d afte r exposur e t o 14 2 an d280-u g methomyl/L . Parenta l generatio n growth , i n term s o f we t weight , showe dasignifican t reductio n afte r

8 4 day s o f

exposur e t o 280-^ g methomyl/L . Reproductiv e activity , measure db y day s t o firs t spawn , numbe r o f spawnin g days , numbe r

o f spawns , an dnumbe r

o f egg s pe r spawn , wa s no t significantl y affecte da t

an y tes t concentration . F I generatio n eg g hatchabilit y an d surviva l afte r

5 6 day s o f

growthwer e no t

significantl y reduce dat

an y tes t concentration . F I generatio n growthmeasure db y lengthan db y blotte dwe t weigh t a t 5 6 day s post-hatc hresulte di n significan t reduction s afte r

exposur e t o 14 2 an d 280-u. g methomyl/L , respectively . - . Treatmen t relate dphysica l abnormalitie s include ddistende dabdomen s an dabloate d appearanc e i n th e 280-ug/Lmethomy l tes t concentration . Base do n th e growthendpoint s evaluate di n thi s fathea dminno w (Pimephale s promelets) , ful l life-cycl e toxicity study , th e MAT C fo r methomy l i s 104|ug/L . Th e n o observe deffec t concentratio n i s 76. 0 jug/L , an dth e lowes t observe deffec t

concentratio n i s 142-(j,g/

Lmethomyl . Conclusion : Th e no-observe deffec t concentratio n i s 76.0-u g methomyl/Lbase do n th e growthendpoin t

evaluate di n thi s fathea dminno w (Pimephale s promelas ) ful l life-cycl e toxicit y study . Th e MAT C i s 104-u. g methomyl/Lan dth e lowes t observe d effec t concentratio n (LOEC ) i s 142-u g methomyl/Lbase do n mea n measure d concentrations . 4. 2 Discussio n o f

availabl e informatio n i n th e ope n literature In 1997 , the US EPA Ris k Assessmen t

Foru mproduce daSpecia l Report on Environmenta l Endocrin e Disruptio n (Cris p e t al , 1997 ) i n respons e t o areques t fro m th e U S EPA' s Scienc e Polic y Counci l t o provid e a n overvie w of . th e curren t stat e o f the science . The US EPA report , whic hwas publishe din the ope n literatur e in 199 8 (Cris p e t al. , 1998) , wa s intende da s an interi massessmen t an danalysi s o f

th e environmenta l endocrin e hypothesis , t o b e subsequentl y supersede db y th e Nationa l Academ y o f

Science s critica l revie w (Nationa l Researc hCouncil , 1999) . Methomy l wa s liste di n th e U S EP A Specia l Report a s impairin g avia n reproductio n base do n informatio n containe di n th e Offic e o f Pesticid e Progra mfiles ; however , n o endocrin e mod e o f

actio n wa s cite dfo r

th e observe deffec t

i n thi s document . A sligh t reductio n i n eg g productio n wa s observe di n astandar dregulatory avian reproductio n .study wit hnorther n bobwhit e quai l (HL O 337-91 , 1991) . Th e observation'wa s no t "statisticall y significan t

an dwa s observe donl y a t th e highes t

tes t leve l (50 0 ppm) . However , reduce deg g productio n i s acommo n effec t i n avia n toxicit y studies ; an d base do n asummary o f

th e U S EP A avia n reproductio n database , approximatel y 50 % 

### 3 2DuPont-2983 8

o f studie s withnorther n bobwhit e quai l elici t areductio n i n eg g productio n (Montague , 1997) . Th e frequenc y o f occurrenc e o f

thi s effec t

indicate s tha t

th e endpoint'i s asensitiv e paramete r o f toxicity . Ther e i s n o evidenc e fro mreproductio n studie s t o support

a n endocrin e mod e o f

actio n fo r

methomyl . Rather , astron g cas e . ca n b e mad e tha t

th e observe deffect s i n th e bobwhit e quai l stud y ar e aresul t o f methomyl' s mod e o f

actio n sinc e similar

effect s o n eg g productio n hav e bee n reporte dwit hothe r

acetylcholinestrs e inhibitor s (Gru e e t al , 1991) . Hoffma n an dAlber s (1984 ) reporte dth e result s o f embryotoxicit y test s conducte d withacommercial formulatio n o f methomy l (Lannate L ) whic hwa s applied , externall y t o mallar deggs . Th e stud y employe dtw o method s o f applicatio n base do n th e vehicl e (wate r

o r oil ) an destimate dvolum e o f fiel dapplication , Fertil e mallar d egg s wer e incubate di n atemperatur e an dhumidity-controlle dincubator . O n th e thir d day o f incubation , egg s wer e reporte dt o hav e bee n immerse dfo r 3 0 second s i n 3- 6 geometricall y space dconcentration s o f Lannat e Leithe r

mixe di n wit hdistille dwate r (aqueou s emulsion ) o r

prepare di n a n inert

oi l (aliphati c hydrocarbon ) vehicle . Survivin g embryo s wer e remove dfro megg s o n day 1 8 o f th e incubatio n perio dan d examine dfo r

externa l malformations . Fro mth e tes t

conducte dwithth e aqueou s preparatio n o f Lannat e L , th e author s reporte da n LCs o tha t

wa s greate r

tha n th e concentratio n o f actua l emulsifiabl e concentrat e (>18 0 Ib/A at 10 0 gal/A ) tested . I t

i s importan t

t o not e tha t

thi s reporte d estimat e o f

th e LCs o occurre da t

greate r tha n 200-time s th e maximu mpermissibl e fiel dleve l o f

application fo r

methomy l a t

th e tim e o f

th e report . Malformation s i n survivor s o f

egg s treate dwit hth e aqueou s emulsio n o f Lannat e Lwer e reporte da s blister , edema , neck , an dstunted . However , th e numbe r o f abnorma l survivor s o r

th e treatmen t concentration s a t

whic hthes e malformation s occurre dhav e no t bee n reporte di n th e article . Th e author s reporte dan LCs o o f 10. 3 Ib/ A at

1 1 gal/ A whe n mallar degg s wer e expose dto th e oi l preparatio n o f Lannat e L . Thi s LCs o wa s reporte dto b e 103.8-time s the maximu mfiel dleve l of applicatio n at

the tim e of

the report . - Malformation s reporte di n th e survivor s fro mthi s tes t wer e blister , brain , gastroshisis , joints , skeleta l (axial) , stunted . Th e observe dgreate r toxicit y o f

Lannat e Lwhe n mixe dwit hth e oi l vehicl e wa s postulate dt o hav e bee n du e t o greate r penetration o f th e shel l an dmembrane s conferre db y th e oil . 

### 5. 0 DuPoN T POSITIO N AN D CONCLUSION S 5. 1 Potential for

Methomy l to Interac t

wit hth e Endocrin e Syste

m Methomy l ha s no t

bee n specificall y evaluate di n th e 1 1 assay s comprisin g th e Tier 1 EDS P battery . However , existin g toxicolog y studie s supporting th e registratio n o f thi s produc t

indicat e tha t

exposur e t o mammal s o r

wildlif e specie s result s i n n o effect s suggestiv e of

interferenc e wit hestrogen , androgen , or thyroi dhormon e function . A fairl y comprehensiv e evaluatio n o f th e structur e an dfunctio n o f endocrine-relate dtissue s ha s bee n don e i n th e combinatio n o f subchroni c an dchroni c studie s i n rats , mice , an ddog s an di n multigeneratio n reproductio n an d developmenta l toxicit y studie s i n rat s an drabbit s (Tabl e 2) . Thi s ha s include d 3 3

### DuPont-2983 8 adrenals , pituitary , thyroid , testis , epididymis , prostate , ovary , fallopia ntubes , uterus , vagina , cervix , an dmammar y gland . I n non e o f thes e studie s wer e ther e treatment relate deffect s o n eithe r

organ weigh t

o r microscopi c patholog y tha t suggeste d endocrin e syste mdysfunction . I n long-termchronic/oncogenicit y studies , n o tumor s o f an y kind , includin g n o rumor s o f th e endocrin e tissues , wer e induce db y methomyl . Methomy l ha s bee n evaluate dfo r effect s o n prenata l developmen t o r reproductio n i n thre e studies . I n th e prenata l developmenta l toxicit y studies , n o effect s wer e observe d o n th e maintenanc e o f

pregnancy , numbe r

o f pups/litter , feta l siz e (crown-rump length) , o r feminizatio n o r

masculinizatio n o f

pup s (sex ratio) . Th e onl y effect s note d wer e decrease s i n bod y weigh t an dfoo dconsumptio n a t

th e hig hdos e (40 0 pp mi n feed , equivalen t

t o 33. 9 mg/k g bw/day ) i n th e rat . I n th e rabbi t study , th e observe d effect s a t th e hig hdos e (1 6 mg/k g bw/da y b y gavage ) include dclinica l sign s an d mortalit y consisten t

wit hcholinesteras e inhibitio n a s wel l a s a n increas e i n bod y weigh t gai n resultin g fro mincrease dfoo dconsumptio n afte r th e tes t substanc e wa s n o longe r administered . Th e outcom e o f

amultigeneratio n reproductio n stud y i n rat s als o demonstrate dalac k o f a n advers e effec t

b y methomy l o n th e hormona l contro l o f sexua l development , growth , an dreproduction . Methomy l produce dn o advers e effect s tha t suggeste dth e endocrin e system s associatio n withreproductio n an ddevelopmen t wer e negativel y impacted . Ther e wer e n o effect s o n reproductiv e parameter s suc ha s mating , fertility , gestatio n length , lactation , an dweaning . Ther e wer e als o n o dat ai n th e stud y t o indicat e tha t

sexua l differentiatio n (se x ratio) , puberta l development , productio n o f germcells , an daccessory se x organ s ha dbee n adversel y affected . Spernrcount s i n th e two-generatio n stud y fo r al l methomyl-treate dgroup s wer e similar t o contro l values . Th e primar y effect s note di n thes e studie s wer e bod y weigh t decrease s an d reduce dfoo dconsumption , whic hwer e consisten t wit heffect s observe da t

hig hdose s o f methomyl . I n addition , areductio n i n pu p surviva l an dpu p weigh t a t th e mi dan d hig hdose s (60 0 an d120 0 ppm ) occurre di n th e two-generatio n stud y an dwer e consisten t withth e sever e bod y weigh t effect s observe da t

th e sam e dose s i n th e parenta l animals . Histopatholog y o f

th e ovary , testes , an daccompanyin g accessory reproductiv e issue s hav e bee n evaluate di n anumbe r o f studie s i n rats , mice , an ddog s followin g subchroni c an dchroni c exposure s t o methomyl . Ther e wer e n o histologica l abnormalitie s i n an y study . Gonada l activit y wa s eviden t b y th e presenc e o f

follicle s an dcorpor alute ai n th e ovary an dth e presenc e o f spermi n th e teste s an d epididymides . I n al l o f

thes e studies , ther e ha s bee n n o indication'tha t methomy l adversel y affect s estrogen o r androge n dependen t

processe s i n thre e species . Therefore , th e overwhelmin g evidenc e fro msubchronic/chroni c toxicity , multigeneratio n reproduction , an ddevelopmenta l toxicit y studie s conducte di n rats , mice , dogs , an drabbit s indicate s tha t methomy l produce s n o advers e effect s o n th e endocrin e syste mo f

mammals .

3 4DuPont-2983 8 A s i n mammalia n toxicit y studies , n o reproductiv e effect s wer e observe di n mallar d ducks , rathe r

th e NOE C wa s base do n parenta l effect s o f decrease dhe n an ddrake weights . Fo r

bobwhit e quail , anon-statisticall y significan t reductio n i n th e numbe r

o f egg s lai dat

the highes t

leve l teste d(50 0 ppm ) was observed . No othe r tes t substance

relate deffect s wer e observe do n reproductiv e parameters . Thu s th e potentia l effect s o f chroni c exposur e t o methomy l i n bird s ar e no t likel y t o hav e reproductiv e impact . Th e result s o f

th e fis hreproductio n studie s showedtha t reproductiv e activit y o f

th e parenta l generatio n wa s no t significantl y affecte da t

an y tes t concentration . Th e F I generatio n eg g hatchabilit y an dsurviva l wer e als o no t significantl y reduce da t

an y tes t concentration . Th e traditiona l guidelin e toxicolog y studie s (subchronic/chroni c toxicity , multigeneratio n reproduction , an ddevelopmenta l toxicit y studie s conducte d i n rats , mice , dogs , an drabbits ) conducte dwit hmethomy l als o provid e compellin g evidenc e tha t

methomy l i s no t

expecte dt o produc e advers e effect s o n th e endocrin e

. syste mo f

wildlif e species . 5. 2 Functionall y Equivalen t Informatio n Fulfillin g th e EDS P Tie r IBatter y EP A outline di n it s Marc h26 , 200 9 approachfo r considerin g OSR I unde r

th e EDS P tha t i t wil l mak e decision s a s t o whethe r existin g informatio n satisfie s al l o r part o f th e Tie r 1 Tes t Orde r base do n th e weigh t

o f th e evidence . Base do n th e availabl e informatio n fro mmammalia n toxicolog y an decotoxicolog y studie s supportin g th e registratio n o f

methomy l a s wel l a s informatio n fro mth e ope n literatur e an d ToxCastTM , DuPon t

propose s tha t

th e primar y purpos e o f

th e Tie r 1 EDS P screenin g battery , t o asses s th e potentia l t o interac t wit hestroge n androgen , o r thyroi dhormon e systems , ha s bee n fulfilled . Th e EPA' s Tie r

1 estroge n an dandroge n recepto r bindin g assay s an destroge n transcriptio n activatio n assa y ar e designe dt o identif y substance s tha t

eithe r bin dt o th e estroge n an dandroge n recepto r o r bin dt o th e estroge n recepto r an dactiv e th e subsequen t

downstrea mtranscriptio n o f areporte r gen e product . A s note di n Sectio n 3.2. 1 above , methomyl , a s wel l a s it s structura l analogu e oxamyl , ha s bee n teste di n anumbe r

o f estroge n o r

androge n transactivatio n assay s i n whic hth e majorit y o f

th e result s sugges t

alac k o f agonis t an dantagonis t

activity . EP A ToxCast TM progra mals o include s anumbe r o f estroge n an dandroge n bindin g assessments'i n whic hth e reporte dresult s sugges t alac k o f activity (Reif, 2009 ) o f bothproducts . Further , th e i n viv o mammalia n toxicolog y databas e fo r methomy l provide s considerabl e evidenc e tha t estroge n an dandroge n dependen t proces s i n rats , mice , dogs , an drabbit s ar e no t adversel y affected . Therefore , i t i s DuPont' s positio n tha t i t i s unnecessary t o conduc t furthe r estroge n o r

androge n bindin g studie s wit h methomyl . Th e purpos e o f

th e Tie r 1 steroidogenesi s an daromatas e assay s ar e t o identif y substance s tha t

eithe r interfer e wit hsteroi dhormon e synthesi s resultin g i n

. testosteron e an destradio l productio n o r whic hinhibi t

th e catalyti c activit y o f

th e enzym e responsibl e fo r

th e conversio n o f

androgen s t o estrogens. I n th e absenc e o f an y advers e effect s i n th e i n viv o mammalia n toxicolog y studie s conducte dt o date ' . wit hmethomy l tha t

woul dsugges t

interferenc e o f

steroi dhormon e productio n (e.g. , adrena l an dreproductiv e orga n weigh t o r histopathology , fertility , gestation , 3 5

### DuPont-2983 8

se x ratio) , i t

i s DuPont' s positio n tha t

n o furthe r testin g o f methomy l i n th e steroidogenesi s andaromatas e assay s is necessary . Th e uterotrophi c an dHershberge r assay s ar e intende dt o determin e th e potentia l o f substance s t o induc e uterin e o r mal e reproductiv e organ weigh t change s consisten t withagonist s o r

antagonist s o f

natura l estroge n o r

androgen/5a-reductase inhibitors , respectively . Uterin e weigh t ha s bee n assesse di n 90-da y studie s i n rat s an dmic e an d i n th e multigeneratio n reproductio n stud y conducte dwit hmethomyl . Histopatholog y o f anumbe r o f femal e reproductiv e tissue s ha s bee n evaluate di n on e o r mor e o f th e th e studie s summarize dabov e (ovary , fallopia n tubes , uterus , vagina , cervi x an d mammary gland) . Teste s weight s wer e measure di n thes e studie s a s well . Som e o f - thes e studie s als o include depididymi s and/o r prostat e weights . Histopatholog y o f th e testes , epididymis , an dprostate were include di n mos t o f th e subchroni c an dchroni c 'studie s a s wel l a s th e multigeneratio n reproductio n study . I n th e absenc e o f an y advers e findings , th e weigh t o f th e evidenc e suggest s tha t methomy l doe s no t

produc e an y effect s consisten t

wit hestroge n o r

androge n agonist s o r

antagonists . Therefore , i t i s DuPont' s positio n tha t

ther e i s n o nee dt o conduc t eithe r th e uterotrophi c o r Hershberge r

assay wit hmethomyl . Th e inten t o f th e mal e an dfemal e puberta l assay s i s t o identif y substance s that interac t wit hth e endocrin e syste mb y assessin g endpoint s associate dwit hpuberta l developmen t

an dthyroi dfunctio n i n mal e an dfemal e animals . A s note di n th e abov e discussio n regardin g th e uterotrophi c an dHershberge r assays , orga n weight s and/o r - histopatholog y o f

afairl y larg e numbe r

o f reproductiv e tissue s hav e bee n assesse di n man y o f th e existin g mammalia n toxicology<studie s conducte dt o date . Adrena l weight s an dpituitar y weight s an dmicroscopi c patholog y hav e bee n include di n th e majorit y o f

thes e studie s a s well . Th e multigeneratio n an ddevelopmenta l toxicit y studie s hav e reveale dn o concern s fo r a n advers e effec t

o n puberta l development . Thes e studie s hav e assesse dsuc hendpoint s a s matin g behavior , fertility , maintenanc e o f pregnancy , gestatio n length , offsprin g se x ratio . I n addition , sperm , count s wer e evaluate di n th e F O an dF ] generation s i n th e reproductio n stud y an dorgan weight s an dhistopatholog y o f

anumbe r o f reproductiv e tissue s wer e evaluate di n th e F I an d p2pup s a t weaning . Therefore , unde r thes e circumstances , i t

i s DuPont' s positio n tha t th e evaluatio n o f

methomy l i n th e femal e an dmal e puberta l assay s i s no t necessary . Th e amphibia n metamorphosi s assay (AMA ) i s designe dt o empiricall y identif y substance s whic hma y interfer e withth e norma l functio n o f th e HP T axis . Thyroi d hormone , throug hth e HP T axis , regulate s man y processe s i n a n organis mincludin g somati c growth , development , metabolism , an dreproduction . I t i s DuPont' s positio n tha t th e existin g mammalia n toxicolog y studie s sufficientl y demonstrat e tha t methomy l doe s no t

interac t wit hth e HP T axis . N o treatment-relate deffect s wer e observe do n th e thyroi dglan di n th e man y studie s supportin g registratio n o f methomy l an dsummarize dabove , whic hinclude da n evaluatio n o f organ weigh t and/o r histopatholog y i n rats , mic e an ddogs . Man y o f th e importan t

feature s o f

th e thyroi dsyste man dth e HP T axi s ar e conserve dacros s al l tax a(OECD , Detaile d Revie w Pape r

o n Thyroi dHormon e Disruptio n Assays , Apri l 2006) . Therefore , give n th e degre e t o whic hth e thyroi dha s bee n evaluate di n thre e mammalia n species ; ther e i s n o nee dfo r

furthe r testin g o f methomy l i n th e Tie r

1 AM A screen . 3 6

### DuPont-29'83 8 Th e fis hshort-ter mreproductio n assa y i s designe dt o measur e th e reproductiv e performanc e o f

fathea dminnow s a s aprimar y indicato r o f potentia l interactio n o f xenobiotic s wit hth e endocrin e system . Th e endpoint s examine di n thi s assay includ e survival , fecundity , fertilizatio n success , change s i n morphology , an dbiochemica l endpoint s whic hmigh t

b e indicativ e o f

disturbance s i n estroge n an dandroge n dependen t processes . I n comparison , th e parameter s examine di n th e fis hful l lif e cycl e stud y wit hmethomy l includ e growth , change s i n morphology , an dreproductiv e activit y suc ha s tim e t o firs t spawning , numbe r

o f spawns , an dnumbe r

o f egg s produced . I t

i s DuPont' s opinio n tha t

th e existin g fis hfull-life cycl e stud y togethe r . wit hth e earl y life stag e stud y wit hmethomy l provid e sufficien t informatio n t o addres s th e intende dpurpos e o f th e fis hshort-termreproductio n assay . 5. 3 Overal l Conclusio n

Methomy l i s acarbamat e insecticid e whic hexert s it s biologica l activit y b y inhibitin g acetylcholinesteras e i n th e nervou s syste mo f insects , mammals , an dothe r vertebrat e

. . species . Methomy l has . hig hacut e toxicit y b y th e oral , inhalatio n an docular route s o f exposure . Th e primar y toxicologica l effect s observe di n toxicolog y studie s ar e thos e associate dwithcholinesteras e inhibition . Thi s substanc e i s no t acarcinoge n o r genotoxin , no r

i s i t areproductiv e o r

developmenta l toxicant . A s summarize dabove , ther e i s n o evidenc e i n th e availabl e mammalia n an decologica l toxicolog y studie s t o sugges t tha t methomy l directl y interact s withth e functio n o f estrogen , androgen , o r thyroi dhormon e processe s t o caus e endocrin e dysfunction . A similar toxicolog y databas e exist s fo r

oxamyl , anothe r

DuPon t carbamat e insecticid e whic hshare s clos e structura l analog y t o methomy l an dwhic hals o demonstrate s lo w potentia l t o interac t wit hth e endocrin e syste m(se e DuPon t respons e t o Tes t

Orde r EDSP-103801-63 , DuPon t -29827) . Methomy l is currentl y bein g regulate dby EPA usin g an extremel y conservativ e mammalia n toxicolog y ora l endpoin t base do n subtl e change s i n bloo d an dbrai n acetylcholin e esteras e activit y a t ver y lo w dos e levels , substantiall y lowe r tha n thos e evaluate di n th e registratio n studie s cite dabove . Likewise , th e aquati c ris k assessmen t

fo r methomy l i s base do n toxicit y observe di n aquati c invertebrate s (Daphni amagna) , bein g th e mos t sensitiv e species . Therefore , DuPon t consider s tha t " inten t of the Tie r 1 EDS Pscreenin g battery has bee n fulfille dfor methomy l base don th e availabilit y o f

alarg e databas e whic hindicate s alac k o f adirec t advers e effec t

b y thi s substanc e o n th e endocrin e syste mo f mammal s an dwildlif

e species . 

### 6. 0 REFERENCE S

Andersen , H . R. , A . Vinggaard , T . Rasmussen , I . Gjermandsen , E . Bonefeld

Jorgense n (2002) . Effect s o f Currentl y Use dPesticide s i n Assay s fo r Estrogenicity , Androgenicity , an dAromatas e Activit y I n Vitro . Toxicol . Appl . Pharmacol . 179:1-12 . Burin , G. , L . Chitlik , S.L . Makris , an dJ.N . Rowe , (1993) . Reproductiv e toxicit y studies . Standar devaluatio n procedure . HealthEffect s Division : Draf t Offic e o f Pesticid e Programs . U S Environmenta l Protectio n Agency , Washington , D:C. , Octobe r 1993 .

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### DuPont-2983 8 Celik , L. , J . Lund , B . Schiott (2008),Explorin g interaction s o f endocrine-disruptin g compound s wit hdifferen t

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No . 33 , Jul y 1996 . Brussels , Belgium . El-Medany , A, , G . El-Medany , an dA.E . Rahm y (1993) . Evaluatio n o f th e effec t o f v

### methomy l insecticid e o nth e testicula r functio n i nnorma l albin o rat s (hormonal , histologica l an dhistochemica l study.) . J . Med . Res . Inst . 14 : 165-i87 ! Gruggs , C.J. , J.C . Perkham , an dK.D . Cat o (1983) . Mammar y carcinogenesi s i n rat s i n relatio n t o th e ag e a t

tim e o f N-nitroso-N-methylure aadministration . J . Natl . Cance r Institute . 70:209-212 . Grue , C.E. , A.D.M . Hart , an dP . Minea u (1991) . Biologica l consequence s o f depresse dbrai n cholinesteras e activit y i n wildlif e in : Cholinesterase-inhibitin g insecticides : Thei r

impac t o nwildlif

e an dth e environment , ed . P . Mineau . • Elsevie r Scienc e Publisher s B.V. , Amsterdam , pp . 151-209 . Hayes , W.J . Jr . (1975) . Toxicolog y o f Pesticides . Baltimore , Th e William s an d WilkinsCo . Hoffman , D. J an dAlbers , P.H . (1984) . Evaluatio n o f Potentia l Embryotoxicit y an d Teratogenicit y o f

4 2 Herbicides , Insecticides , an dPetroleu mContaminant s t o Mallar dEggs . Arch . Environ . Contam . Toxicol . 13: , 15-27 . Hseuh , A.J.W. , M.L . Dufau , an dK.J . Cat t (1977) . Gonadotrophi n induce dhormon e receptor s an ddesensitizatio n o f testicular 3',5'-cycli c AM P an dtestosteron e responses . Proc : Natl . Acad . Sci . US A 74:592-595 . Jiao , B . e t al. , (2008) . Establishmen t o f aTransgeni c Yeas t

Screenin g Syste mfo r Estrogenicity an dIdentificatio n o f th e Anti-Estrogeni c Activit y o f Malachit e Green . J . Cell . Biochem . 105:1399-1409 . Judson , R. , e t

al , (2009) . I n vitr o screenin g o f environmenta l chemical s fo r targete d testin g prioritizatio n - Th e ToxCas t Project . Environ . HealthPerspect . Online , Decembe r 14 , 2009 . Availabl e at

http://dx/doi/org . Judson , R. , e t

al , (2009) . I n vitr o screenin g o f environmenta l chemical s fo r targete d testin g prioritizatio n - Th e ToxCas t Project . Environ . HealthPerspect . Online , Decembe r 14 , 2009 . Availabl e at

http://dx/doi/org . 3 8

### DuPont-2983 8 Kavlock , R. , e t

al. , (2009) . Biologica l profilin g o f endocrin e relate deffect s o f chemical s i n ToxCastTM . Poste r presentatio n availabl e at http://www.epa.gov/NCCT/toxcast/files/summit/40P%20Kavlock%20TDAS.pdf . Klotz , D . M . S . F . Arnold , an dJ.A . McLachlan. , (1997) . Inhibitio n o f 1 7 Beta

Estradio l an dProgesteron e Activit y i n Huma n Breas t Cance r an dEndometria l Cance r Cell s b y Carbamat e Insecticides . Lif e Sci . 60:1467-1475 . Kojima , H . e t

al. , (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 Ovary Cells . Environ . Health . Perspec . 112:524-531 . Mahgoub , A . A . an dA.H . El-Medan y (2001) . Evaluatio n o f chroni c exposur e o f

th e mal e rat reproductiv e systemt o th e insecticid e methomyl . Pharmacologica l Researc h44 , 73-79 . Montague , B . (1997) . Brian.db f database , U S Environmenta l Protectio n Agency , Offic e o f Pesticid e Programs , Augus t 13 , 1997 . Nationa l Researc hCounci l (1999) . Hormonall y Activ e Agent s i n th e Environment . Nationa l Academ y Press . Washingto n DC . 43 0 pages . http://www.nap.edu/books/0309064198/html / Nishihara , T . e t

al. , (2000) . Estrogeni c activitie s o f 51 7 chemical s b y yeas t

two hybri dassay . J . o f

HealthSci . 46 : 282-29 8 Nendza , M . an dWenzel , A . (2006) . Discriminatin g Toxican t Classe s b y Mod e o f Action , 1 ) (Eco)toxicity Profiles) . Environ . Sci . &Pollut . Res . 13:192-203 . Porter , W.P. , S .M . Green , N .L . Debbink , an dI . Carlso n (1993) . Groundwate r pesticides : interactiv e effect s o f lo w concentratio n o f

carbamate s aldicarb an d methomy l an dth e triazin e metribuzi n o n thyroxin e an dsomatotropi n level s i n whit e rats . J . Toxicol . Environ . Healt h40:15-34 . Reif , D. , e t

al. , (2009) . Endocrin e profilin g an dprioritizatio n usin g ToxCas t Assays . Poste r presentatio n availabl e a t http://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=218187&fed_or g _id=1267&SIType=PR&TIMSType=Presentation&showCriteria=0&address=co m ptox&view=citation&orderByl=FY%20desc&orderBy2=ID . Roncaglioni , A. , N . Piclin , M . Pintore , an dE . Benfenat i (2008 ) Binary classificatio n model s fo r

endocrin e disrupte r

effect s mediated.throug hth e estroge n receptor . SARan dQSA

Ri n Environmenta l Researc h19:697-7 3 3 . Stevens , J.T. , A . Tobia , J.C . Lamb , C . Tellone , an dF . O'Nei l (1997) . FIFR A Subdivisio n Ftestin g .guidelines : Ar e thes e test s adequat e t o detec t potentia l hormona l activit y fo r

cro p protectio n chemicals ? J . Toxicol . Environ . Health 50:415-431 . va n Ravenzwaay , B . (1992) . Discussio n o f prenata l an dreproductio n toxicity stud y o f Reg . No . 83-25 8 (Vinclozolin) . Dat asubmissio n t o U S EP A fro mAS F Corporation , 425813-02 .
3 9

### DuPont-2983 8



### TABL E 1 CITE D METHOMY L TQXICITYSTUDIE S SUPPORTIN G

REGISTRATIO N

MRI D NO . 0000719 0 Unknown , repor t supplie d Unknown , repor t supplie d 0000901 0

0007836 1 0007024 1 0007842 3 0000709 1 4325070 1 4376940 1 0000862 1 0013125 7 4189860 1 4189860 2 0013125 5 4601530 5 4307210 1 REPORTN o

MRO-84 8 Hazleto n 201- 1 5 1 52 B 101-630-7 8 52 E 004-630-7 9 HL R 168-6 7

HL R 235-8 1 HL O 253-8 1 Hazelto n 201-51 0 Hazleto n 20 1 -16 5 HL O 5 1 9-9 5 HL R 498-7 8 HL O 331-8 3 HL O 336-9 1 • HL O 337-9 1 HL R 528-8 2 HL O 702-9 1 HL O 47-9 3 TITL E Thre e mont h dietaryadministratio n - rat s

insecticid e 117 9 Asubacut e (9 0 day ) evaluatio n o f Nudri n i n th e Fischer-34 4 ra t fo rShel l Oi l Compan y Asubacut e (9 0 day ) evaluatio n o f Nudri n i n th e B6C3F 1 mous e fo rShel l Oi l Compan y Three-mont h feedin g stud yo n dog s wit h S-methy l N-[(methylcarbamoyl)oxy ] thioacetimidat e [Lannat e Methomy l insecticide ;

INX-1179 ] Long-ter m feedin g stud yi n rat s wit h S-methy l N-((methylcarbamoyl)oxy ) thioacetimidate )

(methomyl ; INX-1179 ) 104-wee k

chroni ctoxicit yan d carcinogenicit y stud yi n mic e methomyl ; H- 1 1 , 1 3 5 Two-yea rdietaryadministratio n - Dogs : - Lannat e methomy l insecticid e (S-methyi-N - ((methylcarbamoyl)oxy ) thioacetimidate ) Nudri n 2 generatio n reproductio n stud yi n rats . Ora l teratogeni c stud yi n rat s wit h Lannat e

(INX-1179 ) Embryo-feta l toxicit yan d teratogenicit ystud yo f

methomy l i n th e rabbit . Aone-generatio n reproductio n stud ywit h th e mallar d (Ana s platyrhynchos ) Aone-generatio n reproductio n stud ywit h th e norther n bobwhit e (Colinu s viginianus ) Earl yLif e Stag e Toxicit yo f methomy l t o

fathea d "minnow . Flow-through , 2 1 -da ytoxicit yo f DPX-X 1 179-42 3 (Lannat e 20L ) t o rainbo wtrou t (Oncorhynchu s mykiss) Ful l life-cycl e toxicit yo f DPX-X 1 179-39 4 (methomyl ) t o th e fathea d minno w(Pimephales promelas ) unde rflow-throug h condition s REVIEWE D B YEP A ( Y O R N )

•Y Unknow n Unknow n Y

Y Y Y Y Y Y Y Y Y Unknow n Y EP A

CLASSIFICATIO N Acceptabl e Unknow n Unknow n Unacceptabl e (waive d du e t o acceptabl e chroni c study ) Acceptabl e Acceptabl e Acceptabl e Acceptabl e Acceptabl e Acceptabl e Acceptabl e Acceptabl e Acceptabl e Unknow n Acceptabl e

4 0

### DuPont-2983 8



### TABL E 2 ENDOCRIN E TISSUE S EVALUATE D I N MAMMALIA N TOXICOLOG Y

STUDIE S CONDUCTE D WIT H METHOMY L

TISSU E Adrena l

. Pituitar y Thyroi d Teste s Epididymi s Prostrat e Ovary . Fallopia n tub e Uteru s Vagin a Cervi x Mammar y glan d 90- D

RAT W , H W , H H W , H H H H 90- D

RA T W , H H H W , H W , H H W , H W , H W, H H 90- D MOUS E W , H H W , H H W , H W , H 90- D

DO G W , H W, H W , H W , H

. H W , H H H H

H 2-Y R

RAT W , H W , H H W , H H H H H

H 2-Y R MOUS E W , H W , H H W , H H H H H

H ' 2-Y R

DO G W , H H W , H W , H

H H 2-GE N REPR O W , H H

' H W , H H W , H W, H H H H RAT

, DE V

G G RABBI T DE V

G G , W

W= orga n weight ; H = histopathology ; G= gros s patholog y onl y



### 4 1