### SllllJECT:

FROM: TIIRO U GII: TO: UNITED STATES ENVIRONMENTAL PROTECTI ON AGENCY WASHINGTON, D.C . 20460 

# OCT 0 6 70 15 O F F I

CE OF CHEMICAL SAFETY AND POUUTt ON PREVEr-ITION 

### Review o f**t-. to** **~u ur**  

### Char.u: tcri z;at ion O ; at ; a. ProlJ: in Exp ri.' SS ion Ana l yses 

### o

nd Hunton llc.:t l th**AS.SC~'i.nlCnt** 

### for 

### r• Jant-lncorpor:ltc d Protcc t ant (P IP ) 

### PnJ"lya Ring spo t Viral Coat Protei n (PRSV - CP) for usc i n X 17 - 2 Papaya (Carica papaya L. ) 

### [EI' A 

### Res. No. 

### 84427-R: OECD 

### U n

i q ue ID 

### UFL X 1 7CP · 6) exp ress in g Popnya. Rin gspo t Virus Coa t Pr o t

ei n. in su pport t O

r FIFRA 

### Se<t ion 

### 3 Registr:Jtion [ Dec is ion No: 

### 497668. [)P 

### llnrcode: 4248&4: Subm issio n No: 

### 9604 23. MR ID Nos : 

### 4949320 1 . -

02.-0-1 . - 05 ] :**ubm U~!** **d** 

### by 

### Un iversi ty 

### of F l orida.**ln. utc**  

### of**ood** 

### and A g riculturnl . 

# n J Sc i e n ces (IF AS ) , J 

# if" 

# t , ;;; < 

# -1 .t)u ,tJ v**c~t r**  Wi1 l ,SClJ1 . 

### Uiolo s is t**tN ~**  

### v 

### V (/ Micro b i n

i Pe s t ici de s Br.tnch. BiofX.<>sticidcs and

# ~:~~:~::**:~ :~~n;:~~)~o::i** **:i::e~e(:~: tii** **)\'::;I;::ANlJ** 

# . 14' 'r 

# Q A 

### Mi c rob ia l P es t icides Branch.

# o~**ti** des 

# 0 ...,' P

o llu t io n l)re\'cntion Dh is ion (7 511 P ) Michael Mt'ndc:l sohn. R cg ulntory Action Leader Microbial Pesticides Branc h. Biopcsticidcs uml Polluti o n l're ven t io n Divi s ion (75 11 P ) ACT I O X ltf:QUESTEI>: To r e view 

### th e d;ua s u bmi tted 

### by 

### U n i

versity ofFiorida .ln . nitu tc of 

### F'oo d and Agricultu13J Sc i ences (IFAS ) in s up port of a**~ction** 

### 3 reg is t 13t ion for the uctivc ingredient Viro l Coatl 'ro tein of Papaya Ring.spot Viru s (P RS V - CP). with an end-usc p roduc t 

### name . X 

### 17 · 2 PopJ)-;J [ QECD 

### Uniq u e ID: UI ' L - X 

### 17 0 '· 6].**f:C OM~!F;** **;" \** 

### TIO I'/ : The s ubmiucd dai.U 

### :1 rc dn ss i lictl as ACCF.rTABLt: 

### A s imi lar p r otei n t o t he p rcsc: n t a ctive in g re-d ient Viml Coot P r o tei n**ofP~tp:ty n**  

### Ri n g s pot Viru s ( PR .SV-CP) . has 

### p rc , ious ly be en**grn nt** **~** 

### a pcmlancnt t o l cmn c c exempt i o n**[~ 0**  

### CFR**7~.5 5].**  I

### DATA (;VALUATION RE CORD 

### Primary**R~ vicwc** **r:** 

### Jennifer \Vin gcn n. Oiotogist EPA Sccond : 1r )' Re viewer: John Kough. Ph . D . . S cni(IT Sc i entist STUI>Y TYI'E:

ACT I VE ING R ED I ENTS : M R IIl NO: DPBARCODE: DECISION NO: SUIJMISSION NO: TEST MATE RI AL: STUDY NO: S I 'ONSOR: TESTING FACILITY :
AU THORS: STU ll \' COMPLETED: 

### Product Identity . Manufacturing P rocess . l)cpositjon of Snmp l c. Fonn . :Hion ofUnintcnt ionallngrcdic:n t $. Analysis of Samples. Ccrt i liClltion of Limi ts. Acute Oral Toxicity. Acute lknn:.'IJ Tox icity. Acute Inh a lat io n Toxicity. Acute Ey e: lrritntion. l'r ima r y Dcnna l Irri tat ion . :\cute**Toxicit~ /Pn** **th ogcnicity** **.** 

### : \ cu te Pulmon:uy 

### Toxici ty !Pm h o0



### '<:n i<ity (OCS I ' r 885 . 1 100 . 885.1200. 88 5 .1 300. 885.1400. 885.1500. 870. 1100 . 870.1200. 870.1300. 870.2400. 870 . 2500. 88 5.3050. 885.3150 

### respccti\ ·c l y) Pl:mt Inc orporated rrotcctan t (PIP) Pap.'Y" Rin gspot V irus Coat Protein ( PRSV - CP) and t he gen eti c matcri:1l nc."<.:c S

.. • > .. 

### 'l.ry for it s p roductio n nnd the mnrkc r gene (N I) TII) . 49493201.49493205 4 2488 4 4 97668 

### 960423 

# P•J>'Ir• R i n

sspo t V iru s Coat Protein (XI7 · 2 } ( EPA 

### Reg. No.**8~~27 )**  

### IR ·4 

## PR No . 0537B 

### ?-.·t i chl u:l Bnh ' emKH'I. 

### l'h.D. , IR·4 P roject 

### Headquarters. 500 College RoJd East. Suite 201 W. Princet o n . NJ 

### 08540 U

n

i"crsi ty of Fl oric.ID . Ins t i tu te of food 

### and Agricult ural Sc ience (UFL- IFAS ). C itm s R ese n

rch nnd Education Cl!nter . L.akc Alfred . Florida !\'

lichacl Brnvc:nnan. Ph.D .. William P . Barne y and Kri sta 

## D. Colcmllll Octob..-r 7 . 2014 2

### CONF I DEKTIA LI TY C LAIM S : GOOD LA BORATORY PilJ\CT ICE (G LP ): STU D Y SUMMARY :

CLASS I FI CA TIOK:**n~u. troun** **l** 

### Konc Signc:d n nd Unlcd S llltcment: 

### Non GLP - comp liant 

### unde r -10 

### CFR Pun 1 60 

### Universi t y of Florida. Ins t i t

ute of Food nnd Agricuhurnl Sciences (IFAS) has subm i ucd datu a nd inform at io n on th e Pl a nt 

### lncorpor:th."<< J "t rotcctruu ( I .. IP) Papaya Rin gspo t Viru s 

### Co3 t Protein (I'RSV - CP). An a r gument w ns m nde 1 0 cons i de r the F l orid:t s t rni n 

### of PRSV - C P 

### cxprcssc:d in X17-2 being in clud ed in the ex i st in g tok r:mcc exemp t ion for PRSV.CP [ 40 CFR § 1 74.515). The end-usc prod u ce tradc -nn.med X f 

### 7-2 

### confers res i stance to pap:t yn ringspot v irus (PRSV). 

### The X 17- 2**rc sisu~nc** **c** 10 

### PRSV i s clnjmed to be 

### t he 

### r esu lt of posH.rnnscrip t ion.aJ ge n e si len c in g 

### due to the presence of 

### t he PRSV - CJ ) gene in th e p::!J\3)'0. 

### genome. Tmn sfonn: :ui on E\'cnt X 17·2 (pB I1 21 fs ): The inse rtio n c\ ·cnt cons i sts of a NOS p r omote r clement. nptll 

### gene:. CnMV 35-S promotc r.I'RSV coat 

### rrotcin gene, 

### and a NOS temtin:uor . ACCEPTABLE

U

niversity of f lor id a . In st i t ute of Food and 

### Agricu lt ural Sciences has subm i ucd data n nd in l (mntuion on th e PJant lncorpomtcd Prote.; t a nt 

### ( PIP) Papaya ltin gs pot Virus Coat Protein ( PRSV ·CP). An arg um en t w:ts mode to c onsider the Florida strJi n of PR SV-CP expre sse d in X 17 · 2 being in cl uded in th e exi s t ing t o ler:u t cc exempt ion lb r J >RSV-CP [ 40 CFR § 

### 17-1.5 15[ . The tmdc· name for 

### the end-usc product i s**X17 2.**  

### a tran sg enic pJp ; a yu t ha t**co nl~** **rs** 

### resis t nncc 

### t o**pap:~.yn** 

### r in gspot 

### virus (PRSV). 

### The basis of the exist in g tolcr:mcc exemptio n 

### i s that the co;at p r otein 

### i s part ofthe virus th at i s 

### nnturnHy present in i n fected**p.:1p~ yt:1**  

### nnd i s 

### alrc:tdy con s umed when c.:at ing**rus~** 

### intCctcd 

### PJJXl)'n. 11lc r cfo rc . 

### tlte presence 

### of t h e v iral 

### co-11 protdn in I'RSV 

### in Flor id a 

### docs not consti t u t e novd 

### dicmry**c~posun :.**  

### The X17-2 resistance to PRSV may be 

### the re s ult 

### of postt ranscrip ti onal gene si l encing due to the insertion of the PRSV-CP gene in t o 

### the p:lJXl) 1.1**g~nomc.** 

### · ntc following sec t ions summ3lizc 

### the d ata on 

### the: production 

### and 

### s.:t l"';.: t y of 

### the X 17 ·2 transscnic p.::sJX!y !l 

### l ines. Pap:tyn (Carkupapt'J.rt) 

### L.) be longs 1 0 piMt fnmily Caricnccac and is a po l yg a mous**hcrbn ccou.._~**  

### perenn ia l 

### p lant. Papay-a ringspot virus ( PRSV ) . u potyvirus, i s 

### highly**p:tth og~** **ic** 

### to pap:tya 

### of all \'aril!ties and m:tjor fhctor 

### limiti ns g lobal commercia l papay.:a production . PRSV. i s the l ead in g cause ofp: 1 p:ayn 

### loss in Florid:a. Typica ll y. n heo hh > ' papaya plant can survive up to 20 

### year s . 3

### ho\\cv.:r. ifinfected with PRS V. t h:tl 

### li f e sp.m is n:duced to 4-5 

### yem . 'The liniwrsi ty offtorid3 h:l.:,

**~<\ elop..--<.1**  

### the X t7-2

**p:ap..1~-nlinc** 

### ba.._<.ed 

### on technology

**simil;~.r** 10 

### t h.1t used 

### by Dennis Gonsal\'CS ;and hi s le3m 

### in the dc\'d opmcnl 

### ofthe ll o.waiian 

### fbinbow Pl.lpa)a.

**Th~** 

### X 17·2 uses 

### the: snmc 

### Agroboctt •rium-mC'diatc:..J tnlJlSIOm 1 atio n and 

### n:gcw:rotion of

**p:~paya** 

### plants from so nuuic embryos as outlined inn study by Davis nod Yin g (2QO...l):. The

**so ur\!~:**  

### oft he co3t pro tein s ene i n t he I fn wa i ian a n d Florid iun c-assettes nrc diflCrcnt nn..J h : w .: s l igh t ly different mnino n cid seq u e n ces: (Hawa i ian S lrn i n (h! n cbank , \ ccc.ss ion II FJ-167933) 

### nnd Fl oridia n s 1 mi n (Ocncb:: mk Acce ss i on #AF 196R39 ). ll owcvc r . the: ro:n

**protein. ~**  

### nre the s:t mc stroc lu!.'lly 3nd functi o nally. The FL and HI vmi1mb 

### ore 

### indica t ive of different PRSV strajns which \'al) ' 

### gcogr.sphicall)' :LI1d 

### 1xuh

**~usc** 

### di sc3Sc in papaya. l11c J 

### ftm":1iian tr.lnSt;tnic

**P=Jp:l~<l** 

### dCk."":S not prolct"t

**3g;~inst** 

### 1hc: Floridi.ln J•RSV str.Un. HO \\c\ ' cr.lhc slish- t ::unino acid dirfc:n.-nccs do not indicate: 

### o.ny signifie:un hc::thh

**iss\~S** 

### rd n ted to toxicity o r 

### :dlc:rg.cnicily during conswnpcion or othe r txposurc5 . The X I

**7~1** 

### line presently 

### corui stS prinu.rily 

### of n

**r::~.mily** 

### or 

### four bre'l!'djng lines 

### tllnt nrc 

### di.!'SCendants of cros ses 

### between th e originnl U"3.nsgc:nie plant 

### (l{a sencrntion) 

### and fom elite n on -lmn sg c:nie: p DpDyn ge n otype$. 

### namel y: So lo Sunrise. P u cno Ric o 6-65 . T3inung No.5. and So lo -' 0 (Da\'is 

### :.nd Yin g. 2004f. 

### A J >roccs.s o f rccum: nt

**backcr OS$**  **in ~**  

### nnd se lect io n for de s ired p h eno t y piC: chnr 11ctcri.s 1 ics. i n cl udi n g re s i s tru1ce t o PR SV, h ll\

' C bee n us ed t o de\'c lop l hc,:.c a dvnnccd b r cc:ding l i n

es nnd to sta bi l i7.c their charJctcri S. Iic S. I' C K 

### u nd Southern Ana l y ses eontim led the 

### positi\'c expres s ion 

### of the npl ll 

### and PRS V-CP for :til 

### four 

### e l ite: p3J'I.l) ' Q 

### lines expre ss

**in ~;**  

### PRSV-CP trnnsgcnc: s. The: cbt3 

### sub mi ned 

### to lht 1 \ gcncy 

### for X1 7 - 2 is from 1hc six 1h b.1cl::cros.sc<l gcncr.1tio n

**(~).** 

### Tronsfcr DNA (T -DNA). "h ich inc luded lhc OO>l 

### pcotcin (C P ) g ene 

## orPRSV isolnte HIK W:l S 

### modifit"d b) ' 

### i ne orpor.uing the nu cleosi de thymidine . . **n:~uhin g**  

### inn fr..unc-shifi mutation nnd the: nplll 

### gene . 

### This cassette wns

**tr nnsl~rrcd**  

### to 

### the geno me of 

### in<lividuaiJXlp.1)'U so nuuic embryos und p l u

nt s

**n:gcn~ro cd**  

### from t he 

### uan .s l b n ncd e mb r yos. X 17 - 2

**~xp~ s.scs**  

### two pro t eins not found in t ypicnl papaya: the PRSV -C I ' nnJ n eomy cin p h

os pho trnns l cm.<c (NI> ' rtl) . N l 'l'll pro te i n an d the n

p tll 

### g.;nc urc no t kn own 

### t o en usc 

### nd\'c rsc effects 1 0 

### mu mm nl ian s peck s and 

### ha \'e been 

### pr.:viously gr.unc d 

### exemption f r

om th e 

### l'l"qUiremcn t of a t o l

crnnec 1 -10 

### C FR § 

### 174.521 ] when used in nil

**D~ricuhur:1l** 

### commoditi es.

**c»"1~transcriptio nal**  

### sene silenci ng

**:~1.-;.o nown**  :IS 

### R...'JA - intcrfcn:ncc (hcncdorth referred to 3S 

### R. N , \i ) occurs in ntOSI

**eu~ 'O**  **cs .**  

### Doublc: - sar.mdcd RNA (d( R NA) 

### is recognized and

**p~** 

### by 3 

### nuclcasc·dic cr into small 

### in h."tli: rins Rt'lt\ 

### (si RNA ).

**~** 

### si RNA s incorpor.uc into the

**Ar~on.aut** 

### prot ein and 

### mcdi-:tcc

**dc ~md.n**  **ti n**  

# or

**me sse**  **ng~r** 

### RNA (mRN 1 \ ) '"ith highly spe-c-i t ic s -.'<lucncc complimentarily to the ds R NA. The obse r ved r esult of

**hi ~**  

### n nti s cnsc technology i n Pll ' s

**~hows** 

### plants re- s i stant t o infec t io n 

### by P!) R V . 3.11 

### R NA \ ' i

ru s. Rc sis tnncc co n ferre d by t he coat pr o t

ei n 

### gem: of a

**p<) )'\'**  **n.t.~** 

### i s

**~\tA- mcdintcd**  

### {L i nbo ond L> o ughc n y. 1992)#,>. E..'lrly ellOrt$ t o mod i I )' pl30ts . insect s.

**nmn .**  

### o n

d various 

### o th e r organi s m s 

### wi th tro l tsgcnc s or an t is en se contructs used s ingle 

### strand ed RNA (ssRNA). n lm os t exclusi\'cly. Ln l er

**~1udies** 

### r.: \' caled th:lt 

### substi tuting double - Stranded RNA (dsRNt\) for ss RNA produc<d an cnh,.c<d potency eqWII t O 

# •

### 1 0 t i mes t he i m cndcd cft\.-ct on s upp ress i ng gene exp r essio n (EPA, 2013 )' . S i nce th is d i scon r y . r esea r ch into sclccth ' i

t)' , eflic ncy n n

d human toxicology of u tiliring RNt \ i i n I Ood commod i ties ha. <O**~><e n**  

### o ngo ing. 

### Wh i l e \' :t ri at ion s of 

### b iochem i ca l detDil s of 

### RNAi**pathwa~ s** **~xist** 

### among 

### organisms of ditl'eri n

g ta.x o no my.lhc: modes of net 

### ion a rc 

### s i m

i lar and core 

### c o mpon en t s n r c**rctain~d :**  

### Arg.On ! H J !c ( Ag o) 

### :m d Di ccr (Ocr) . S ome 

### e xample s of coat 

### protein s ex p re ssed in p lants 

### i nclude Zucch i n i Yellow Mosaic.**Wat~mtclon** 

### ?\ ·los.1ic-2 . and Cucwnber 

### t>.· fo sa ic Viru. 'iCs 3 $ 

### expre sse d i n squas. h (Cm · urbitu fh:po) 

### p l::

m

t s .linc ZW - 20 

### n nd vnrious 

### melons. 

### These \' ir.sl co:n 

### p

ro tein s were g.rnntc d 

### toleran ce exempt ions ba..54!d on r.uiona l c ofth e 

### ubiq u ity of 

### p lan t v i ru ses in 

### the 

### ngricultural en vi r o nment. lac k of any indicat ion 

### of m:unmaJian toxici t y 

### w i th c urrent 

### dictnr y co nsu mptio n of the 

### ent i re \'ir'US 

### incl udi ng th e 

### p lan t vim ! co;u p r

otei n. 1md lnck of m n

mmn l ian p :nh og cnici ty for p l ru

ll viruses. (40 C FR 

### 147.5 1 4 :md 40 CFR 

### 1 74.5 1 5. r <SJl< C i ivc l y ] . 

### Add iti ona ll y. the 

### pre s ence of p lant vinLSCS. 

### t md there fo re ' ' iruJ co : u p r

Oi c i ns . is h ig he r i ll 

### natur.tlly i nfec te d 

### c r ops th;m in the t rrut.S gcn i c Jine s of JXlp.lya T il"$ t M rth od§ and**t~ ul** **t'i**



## · n1e fO ll owing t ab le con t a i

ns t h e tc.)1 

### matc: riaJs an d method s i n s uppor1 of 

## .;o CFR § 

### 1 58 .2130 Microbial Pwicidt·s Product Anai) ' SI

.<

/Jara <;uiddin,· . <: 

### 885.1100. 885.1200. 885.1250. 885.1300. 885 .1 400 . 885 . 1 

# soo. Pr oduct C h em is try an d Compo s i ti on 88S.UOO T h e X 17 - 2 l i ne pre se nt l y co n sists p rim a r i l y of a fami l y of fo ur**br~eding** MR I O: P rodu ct Id entity li nes lhJt Jre des.cendJnts of crosses 

### between the or i g i nal 

### tr ansgenic 49 4 93201 p l ant { Ro ge n eration} and fou r e li te nont r ansge ni c. papaya ge no typt\s , 

### na m ely; So t o S unris.e. P uerto Ri c o 6--GS. 

### Ta inu n g No . S, and SO lo -1 0 . 

### Transforma t ion Event X17 · 2 (p8 1 12lfs} : 

### One i ns e r t i

on eve nt 

### cons i sts of a N O S promote r e l e ment , np tll g e ne, C aMV 3S·S p r omote r, PRSV coa t p r ote i n ge n e. and a N O S te rmi na to r.**~c** F i g ure 1 for comp l ete D

NA co m po n e n ts i n v e cto r p8 1 1 21f s . The p ri mers used t o dete< t the tr ansgene construct w ere as f o ll ows : F

OI (S ' ·AGAACTAGTCCCGGGT GGTCAGTCCCTrATGTTC CAMM T GM G CT GT GGAT G · 3') ROl (S '· TACAGATCTA CC C T CACT A T AAAATAGAAGC · 3 ' 1 Th e c l o n ed CP ge n e wa s u s.e d as a temp l ate to a m p l i fy the ONA sequence o f t he m odifie-d C .P by P o 1

y

mc r ase Cha i n**Re~ction** ( P C R ) .**B~sed** on previous s tudi es a n d t r ial s , the u l dA l eader was found t o be more effect iv e t h an a h o m ologous virus l t.'l d e r to enhan ce th e exp r enion of t h e PRSV· C P. T he r efo r e. the u l dA Seader w i t h an in itiat i on codon (S' · CT A

GTCCC GGGTGGTCAGT CCC TIA T G-3 ' ) was p l a ce d at the S ' end o f the PRSV · C P construct by i n dus i on of the sequence i n the 

# rot primer . PCR was**~rfo rm** **d** a n d the f r a gmen t wit h the added uidA I Nde r an d start codon wa s confirmed bv 

## sseoquencir 'l - The bfN ry

**~OC"** pBi lll was doub6Hi lg e1ttd 

### wit.h Stnol a nd 

### Sect blunted with T4 DNA

**po lym~ras.e**  and thtn rtli &ittd 

### wit.h T.: DN A

**ga~** to produce p8112lzy.lineariztd p8112tzy w.n produced by d i ge.lt io n with X bol and SamHI. The PRSV · CP fra gmen t w as

**separ at~d**  by

**agaros~** gel e l e< H o phore .sts. isola ted fro m t he g e l .
**doubl o-**  **g~ st~d**  with Bgtu and Spr l , and t h en

**don~ d**  I nto the li nearized p9 1 121ty to p r

od u te pB I 1 2l f .s . During a norn 'l a1 virus I nfection. CP of PRSV Is encoded as pi'lrt of a potypr otel n . The CP codi n g region th er efore l acks a S' -unuanslat e d r egk m and a translat ion lnl tlat lon co don . The con.sttuct developed for t raru formation of papayil

**rov~cs** these eu.entia1 sequences for

**appropri.lu~** pl.ant6p res slon . Trans.c:ription of the modififif coat prot ei n gene i n

**transform~** ptanu is d i rrtted by the 35S promoter of 

### tnt

**C~u li**  **flower** Mowk Virus (CAMV) ;and termi nated by

**t~** NOS tetmiNtor sequence of A. 

### tumr/oc k n.s . The npC11 gene f rom the tro1n.sposon TnS of 

### the bacter i um £Jchtr i cltio coN wa . s introdu ced I nto

**P.l~ya** as a select<1ble marker . The &ene codes

**forth~ ~nzymc**  neomyci n

**pho photransfer**  **a~** th at confers resist an ce to the c ommon amlnoglycoslde ant ib i otic kan amycin . The np rll ge ne l.s under control of a bac-te ri 31 promo te r ; NOS termln ator of A. wmefocftn s. 

### CLASSIFICATION ; ACCEPT ABLE 8&5 . 1200 The Xl7-2 l ine p1escnt ly cons i sts primarily of a fam i l y of four bree-ding 

### M R I D :

**Manuf~ctu ri**  **ng** l i nes th at

**au~** descendants of

**cros~s** between th e

**n~1 lr**  **ansge nl**  **c** 4949310 1 Proceu

**plan (Rlgene~**  **on)** and four t' li te

**nontr.~ru.ctnk** papaya genotypes . n.ametv SoJo Sun r t st, Putno Rico 6-65 , Tainunz: No . s . and Solo 40 . 

### The vector ( p8112lfs ; Figure 1) used to

**tr.~ru.fer** the &enes to p,;JJNY OI plants was constructed from 

### the binary ve ctor p8 112 1, a derfv a tlve of p81f'll9 by repiJ ci ng 

### the GUS gene with a connruct of the CP gene of PRSV . The complete stque n ces or pS I N19

**~nB3 k**  ;,ccenlon 11U09365) •nd pBilll (G•nBan k Mf48578l) or< pub lis h e d ond

**~wai!Jble** to th e genernl public- . T he transforma ti on tk'ments i n th e vec t ors were derN cd from part of t h e 

### Agrobo ctetlum tu mot l n d u d ng plasmid pTiT 37 . The mC!'thod for Ag robo rt trfum -med l a ted transformati o n a nd re ge nC!'rJ ti on of papaya p l ana from MJma 1k embr)•os conduCled by Ying et al. (1999)1 > , was used to dev elop lilt Xl7 · 2 11 ne. T •ONA. wh i ch J ncJuded the CP gene of PRSV k.o1;Jtt HlK mod i fied by anc orporation of thymkfine to UNte I fr.tme · shi h mut.Jtion and 

### the n p:U gene, was

**tr.uuJ~rr~** to 

### the genome of incfrvkfual pap.lyiJ somat i c embryos and p l ants regtneliJttd from 

### t he tr.li ns form.ed embryos und er Una m ydn selection . (See rt s ure 1: schematic

**lll strat~n**  of p linm !d

**v~or.** 201 •1) 11 

### C

lASSIFICATION : ACCEPTAB l E 885.125 0 T he cloned HlK se quence Is depos i ted r n G enB:t n k as 

### MR I D : De p os i ti on of A CCC!'ss lon li

**96839.** The H lK cult i var was b;ued ofr s im ila r 494 93201 samp l a In a tech nology d eveloped by Dennts

**Gon~ ves**  

### tt a1 . • for u se the PRSV nationall y resis tan t HawiiiJn

**P•J»~ .**  While PRSV · C:P was shown to be tlp r ei.se d recocniled in the H a w J ii an pap•ya, It Is now beli eve-d that no rm al

**viN~ te~lit.'llnce**  re sponses in planll uhllllng R N Ai pathways were probibty

**~lso** In play.



### 6cui c urt- Bi olnfo r matK: a n alytica l**co mp** **ri~on** of th e two a m ino aCid sequences collection of HlK and the H awaiian Rol i nbow P apaya via a BLAST ana l ysis yielded 

### a 96 % homology. (See F i gure 

### 3) ' CLAS51ACAnON : ACCfPTABLe 885.1300 R esista n ce to ka n amycin i n t he trans formed cell tines I ndicated 

### MRID: Discussion of e xpr essio n of the nptll gene i n theX17 · 2 1i ne. T h e PRSV·CP is also 494 9 3201 forma tion of expres-sed but 

### at low t e.. ·e ls compared to coat protein l evels in papaya unintentional n aturally inf ected w it h PRSV. fnzy-me·linked lmmunosorbent Assay ingredien ts {ELISA) usi n g polydonal a n t.i-PRSV serum in dicated a low co n centration of the P RSV· CP In uninfec ted X17 and 

### 06 b r eed i ng l i n es . Similar res u lt s were obta in ed using another po lyc l oM I P RSV antiserum fWMV·l ant i serum) obta i ned from the Univers ity of Flor i da. Addltlonally, the expected 3S kOa PRSV CP wJs detected by Western - blot analyses us i ng t he same two antisera. The 3S kOa s i ze of the express ed PRSV 

### CP In X17 · 2 Jnd ot her wmsgen l c l i nes was the s.ame as predkted from t he nucleotide se-quence of PRSV RNA . The natura l PR.SV<.P i s one of at least se\•en prote i ns clea ved posHranslatlonally from the ea:pressed poly p rote i n of PRS V i n i nfected plants. The 

### C9 co n structs i n the X17 · 2, 0&, a nd 09S l i nes we r e de signed to b e**no ~trans** **atab e**  into P RSV coat protein du e to i n sert i on of stop· codon or f rame · s h ift mutatio ns at the 

### 5 ' ·end of t he CP ge n . T he Western b l ot resu l ts , howeve r , I ndicate th at t he m ut .lt ion s were r e.ld t hrough or repa i red resu l ting i n expression of the CP ge n e. { S ee F i gure 2) 1 1 No s i g n ifica nt imp a cts or un i ntentiona l cffecu arc expKted, as both the PRSV - C P and th e NPT II prote ins were previous l y found to p re sent a reasonable certainty of no h arm grante d to l e r ance t> x e mpt ions i n 

### all food commod it i es when used 

### a.s a pl an t· in corporated p rotectant; 40 CFR §174.SlS, 40 C F R §174 . 521, respective ly . ClASSIFICAT I ON : ACCEPT ABL£

The rollow l n . e table con t ains t he rationale along w it h t est ma t eria l s and m ethods i n suppo rt of 40 CFR §158 . 2140 Microb i al Pesticides ToJtkology Dato Rt:qu l trmtnls Tobfe & Gu i ctelin es; 885..30 50 , 88S.:HSO , 885 . 3200, 870. 1100 . 870. 12 00, 870.1300,840 . 2400 . A tier Ill study was submitted as 

### part of th e Sec. 3 R

egis tration and 

### h as b ee n o mi tted , as it 

### J s not 

### a required study or gu id eline for P l a nt - I ncorpo rated Pro tectant r eg i st r ations 1 40 CFR §158.2 140 , 885 . 3650 Rt'product t.. •e fertility EHe<ts) . Mi trobial Ptstlddts ToldCQl ogv pua RtQu i remt'nt s (OPPT 885.3050 , The Un iversity of Flo rida has subm f tt ed data to su pport th eir 

### MRID: 870.1100) d.ata waiver requt'st s based on t h e following rationa l e. Who l e 49493202 vir uses are ub l auitous i n the a,::ric ul tural environment. Virus 7Acute Or.ll Infected plants 'o\oill express tM enHre virus

**par1id~.** as 

### wd .11 Toxldtv/P.ltho&tnlcl ty vl101l coat PfOtein, at levels h i ghtr than w i U 

### be present In tromsgenk plants. Virus in!ecttd plants hive historic:alty M-en a

**~n** of 

### the

**hu~n** and dornet.tk

**<~nlmll** food suppty with no obse:tved adverse etft"CU to human Malth and •nf.lr'lls upon consumption ( EPA 2007 )) . The Agency del . ttmintd 

### the

**co~t** protein of papaya ringspot virus mut lht:

**stt~nd.ncf** of presenti n g a reasonable cenJlnty of no harm ;nd

**;~re** exemp t from the requi rem ent or a to ,er.1nce when ustd as a pbnt· inco rpora ted protectant I n or on all food commodltles.l72 FR. 20-13.4, Apr. 25 ,

**2007) Ge**  **et call**  **y** m odified PRSV re sista nt papJy.l has a l ready b fJ'e n commer cially availab l e and co nsumed s i nce 1998 wit h t h e In troduction o f th e Or st t ra n sgcmk: Hawal i an Rainbow Papaya , de veJope d by J collabora ti ve e ffort between th e Unlver slty of H awa i i and Corne ll Uni versi ty {Gonsalves, et .1 1. }'. A relevant w ho l e food reed i ng s t udy submitted by IR · 4 in suppon of a data waNer for 40 CFA §1SS . 2:140, eva l uate d the comparative toxicitY efferu of devel ope--d

**r.t ~genk**  lines of tht': fruits of tW'O batkcroutd tra

**n~genlc** pap1y1 line-s ('-tr.1in.s; 2210, 823) and one hybrid tine (821·2210), to c-ompare the i r

**@'nt** non·tlinsgenit {TN-2)

**counte~paru ln**  lh-e c-itt'd study, a 

### sUbtlity analysis for PRSV·C.P wu conducted v i 1 dfs:est . ion Slibiflty assays in simuJ.Ited

**~~uric** Rukb (SGf) wt1h a pH • <2.0 .. simulated intestinal 

### fluid (SIF).

**<~nd** b il e Silts co detect the CP fragments. Resu llS revuf.ed tNt the CP fragments were rapidty h ydrolyzed in SGF and were u ndetectable in organs and sast r of nt estinal const ituents in 

### tats . In;, 28-diV repeated ftcdlng st udy, no death o r abnormality of rats occurred In the nont ransgenlc TN · 2 an d bactcross tr ansgeni c P.lPOlVJ fruit treatmen t group s . Additio n a ll y, no slg n 10cJ nt d U ference of feed e ffi c acy was observed i n a ll t rea te d g r o up s I lin , ot al . 20 13 ) 1 • ClASSIFICATION: ACCEPTABLE Acute Dermal Toxldt y Du e to the nat ure of vlrJ I co-at proteins produced in p la nt s a s MRID: (OPP T 

### 870.1ZOO); part or a PIP , expos urt- th rough any rouH! o t he r than dietary i s 

### 49493202 Acute Dermal unl i k e l

y to occur. PhyslcJI con t act with the plant or raw Ir

rit ation (OPPT agricult ural food from the plant 

### mav

**pr~sen t**  **~me** limited 

### 870 . 2500) •nd Acut o opportuni ty for dermal e l(posure . .. owevt'!r. on .1n lndMdu.1 l Otrm.al buls, the

**potent~l** Jmount\ i.nvolved In this t_l(posure are Tokl<lOV/P•thotorv ne-gligible in

**com~risonto** e k posure through the diet1ry route. (OPPT 

### 815 .ll00 ) Additionally, viral coat proteins

**ptod'U<~** I n pbnt.s

**~~** p.ut of a PIP are

**unh~ty** to penetrate tht>

**e-piderm~IIJ~r** of the. skin (EPA. 1997),.As prevlousty nated . tht c01t ptotein of JNpaya ring.spot virus are ex-empl 

### fro-m 1M requirement

**of~** tolerance when 

### use-d as a pl.ant·lncorporattd pro · tect.Jnt I n or on all food commoditMos. [72 FR 20434, Apr . 25, 

### 2007I ' Un •••t . , determined ln the i r study through r epeated

**uposuu~** that no sfgniftc<Jnt dermall tsions were observed In either m a l e or f emale rats )•l The Food and Orug

**Admln t~tton**  lFOA) 

### 8determined th.at based on the safe ty and nutriti onal asse.ssmen t , papayas are not compositio n ally different from p apayas cur r e ntly on the market and th.at t he gcn et i c . ally eng in ee red l ine Xl7 · 2 does not raise nove l issues t hat would requi re premarScet approva l by I' DA . IlDI ClASSIFICATION: ACCEPTABlE Acute lnh a l .3tion The**occurrenc~** of resp ira tory exposure of vi ral coat ptotein MRID : Toxicity (OPPT produced In plants .1s part of a PIP Is neg Ug ib l t" In compJr i son to 49493202 870 . 1300) and Ar:ute po t entia l exposure through t he dietary route . V I ra l coat Pu l monary protei n s, w h en present In polfen, are un l ikely to cross the**Todcity/Pathog~nidty** barr i e r provided by the mucous membrane of the respiratory (OPPT 885.3150) tract. The r e is n o sc l entific ev i dence, to date. wh i ch suggests that p u lm o n ary exposure to who l e plant v i rvses by i nh-a l at i on resu lt s In a n y ad verse effects to mammals, to in cl ude infants and child ren. Additiona lly , the fin a l product Is J sys t em i c vira l res i stance and**d~s** not h .avc a sp r a y.1bl c compont"nt; thus rendering the r i sk of pa r ticu l ate i nhalat i on <"xpos ur e pract i e:31ly non-existent . ClASS I FICATION: ACaPTABLE Acute Eye Irritation All available i nformatJ on Ind icat es that v i ra l coat proteins In MRID: (OPPT 870.2400) food have no hum an t oxic ity . Data regard i ng Acute Eye 49493202 Irritat i o n 

### ts n ot r eq u i red fo r v i ra l coat prote ins**~ince** th e re**~** a long history of m3mmat ian d erma l ex po s ure of th{' p l ant v i rus part i cl es by handli n g food, w i thout causing a n y k n own de l ete rio us huma n hea l th e ffects or any evidence of toxicity. T he Xl7·2 Line of TrJnsgeni<, P.lpJy.l R ingspo t ·Res i stJnt PJp3ya does not repres e nt a source of novel potentia l allergen i c or ant i ·nutrlent prote i ns . CLASSlF I CATION: ACCEPTABLE



### Conclu sion: The X17 · 2 line pre se ntly cons i sts pr i marily of a fam ily of four breeding lines that are descendants of cro sses b e t ween the ori gina l tran sge n ic plant (RO**gcn.c-r.~tion}** and four elite non- t ransgenic papaya geno typ es , nam e l y Solo Sun rise, Pu e rto Rico 6 - 65. T al nune No . 

# s. and So l o 40 (Da v i s an d Vlng, 2004) 1 • A pr

ocess of r ecu rring se l ective backcrossing fo r desired phenotyp i c charJcteristic.s, i nc l udi n g r esistance to PRSV, yielded advanced b r eed i ng lines and stab i l i:ed th e i r characte ris t k.s . They a re currently in the sixth ge neration {R4) of th e p r ocess. In th e R s gencrcltion of**Xl7 2.**  a 

### 493S base pair PCR f r ag ment contAining a ll but 

### 336 base pai r of the T·DNA i nsert was clon ed an d seq u enced. The expected ver sus ob s-erved va l ues i nd icated 99. 7% hom o l ogy. PCR a nd Southern Ana l ys i s confirmed the pos i tive expression or the 

### 9nptll Jnd PRSV · CP for all four PRSV<P

**aransge~s.** Mendel ian

**stgreg~tlon** va1ue:S of the transgenes in the Xl7 ·2 liM Wf'rt as

**~pecttd** for a singte insertion site of 

### the tnns.genes; 1:1 for the outuosstd Rt gentttltfon (50 . 1" pos1tfve). and 3:1 for R 1 generation (n.9,.) . Tht It, gentrat . ion 

### won. 1-t ill doSt" to the tllpt<ttd rrequtnty of 7S % with a vo\Jut' of 3 : 1 (79 . 8%). Althou&, the Xl 7·21ine e xpresses two proteins, the concentration fn relation to I'IM-lt.lnsgenk papaya b e.-tremely ktw . ltle expression levels of PRSV·CP were net dete-ctable vtJ EllSA, but only Western 8Jot Anlllys es: indfc.Jtl ns the tonuntration in the transgen k: cultivar 

### to hh't 

### a kM'er PASV · CP concentration (O . D . of 0 . 18710 . 072), than naiUrally inf•Cl«l PRSV pap.>yale•f tiSiut (0 . 0 of 2.54110 . 018) . The aforementioned transgenic 

### ELISA values a re median concentr3tions and standard d@vlation v.J i ues. ca l c ul ated from th e four e lit e brfoeding lines of 

### X 17 · 2: Solo Sunrise progc!nles. Tai nun g No .S progenies, Pu e rto Rico 

### G · 6S progenies. a n d 

### 06 . The 

### X17 ·2 res is tance to 

### PRSV is believed to be the result of post-transcriptiona l gene sile ncin g du e 

### to t he lnser,l on ot th o PRSV · CP gene Into the papaya genome . PosHranscrlptlonJ I gene sile ncing. known as RNA · Intorfcrcnco or P.NAi, (referred to hereifter as RNAl) occu r s In most eu\aryotes.. Oouble · st r anded RNA (dsRNA) 

### ls d<ed by 01 nU<:Itase-dicer into smatl

**in t~rfet**  **ing** RNA (s i RNA) tllat media te 

### a h ighty spedflc: iequence degrJditlon ot meS$Cnger RNA {mR NA) and prtvel'lt translation of that mRNA in to prote in. 

### The

**obienr~** result of this antisen}e H:chnotogy In PIP'S Is the plants r esistance to infecH on by RNA vlruses. ReSistance conferred by the coat prote in 

### gene of 

### a potyvfrus 

### is RNA·mediated (Un bo and Dougherty. 1992) '. 

### E.Jrty efforts to modrfy plants. lnsr< t.s. mammals. and Viriousother orsanUrm w i th transsenes or

**otnt~n.sc** cont.ructs used only sins,le stranded RNA (ssRNAJ. otlmost e x

clusn.-ety. Later studies r eve.1led tholt substituting double-strand e d RNA {dsRNA} f or uRNA produced J tenfo J d ii'Ktease fn potency for 

### th-e Intended effect of gene suppression 

### tEPA, 2013) 4 • Since the discovery cf 

### RNAf i n p la nts, r csc;uch Into selectivity. etr.cacy and human to);lcology of utlllllng ANAl In food commoditje s has been cngclng, While variations of biochemical detalls of RNAI

**pathw~vs** C Ai st among organisms of differing taxonomv. the modes of actiOn are s i milar and cora components r tte retained : Atgona ute (Ago) and O ico r (Ocr) . Some previous exolmp tcs of coJt proteins cons tr uc t .s in p l o'IOLS Incl ud e Zucch in i Yellow M osaic, W.1termclon Mosalc·2, and Cucumber Mosaic

**Vlru~ cs**  **.:~s** ctprcued 

### I n s.quash

**~Cucurbfto ~po)**  p l ants, li n e ZW·20 and various melons . All of these prev ious vlt1 l coa t pro t ein C'Xprcsslng constructs w e re granted

**•olcro:~nce** exemptions based on rationale of the- ubiquity of plant viruses In consumer agricullur.ll supply,

**I;~** c)( of incliatlo n of ma mm alia n toxic i ty with current plant vi tol l

**co;~t** protein consumption, and l ac k of mammalian pathogenicity for pl.ant vi ruses . (40 CF.R 

### 147 Sl4 and 40 CFR 

### 174 . • 51 

# s. r espectively) , Add i t J onany. the presence of plant viruses. 

### b h. lc, her In natunltv Infected crops than I n tht transcen.lc lines of J)lpaya . furthermore. plant virum

**~re inap.~btc**  of

**reproduc~** in

**mamtn.~ls** and other venebrates. thus elim i nating tht- potential for lnfe<-tion. The transfottned fktrkfa lint of X.l7 · 2 was deregulated by FDA in 2008 and by

**USOA·AP'H~** in 2009 . 

### &sed on 1ht efficacy of 

### the Hawaiian Rainbow PiPiYi that hu been ow• Ua ble since 1998 , there have Moen no obs.rwd 

### COJdcitv cr other issues nsocia · ttd with hum in hei!lth . To confirm s imll.lrtty bJsed off submitted r.ttk>nale for exemptions. the

**awai~n** sequence 

### wou

**compo:~red** vi a BLAST to its florida HUC counterpan. and

**ln dl**  **co:~te d**  a 96% homology. The mlnor v;)ri;,tkln rn 01mlno acid sequence between the t wo strains I s negligib le and cou l d be expected s ii'Ke t he virus phenotypes

**v~ry** gecgtJphlcJ i ty . 1 0

### Kcvicw of n s u bmiucd s tudy f r

om IFAS J•rcnatal J.>cvcloprnent Toxicit!o ' s tudy . OCSPP**guic.l in~**  

### 870 . 3700. wa s omitccd. 11tis submitted st ud y rdt:rs to t he: current Tier Ill guideline OCSPP 

### 885.3650. R eproduct i ve Fe rtility EOCcts w hich is not a requ i rement tOr 

### Sect ion 3 

### rcgistr:H io ns of Pl

unt - lncorporntcd Protcctants ( PIP s) un le ss then: n.re 

### wxicity re s ults or other indication s th a t this s tudy wou ld provide use fUl infonnation .

II

### F igure' 1 : 

### //ill\1111 &oR I

F igur t' 1 : pBt121 

### fs Schem3dc illusuouion of the 

### pt3.Smid \ ' CCtor

**t~l** 

### wastmnsfcrrt'd into

**~robuc:teriwntrmt~fat:i;.• JJ**  

### by 

### dc:cuoporotion.. Various components of the T-UNA 

### of pl)ll2 1 fs ore described in Tobie 2 .

**up** 

### rclle<: tS t he gene l )rder 

### b ut n ot the siz.cs. The T -D NA lx: twe<n the lefi 

### (LIJ) n n

d riSIII (RB) borders has re s tri ct ion s ite s 

### li)r l/ i11d lll n nd 

## EcoR I. In sert i on of t hy mid i ne. ns i nd i cn t.;. 't

l 

### by an O$ t cr

is k. res ul ted in crc : 1ti on of th e und.:rlined stop co.Jon dO\\nsttcn.m. 

### TI1c rcg . ion ou t si de I he 

### RO ::uu l 

### tn is the p l nsmid \'Ci:'tor backbo n e DNA
" Source: USDA -AHPIS. 2009 ' .

12**Fi n**  

### 2: A 

### 8 lll!t 

# • 

# ' ' • 

# • • ' )SI!J 

# • 

# ' ' • 

# • 

# • '

**:~** 

# .. " 

# .,_ • • 

# ,._ 

# .. "

**---~** 

# ........... 11- 21 

# , _ 

# " "'- 

# . . ... 

# .

- . ...... ' 

# ,_ Fq;urC' 

### SA :.tnd 5B , Wotcm 

### Blou oftoul p.1p3yn k:af ptoet:IO 

### from I he 

### X 1 7-2. 06. 

### :wd 095 lr.t n sgC"mc 

### lu)($.. W 

### from no•Hmnst::,<'U I C 

### Solo Sunrise

**pop.l~'D** t h.lt \\ '3$

**hi:~llh)** 

### or utfcctOO w.th 

### ' 'RSV H It( S3mpk$

**COOI:Umn~** 

### M O JI ,Y 

# or p!OI:I;'ID \\('f(' 

### 5q).'lmiC'd

**b~** 

### SOS- I )AGE on 3 

### pr\."QSS 12 '!" NuPAGE.scl ''•th MES

**NMm~** 

### buffer :1nd

**tr:ut~fcm.-d** 

### t o POVF lllC'tnbnma . I n Fig ure SA. th e mC"mbr.mc: \\:1$ 

### probed \\llh 3ni i ·PR.SV saum (A.l;,di:t. lnc. .). :&nd 

### an F•itourt

**.** 

### the mcmbr:mc- 

### '' "u p1obcd \\11h

**~r** 

### :mhwum for I'RSV 

# .o . Pu.rciful. 

# uru,

**r.•ffit~** 

### o.fFlOfacb) lb: 

### :wnplcs

**"n~** 

### (I) X 17-2 hnc: "'Lh Solo SunrL.SC proacrutor . (2) 

### Xl1·1 hnc wnh TautuJlt; 

### No 5 proge:nilor . 

### (3, l)(J 

### hue: \\11h Solo Sunruc Pf'OS\ " Mor . (-I) 

### 061anc \\itb 

### Solo .& 0 

### pn)lc.-:rutor . (5) D9S 

### lu1e 

### Wllh 

### So&o Sunnsc 

### progaulor , ( 6 ) 

### So&o SUN\k': . 

# en Solo Sunnsc mf«Ctd \\llh PRSV 

### "l'bc ;utO\' ttl both fi b'\l tC:S mdctcs tbc

**l~t.ot~** o flk 35 "--.a PRSV C() WI \\!lJ (ou,nd

**01.\1~** 1n

**r.\l'~Sicruc:** htld 

### 'S o urce : USDA·t\ P HI S. 2009 11 •

13**fi re** **~:** 

### , \min o A ci d S l'9U C Ut' (' ffiL\S D 

### D osc ri pJLo .ru;**~n** O'QI;II.Cing '9"'<'rt**~9'"'t""U** OesQJp l •on Max 

### score**ToU~I** StOIC Query 

### covet E v a l ue**Cto\lP'~Ie'on** IC••u**e> t~r•l**  

### Align m e n t s <Ol1 Ot?:f!II'I I C¥1Ca**~AI** 

# ,., 

# , 

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# ..**.** 

# .... ..... . 

# ,.

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# "'**~** ¥\**._5C~l ~~** l Mitl"'.'loC I U G f\110 1 5>... ...

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