SllllJECT: 

FROM: 

TIIROUGII: 

TO: 

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C . 20460 

OCT 0 6 7015 

OFFICE OF CHEMICAL SAFETY 

AND POUUTtON PREVEr-ITION 

Review of t-. to ~u ur Char.u:tcriz;ation O;at;a. ProlJ:in Expri.'SSion Analyses 

ond Hunton llc.:tlth AS.SC~'i.nlCnt for r•Jant-lncorpor:ltcd Protcctant (PIP) 

PnJ"lya Ringspot Viral Coat Protein (PRSV-CP) for usc in X 17-2 Papaya 

(Carica papaya L.) [EI'A Res. No. 84427-R: OECD Unique ID UFLX 17CP·6) expressing Popnya. Ringspot Virus Coat Protein. in support tOr 

FIFRA Se<tion 3 Registr:Jtion [Decision No: 497668. [)P llnrcode: 

4248&4: Submission No: 960423. MRID Nos: 49493201. -02.-0-1. -05]: 

ubm U~!d by University of Florida. ln. utc of ood and Agriculturnl . n J 

Sciences (IF AS) , J if" 

t

,;;; < -1.t)u,tJv c~t r Wi1l,SClJ1. Uiolosist tN ~ v V (/ 

Micro bini Pesticides Br.tnch. BiofX.<>sticidcs and 

~:~~:~:::~:~~n;:~~)~o::i :i::e~e(:~:tii )\'::;I;::ANlJ .14' 'r Q A 

Microbial Pesticides Branch. o~ti des 0 ...,' Pollution l)re\'cntion Dhision (7511 P) 

Michael Mt'ndc:lsohn. Rcgulntory Action Leader 

Microbial Pesticides Branch. Biopcsticidcs uml 

Pollution l'revention Division (75 11 P) 

ACTIO X ltf:QUESTEI>: To review the d;ua submitted by University ofFiorida.ln.nitutc of 

F'ood and Agricultu13J Sciences (IF AS) in support of a ~ction 3 regist13tion for the uctivc 

ingredient Virol Coatl'rotein of Papaya Ring.spot Virus (PRSV-CP). with an end-usc product 

name. X 17·2 PopJ)-;J [QECD Unique ID: UI'L-X 170 '·6]. 

f:COM~!F;;" \ TIOI'/: 

The s ubmiucd dai.U :1rc dnssilictl as ACCF.rTABLt: A similar protein to the prcsc:nt active 

ingre-dient Viml Coot Protein ofP~tp:tyn Ringspot Virus (PR.SV-CP). has prc,iously been grnnt~ 

a pcmlancnt tolcmncc exemption [~ 0 CFR 7~.5 5]. 

I 
DATA (;VALUATION RECORD 

Primary R~vicwcr: Jennifer \Vingcnn. Oiotogist 

EPA Sccond:1r)' Reviewer: John Kough. Ph.D .. Scni(IT Scientist 

STUI>Y TYI'E: 

ACTIVE INGREDIENTS: 

MRIIl NO: 

DPBARCODE: 

DECISION NO: 

SUIJMISSION NO: 

TEST MATERIAL: 

STUDY NO: 

SI'ONSOR: 

TESTING FACILITY: 

AUTHORS: 

STUll\' COMPLETED: 

Product Identity. Manufacturing Process. l)cpositjon of 

Snmplc. Fonn:Hion . ofUnintcntionallngrcdic:nt$. Analysis of 

Samples. CcrtiliClltion of Limits. Acute Oral Toxicity. Acute 

lknn:.'IJ Toxicity. Acute Inhalation Toxicity. Acute Eye: 

lrritntion. l'rimary Dcnnal Irritation. :\cute 

Toxicit~ /Pn thogcnicity. :\ cute Pulmon:uy 

Toxicity!Pmho0'<:ni<ity (OCSI' r 885. 1100. 885.1200. 

885.1 300. 885.1400. 885.1500. 870.1100. 870.1200. 

870.1300. 870.2400. 870.2500. 885.3050. 885.3150 

respccti\·cly) 

Pl:mt Incorporated rrotcctant (PIP) Pap.'Y" Ringspot Virus 

Coat Protein (PRSV-CP) and the genetic matcri:1l nc."<.:cS .. •> ..'l.ry 

for its production nnd the mnrkcr gene (N I)TII). 

49493201.49493205 

424884 

497668 

960423 

P•J>'Ir• Rinsspot Virus Coat Protein (XI7·2} (EPA Reg. 

No. 8~~27 ) 

IR·4 PR No. 0537B 

?-.·tichlu:l Bnh'emKH'I. l'h.D., IR·4 Project Headquarters. 500 

College RoJd East. Suite 201 W. Princeton. NJ 08540 

Uni"crsity of Floric.ID. Institute of food and Agricultural 

Science (UFL-IFAS). Citms Resenrch nnd Education 

Cl!nter. L.akc Alfred. Florida 

!\'lichacl Brnvc:nnan. Ph.D .. William P. Barney and Krista 

D. Colcmllll 

Octob..-r 7. 2014 

2 
CONFIDEKTIALITY 

CLAIMS: 

GOOD LABORATORY 

PilJ\CTICE (GLP): 

STUDY SUMMARY: 

CLASS I FICA TIOK: 

n~u. troun l 

Konc 

Signc:d nnd Unlcd Sllltcment: Non GLP- compliant under -10 

CFR Pun 160 

University of Florida. Institute of Food nnd Agricuhurnl Sciences 

(IFAS) has submiucd datu and information on the Plant 

lncorpor:th."<< J"trotcctruu (I .. IP) Papaya Ringspot Virus Co3t Protein 

(I'RSV-CP). An argument wns mnde 10 consider the Florid:t strnin 

of PRSV-CP cxprcssc:d in X17-2 being included in the existing 

tokr:mcc exemption for PRSV.CP [40 CFR §174.515). The end-usc 

produce tradc-nn.med X f 7-2 confers resistance to pap:tyn ringspot 

virus (PRSV). The X 17-2 rcsisu~ncc 10 PRSV is clnjmed to be the 

result of posH.rnnscription.aJ gene silencing due to the presence of 

the PRSV-CJ) gene in the p::!J\3)'0. genome. Tmnsfonn::uion E\'cnt 

X 17·2 (pBI121 fs): The insertion c\·cnt consists of a NOS promoter 

clement. nptll gene:. CnMV 35-S promotcr.I'RSV coat rrotcin gene, 

and a NOS temtin:uor. 

ACCEPTABLE 

University of f lorida. Institute of Food and Agricultural Sciences has submiucd data nnd 

inl(mntuion on the PJant lncorpomtcd Prote.;tant (PIP) Papaya ltingspot Virus Coat Protein 

(PRSV ·CP). An argument w:ts mode to consider the Florida strJin of PRSV-CP expressed in X 17· 

2 being included in the existing toler:utcc exemption lbr J>RSV-CP [40 CFR § 17-1.5 15[. The tmdc· 

name for the end-usc product is X17 2. a transgenic pJp;ayu that conl~ rs resistnncc to pap:~.yn 

ringspot virus (PRSV). The basis of the existing tolcr:mcc exemption is that the co;at protein is part 

of the virus that is nnturnHy present in infected p.:1p~ yt:1 nnd is alrc:tdy consumed when c.:ating rus~ 

intCctcd PJJXl)'n. 11lcrcforc. tlte presence of the viral co-11 protdn in I'RSV in Florida docs not 

constitute novd dicmry c~posun:. The X17-2 resistance to PRSV may be the result of posttranscriptional gene silencing due to the insertion of the PRSV-CP gene into the p:lJXl)1.1 g~nomc. 

·ntc following sections summ3lizc the data on the: production and s.:tl"';.:ty of the X 17·2 transscnic 

p.::sJX!y!l lines. 

Pap:tyn (Carkupapt'J.rt) L.) belongs 10 piMt fnmily Caricnccac and is a polygamous hcrbn ccou.._~ 

perennial plant. Papay-a ringspot virus (PRSV). u potyvirus, is highly p:tthog~ ic to pap:tya of all 

\'aril!ties and m:tjor fhctor limitins global commercial papay.:a production. PRSV. is the leading 

cause ofp:1p:ayn loss in Florid:a. Typically. n heohh>' papaya plant can survive up to 20 years. 

3 
ho\\cv.:r. if infected with PRSV. th:tl life sp.m is n:duced to 4-5 yem. 'The liniwrsity offtorid3 

h:l.:, ~<\ elop..--<.1 the Xt7-2 p:ap..1~-nlinc ba.._<.ed on technology simil;~.r 10 th.1t used by Dennis 

Gonsal\'CS ;and his le3m in the dc\'dopmcnl of the llo.waiian fbinbow Pl.lpa)a. Th~ X 17·2 uses 

the: snmc Agroboctt•rium-mC'diatc:..J tnlJlSIOm1ation and n:gcw:rotion of p:~paya plants from 

sonuuic embryos as outlined inn study by Davis nod Ying (2QO...l):. The sour\!~: oft he co3t 

protein sene in the I fnwaiian and Floridiun c-assettes nrc diflCrcnt nn..J h:w.: slightly different 

mnino ncid sequences: (Hawaiian Slrnin (h!ncbank ,\ ccc.ssion II FJ-167933) nnd Floridian s1min 

(Ocncb::mk Accession #AF196R39). llowcvcr. the: ro:n protein.~ nre the s:tmc stroclu!.'lly 3nd 

functionally. The FL and HI vmi1mb ore indicative of different PRSV strajns which \'al)' 

gcogr.sphicall)' :LI1d 1xuh ~usc disc3Sc 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 rdnted to toxicity or :dlc:rg.cnicily during conswnpcion or 

other txposurc5. The X I 7~1 line presently coruistS prinu.rily of n r::~.mily or four bre'l!'djng lines 

tllnt nrc di.!'SCendants of crosses between the originnl U"3.nsgc:nie plant (l{a sencrntion) and fom 

elite non-lmnsgc:nie: pDpDyn genotype$. namely: Solo Sunrise. Pucno Rico 6-65. T3inung No.5. 

and Solo -'0 (Da\'is :.nd Yin g. 2004f. A J>roccs.s of rccum:nt backcrOS$in~ nnd selection for 

desired phenotypiC: chnr11ctcri.s1ics. including resistru1ce to PRSV, hll\'C been used to de\'clop 

lhc,:.c advnnccd brcc:ding lines nnd to stabili7.c their charJctcriS.IicS. I'CK und Southern Analyses 

eontimled the positi\'c expression of the nplll and PRSV-CP for :til four elite: p3J'I.l)'Q lines 

express in~; PRSV-CP trnnsgcnc:s. 

The: cbt3 submined to lht 1\ gcncy for X1 7-2 is from 1hc six1h b.1cl::cros.sc<l gcncr.1tion (~). 

Tronsfcr DNA (T-DNA). "hich included lhc OO>l pcotcin (CP) gene orPRSV isolnte HIK W:lS 

modifit"d b)' ineorpor.uing the nucleoside thymidine .. n:~uhing inn fr..unc-shifi mutation nnd the: 

nplll gene. This cassette wns trnnsl~rrcd to the genome of in<lividuaiJXlp.1)'U sonuuic embryos 

und plunts n:gcn~ro cd from the uan.slbnncd embryos. X 17-2 ~xp~s.scs two proteins not found in 

typicnl papaya: the PRSV-CI' nnJ neomycin phosphotrnnslcm.<c (NI>'rtl). Nl'l'll protein and the 

nptll g.;nc urc not known to en usc nd\'crsc effects 10 mummnlian specks and ha\'e been 

pr.:viously gr.uncd exemption from the l'l"qUiremcnt of a tolcrnnec 1-10 CFR § 174.521 ] when used 

in nil D~ricuhur:1l commodities. 

c»"1~transcriptional sene silencing :~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(RNA) is recognized and p~ 

by 3 nuclcasc·diccr into small inh."tli:rins Rt'lt\ (siRNA). ~ siRNAs incorpor.uc into the 

Ar~on.aut protein and mcdi-:tcc dc~md.n ti n or messeng~r RNA (mRN1\ ) '"ith highly spe-c-itic 

s-.'<lucncc complimentarily to the dsRNA. The observed result of hi ~ nntiscnsc technology in 

Pll's ~hows plants re-sistant to infection by P!)RV. 3.11 RNA \'irus. Rcsistnncc conferred by the 

coat protein gem: of a p<) )'\' n.t.~ is ~\tA-mcdintcd {Linbo ond L>oughcny. 1992)#,>. E..'lrly ellOrt$ 

to modi I)' pl30ts. insects. nmn . ond various other organisms with troltsgcncs or antisense 

contructs used single stranded RNA (ssRNA). nlmost exclusi\'cly. Lnler ~1udies r.:\'caled th:lt 

substituting double-Stranded RNA (dsRNt\) for ssRNA produc<d an cnh,.c<d potency eqWII tO 

• 
10 times the imcndcd cft\.-ct on suppressing gene expression (EPA, 2013)'. Since this disconry. 

research into sclccth'it)', eflicncy nnd human toxicology of utiliring RNt\ i in IOod commodities 

ha.<O ~><e n ongoing. While \':triations of biochemical detDils of RNAi pathwa~ s ~xist among 

organisms of ditl'ering ta.xonomy.lhc: modes of net ion arc similar and core components nrc 

rctain~d : Arg.On!HJ!c (Ago) :md Diccr (Ocr). Some examples of coat proteins expressed in plants 

include Zucchini Yellow Mosaic. Wat~mtclon ?\·los.1ic-2. and Cucwnber t>.·fosaic Viru.'iCs 3$ 

expressed in squas.h (Cm·urbitu fh:po) pl::mts.linc ZW-20 nnd vnrious melons. These \'ir.sl co:n 

proteins were g.rnntcd tolerance exemptions ba..54!d on r.uionalc of the ubiquity of plant viruses in 

the ngricultural environment. lack of any indication of m:unmaJian toxicity with current dictnry 

consumption of the entire \'ir'US including the plant vim! co;u protein. 1md lnck of mnmmnlian 

p:nhogcnicity for plrull viruses. (40 CFR 147.514 :md 40 CFR 174.515. r<SJl<Ciivcly]. 

Additionally. the presence of plant vinLSCS. tmd therefore ' 'iruJ co:u prOicins. is higher ill 

natur.tlly infected crops th;m in the trrut.Sgcnic Jines of JXlp.lya 

T il"$t Mrthod§ and t~ul t'i 

·n1e fOllowing table contains the tc.)1 matc:riaJs and methods in suppor1 of .;o CFR § 158.2130 

Microbial Pwicidt·s Product Anai)'SI.< /Jara <;uiddin,·.<: 885.1100. 885.1200. 885.1250. 885.1300. 

885.1 400. 885.1 soo. 

Product Chemistry and Composition 

88S.UOO The X17-2 line presently consists primarily of a family of four br~eding MRIO: 

Product Identity lines lhJt Jre des.cendJnts of crosses between the original transgenic 49493201 

plant {Ro generation} and four elite nontransgenic. papaya genotypt\s, 

namely; Soto Sunris.e. Puerto Rico 6--GS. Tainung No. S, and SOlo -10. 

Transformation Event X17·2 (p8112lfs}: One insertion event consists 

of a NOS promoter element, nptll gene, CaMV 3S·S p romoter, PRSV 

coat protein gene. and a NOS terminato r. ~c Figure 1 for complete 

DNA components in vector p81121fs. The primers used to dete<t the 

transgene construct were as fo llows: 

FOI (S'·AGAACTAGTCCCGGGTGGTCAGTCCCTrATGTTC 

CAMMTGMGCTGTGGATG· 3') 

ROl (S'·TACAGATCTACCC TCACTATAAAATAGAAGC· 3'1 

The clo ned CP gene was us.ed as a template to amplify the ONA 

sequence o f the modifie-d C.P by Po1ymcrase Chain Re~ction (PCR). 

B~sed on previous studies and trials, the uldA leader was found to be 

more effective than a homologous virus lt.'lder to enhance the 

exprenion of the PRSV·CP. Therefore. the uldA Seader with an 

initiation codon (S'·CTAGTCCCGGGTGGTCAGTCCC TIATG-3') was 

placed at the S' end o f the PRSV·CP construct by indusion of the 

sequence in the rot primer. PCR was ~rform d and the fragment 

with the added uidA INder and start codon was confirmed bv 

s 
seoquencir'l- The bfNry ~OC" pBilll was doub6Hilge1ttd wit.h Stnol 

and Sect blunted with T4 DNA polym~ras.e and thtn rtli&ittd wit.h T.: 

DNA ga~ to produce p8112lzy.lineariztd p8112tzy w.n produced by 

dige.ltion with Xbol and SamHI. The PRSV·CP fragment was separat~d 

by agaros~ gel e le<Ho phore.sts. isolated from the ge l. doublo- g~st~d 

with Bgtu and Sprl, and then don~d Into the linearized p91121ty to 

produte pBI12lf.s. During a norn'la1 virus Infection. CP of PRSV Is 

encoded as pi'lrt of a potyproteln. The CP coding region therefore lacks 

a S'-unuanslated regkm and a translation lnltlatlon codon. The 

con.sttuct developed for traruformation of papayil rov~cs these 

eu.entia1 sequences for appropri.lu~ pl.ant6presslon. Trans.c:ription of 

the modififif coat protein gene in transform~ ptanu is d irrtted by 

the 35S promoter of tnt C~uliflower Mowk Virus (CAMV) ;and 

terminated by t~ NOS tetmiNtor sequence of A. tumr/ock n.s. The 

npC11 gene from the tro1n.sposon TnS of the bacterium £Jchtricltio coN 

wa.s introduced Into P.l~ya as a select<1ble marker. The &ene codes 

forth~ ~nzymc neomycin pho photransfera~ that confers resistance 

to the common amlnoglycoslde antibiotic kanamycin. The nprll gene l.s 

under control of a bac-teri31 promote r; NOS termlnator of A. 

wmefocftns. 

CLASSIFICATION; ACCEPTABLE 

8&5.1200 The Xl7-2 line p1escntly consists primarily of a family of four bree-ding MRID: 

Manuf~ctu ri ng lines that au~ descendants of cros~s between the n~1 lransge nlc 49493101 

Proceu plan (Rlgene~ on) and four t'lite nontr.~ru.ctnk papaya genotypes. 

n.ametv SoJo Sunrtst, Putno Rico 6-65, Tainunz: No. s. and Solo 40. The 

vector (p8112lfs; Figure 1) used to tr.~ru.fer the &enes to p,;JJNYOI plants 

was constructed from the binary vector p81121, a derfvatlve of 

p81f'll9 by repiJcing the GUS gene with a connruct of the CP gene of 

PRSV. The complete stquences or pSIN19 ~nB3 k ;,ccenlon 

11U09365) •nd pBilll (G•nBank Mf48578l) or< published ond 

~wai!Jble to the genernl public-. The transformation tk'ments in the 

vectors were derNcd from part of the Agroboctetlum tumot lndud ng 

plasmid pTiT37. The mC!'thod for Agroborttrfum-medla ted 

transformatio n and regenC!'rJtion of papaya plana from MJma1k 

embr)•os conduCled by Ying et al. (1999)1>, was used to develop lilt 

Xl7·2 11ne. T•ONA. which JncJuded the CP gene of PRSV k.o1;Jtt HlK 

modified by ancorporation of thymkfine to UNte I fr.tme·shih 

mut.Jtion and the np:U gene, was tr.uuJ~rr~ to the genome of 

incfrvkfual pap.lyiJ somatic embryos and plants regtneliJttd from the 

tr.linsform.ed embryos under Unamydn selection. (See rtsure 1: 

schematic lll strat~n of plinm!d v~or. 201•1)11 

ClASSIFICATION: ACCEPTABlE 

885.1250 The cloned HlK sequence Is deposited rn GenB:tnk as MRID: 

Deposition of ACCC!'sslon li 96839. The HlK cultivar was b;ued ofr similar 49493201 

sampla In a technology developed by Dennts Gon~ ves tt a1.• for u se the PRSV 

nationally resistant HawiiiJn P•J»~ . While PRSV·C:P was shown to be tlprei.sed 

recocniled in the HawJiian pap•ya, It Is now believe-d that normal viN~ te~lit.'llnce 

responses in planll uhllllng RNAi pathways were probibty ~lso In play. 

6 
cui curt- BiolnformatK: analytical co mp ri~on of the two amino aCid sequences 

collection of HlK and the Hawaiian Rolinbow Papaya via a BLAST analysis yielded 

a 96% homology. (See Figure 3)' 

CLAS51ACAnON: ACCfPTABLe 

885.1300 Resistance to kanamycin in the transformed cell tines Indicated MRID: 

Discussion of expression of the nptll gene in theX17· 2 1ine. The PRSV·CP is also 49493201 

formation of expres-sed but at low te .. ·els compared to coat protein levels in papaya 

unintentional naturally infected with PRSV. fnzy-me·linked lmmunosorbent Assay 

ingredients {ELISA) using polydonal ant.i-PRSV serum indicated a low 

concentration of the PRSV· CP In uninfected X17 and 06 breeding 

lines. Similar results were obtained using another polycloMI PRSV 

antiserum fWMV·l antiserum) obtained from the University of 

Florida. Addltlonally, the expected 3S kOa PRSV CP wJs detected by 

Western-blot analyses using the same two antisera. 

The 3S kOa size of the expressed PRSV CP In X17·2 Jnd other 

wmsgenlc lines was the s.ame as predkted from the nucleotide 

se-quence of PRSV RNA. The natural PR.SV<.P is one of at least se\•en 

proteins cleaved posHranslatlonally from the ea:pressed poly protein 

of PRSV in infected plants. The C9 constructs in the X17· 2, 0&, and 09S 

lines were designed to be no ~trans atab e into PRSV coat protein due 

to insertion of stop· codon or frame· shift mutations at the 5' ·end of 

the CP gen. The Western blot results, however, Indicate that the 

mut.ltions were re.ld through or repaired resulting in expression of 

the CP gene. {See Figure 2)11 No significant impacts or unintentional 

cffecu arc expKted, as both the PRSV-CP and the NPTII proteins were 

previously found to p resent a reasonable certainty of no harm granted 

tolerance t>xemptions in all food commodities when used a.s a plant· 

incorporated protectant; 40 CFR §174.SlS, 40 CFR §174.521, 

respectively. 

ClASSIFICATION: ACCEPT ABL£ 

The rollowln.e table contains the rationale along w ith test materials and methods in support of 40 CFR 

§158.2140 Microbial Pesticides ToJtkology Dato Rt:qultrmtnls Tobfe & Guictelines; 885..3050, 88S.:HSO, 

885.3200, 870.1100. 870.1200, 870.1300,840.2400. A tier Ill study was submitted as part of the Sec. 3 

Registration and has been omitted, as it Js not a required study or guideline for Plant-Incorporated 

Protectant registrations 140 CFR §158.2 140, 885.3650 Rt'productt..•e fertility EHe<ts). 

Mitrobial Ptstlddts ToldCQlogv pua RtQuiremt'nts 

(OPPT 885.3050, The University of Florida has submftted data to support their MRID: 

870.1100) d.ata waiver requt'sts based on the following rationale. Whole 49493202 

viruses are ublauitous in the a,::ricultural environment. Virus 

7 
Acute Or.ll Infected plants 'o\oill express tM enHre virus par1id~. as wd .11 

Toxldtv/P.ltho&tnlclty vl101l coat PfOtein, at levels hightr than wiU 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 

presenting a reasonable cenJlnty of no harm ;nd ;~re exempt 

from the requirement or a to,er.1nce when ustd as a pbnt· 

incorporated protectant In or on all food commodltles.l72 FR. 

20-13.4, Apr. 25, 2007) Ge et cally modified PRSV resistant 

papJy.l has already bfJ'e n commercially available and consumed 

since 1998 with the Introduction o f the Orst transgcmk: Hawalian 

Rainbow Papaya, deveJoped by J collaborative effort between 

the Unlverslty of Hawaii and Cornell University {Gonsalves, et 

.11.}'. A relevant whole food reeding study submitted by IR·4 in 

suppon of a data waNer for 40 CFA §1SS.2:140, evaluated the 

comparative toxicitY efferu of develope--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 their @'nt 

non·tlinsgenit {TN-2) counte~paru ln lh-e c-itt'd study, a 

sUbtlity analysis for PRSV·C.P wu conducted vi1 dfs:est.ion 

Slibiflty assays in simuJ.Ited ~~uric Rukb (SGf) wt1h a pH• <2.0 .. 

simulated intestinal fluid (SIF). <~nd bile Silts co detect the CP 

fragments. ResullS revuf.ed tNt the CP fragments were rapidty 

hydrolyzed in SGF and were undetectable in organs and 

sastrofntestinal constituents in tats. In;, 28-diV repeated 

ftcdlng study, no death or abnormality of rats occurred In the 

nontransgenlc TN· 2 and bactcross transgenic P.lPOlVJ fruit 

treatment groups. Additionally, no slgn10cJnt dUference of feed 

effi cacy was observed in all treated groups I lin, ot al. 2013)

1• 

ClASSIFICATION: ACCEPTABLE 

Acute Dermal Toxldty Due to the nature of vlrJI co-at proteins produced in plants as MRID: 

(OPPT 870.1ZOO); part or a PIP, exposurt- through any rouH! other than dietary is 49493202 

Acute Dermal unlikely to occur. PhyslcJI contact with the plant or raw 

Irritation (OPPT agricultural food from the plant mav pr~sent ~me limited 

870.2500) •nd Acuto opportunity for dermal el(posure . .. owevt'!r. on .1n lndMdu.1l 

Otrm.al buls, the potent~l Jmount\ i.nvolved In this t_l(posure are 

Tokl<lOV/P•thotorv ne-gligible in com~risonto ekposure through the diet1ry route. 

(OPPT 815.ll00) Additionally, viral coat proteins ptod'U<~ In 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 In or on all food 

commoditMos. [72 FR 20434, Apr. 25, 2007I' Un •••t., 

determined ln their study through repeated uposuu~ that no 

sfgniftc<Jnt dermalltsions were observed In either male or 

female rats)•l The Food and Orug Admln t~tton lFOA) 

8 
determined th.at based on the safety and nutritional 

asse.ssment, papayas are not compositionally different from 

papayas currently on the market and th.at the gcnetic.ally 

engineered line Xl7·2 does not raise novel issues that would 

require premarScet approval by I'DA.IlDI 

ClASSIFICATION: ACCEPTABlE 

Acute lnhal.3tion The occurrenc~ of respiratory exposure of viral coat ptotein MRID: 

Toxicity (OPPT produced In plants .1s part of a PIP Is negUgiblt" In compJrison to 49493202 

870.1300) and Ar:ute potential exposure through the dietary route. VIral coat 

Pul monary proteins, wh en present In polfen, are unlikely to cross the 

Todcity/Pathog~nidty barrier provided by the mucous membrane of the respiratory 

(OPPT 885.3150) tract. There is no sclentific evidence, to date. which suggests 

that pulmonary exposure to whole plant virvses by inh-alation 

results In any adverse effects to mammals, to include infants 

and children. Additionally, the final product Is J systemic viral 

resistance and d~s not h.avc a spray.1blc compont"nt; thus 

rendering the risk of particulate inhalation <"xposure practie:31ly 

non-existent. 

ClASSIFICATION: ACaPTABLE 

Acute Eye Irritation All available informatJon Indicates that viral coat proteins In MRID: 

(OPPT 870.2400) food have no human toxicity. Data regarding Acute Eye 49493202 

Irritation ts not required for viral coat proteins ~ince there ~ a 

long history of m3mmatian dermal exposure of th{' plant virus 

particles by handling food, without causing any known 

deleterious human health effects or any evidence of toxicity. 

The Xl7·2 Line of TrJnsgeni<, P.lpJy.l Ringspot·ResistJnt PJp3ya 

does not represent a source of novel potential allergenic or 

anti·nutrlent proteins. 

CLASSlFICATION: ACCEPTABLE 

Conclusion: 

The X17·2 line presently consists primarily of a family of four breeding lines that are descendants of 

crosses between the original transgenic plant (RO gcn.c-r.~tion} and four elite non-transgenic papaya 

genotypes, namely Solo Sunrise, Puerto Rico 6-65. Talnune No. s. and Solo 40 (Davis and Vlng, 2004)1• A 

process of recurring selective backcrossing for desired phenotypic charJcteristic.s, including resistance to 

PRSV, yielded advanced breeding lines and stabili:ed their characteristk.s. They are currently in the sixth 

generation {R4) of the process. In the Rs gencrcltion of Xl7 2. a 493S base pair PCR fragment contAining 

all but 336 base pair of the T·DNA insert was cloned and sequenced. The expected versus obs-erved 

values indicated 99.7% homology. PCR and Southern Analysis confirmed the positive expression or the 

9 
nptll Jnd PRSV·CP for all four PRSV<P aransge~s. Mendelian 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 R1 generation (n.9,.). Tht It, gentrat.ion won. 1-till doSt" to the 

tllpt<ttd rrequtnty of 7S%with a vo\Jut' of 3:1 (79.8%). 

Althou&, the Xl7·21ine expresses 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 

Anlllyses: indfc.Jtlns the tonuntration in the transgenk: 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 are median concentr3tions and standard d@vlation v.Jiues. 

calculated from the four elite brfoeding lines of X17·2: Solo Sunrise progc!nles. Tainung No.S progenies, 

Puerto Rico G·6S progenies. and 06. 

The X17·2 resistance to PRSV is believed to be the result of post-transcriptional gene silencing due to the 

lnser,lon ot tho PRSV·CP gene Into the papaya genome. PosHranscrlptlonJI gene silencing. known as 

RNA·Intorfcrcnco or P.NAi, (referred to hereifter as RNAl) occurs In most eu\aryotes.. Oouble·stranded 

RNA (dsRNA) ls d<ed by 01 nU<:Itase-dicer into smatl int~rfeting RNA (siRNA) tllat mediate a highty 

spedflc: iequence degrJditlon ot meS$Cnger RNA {mRNA) and prtvel'lt translation of that mRNA into 

protein. The obienr~ result of this antisen}e H:chnotogy In PIP'S Is the plants resistance to infecHon by 

RNA vlruses. ReSistance conferred by the coat protein gene of a potyvfrus is RNA·mediated (Unbo and 

Dougherty. 1992)'. E.Jrty efforts to modrfy plants. lnsr<t.s. mammals. and Viriousother orsanUrm with 

transsenes or otnt~n.sc cont.ructs used only sins,le stranded RNA (ssRNAJ. otlmost exclusn.-ety. Later 

studies reve.1led tholt substituting double-stranded RNA {dsRNA} for uRNA produced J tenfoJd ii'Ktease 

fn potency for th-e Intended effect of gene suppression tEPA, 2013) 4

• Since the discovery cf RNAf in 

plants, rcsc;uch Into selectivity. etr.cacy and human to);lcology of utlllllng ANAl In food commoditjes has 

been cngclng, While variations of biochemical detalls of RNAI pathw~vs CAist among organisms of 

differing taxonomv. the modes of actiOn are similar and cora components rtte retained: Atgonaute (Ago) 

and Oicor (Ocr). Some previous exolmptcs of coJt proteins construct.s in plo'IOLS Include Zucchini Yellow 

Mosaic, W.1termclon Mosalc·2, and Cucumber Mosaic Vlru~cs .:~s ctprcued In s.quash ~Cucurbfto ~po) 

plants, line ZW·20 and various melons. All of these previous vlt1l coat protein C'Xprcsslng constructs 

were granted •olcro:~nce exemptions based on rationale of the- ubiquity of plant viruses In consumer 

agricullur.ll supply, I;~ c)( of incliatlon of mammalian toxicity with current plant vi toll co;~t protein 

consumption, and lack of mammalian pathogenicity for pl.ant viruses. (40 CF.R 147 Sl4 and 40 CFR 

174 .• 51 s. respectively), AdditJonany. the presence of plant viruses. b h.lc,her In natunltv Infected crops 

than In tht transcen.lc lines of J)lpaya. furthermore. plant virum ~re inap.~btc of reproduc~ in 

mamtn.~ls and other venebrates. thus eliminating 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•Uable since 1998, there have 

Moen no obs.rwd COJdcitv cr other issues nsocia·ttd with humin hei!lth. To confirm simll.lrtty bJsed off 

submitted r.ttk>nale for exemptions. the awai~n sequence wou compo:~red via BLAST to its florida HUC 

counterpan. and lndl co:~ted a 96% homology. The mlnor v;)ri;,tkln rn 01mlno acid sequence between the 

two strains Is negligible and could be expected sii'Ke the virus phenotypes v~ry gecgtJphlcJity. 

10 
Kcvicw of n submiucd study from IF AS J•rcnatal J.>cvcloprnent Toxicit!o' study. OCSPP guic.l in~ 

870.3700. was omitccd. 11tis submitted study rdt:rs to the: current Tier Ill guideline OCSPP 

885.3650. Reproductive Fertility EOCcts which is not a requirement tOr Section 3 rcgistr:Hions of 

Plunt-lncorporntcd Protcctants (PIPs) unless then: n.re wxicity results or other indications that 

this study would provide usefUl infonnation. 

II 
Figure' 1: 

//ill\1111 &oR I 

Figurt' 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)ll21 fs ore described in Tobie 2. up rclle<:tS the gene l)rder but not the siz.cs. The T-DNA 

lx:twe<n the lefi (LIJ) nnd riSIII (RB) borders has restriction sites li)r l/i11dlll nnd EcoRI. 

Insertion of thymidine. ns indicnt.;.'tl by an O$tcrisk. resulted in crc:1tion of the und.:rlined stop 

co.Jon dO\\nsttcn.m. TI1c rcg.ion outside I he RO ::uul tn is the plnsmid \'Ci:'tor backbone 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 17-2. 06. :wd 095 

lr.tnsgC"mc lu)($.. W from no•Hmnst::,<'UIC Solo Sunrise pop.l~'D th.lt \\'3$ hi:~llh) or utfcctOO w.th 

''RSV HIt( S3mpk$ COOI:Umn~ MO 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 to POVF lllC'tnbnma . In Figure SA. 

the mC"mbr.mc: \\:1$ probed \\llh 3nii·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 fib'\ltC:S mdctcs tbc l~t.ot~ oflk 35 "--.a PRSV C() WI \\!lJ (ou,nd 01.\1~ 1n r.\l'~Sicruc: 

htld 

'Source: USDA·t\PHIS. 200911 • 

13 
fi re ~: ,\mino Acid S l'9UCUt'(' ffiL\SD 

DoscripJLo.ru; 

~n O'QI;II.Cing '9"'<'rt ~9'"'t""U 

OesQJpl•on Max 

score 

ToU~I 

StOIC 

Query 

covet 

E 

value 

Cto\lP'~Ie'on IC••u 

e> t~r•l 

Alignments 

<Ol1 Ot?:f!II'I IC¥1Ca ~AI 

,., , .. "' 

~t-10 I< 54GJ%.tjl~n )Col t.I ""'OC:rd ~ 1 

AOf"?C' l 1) tc- )1)4. 

.. . .......... 

,. 

,,_ . ~Otol\l JWI 000 COII'C)OU>7'ltiNII'\• ~!AI ~96'() 281.~96~1 217W\O"JC.I 

r~*'"" 

~(l'"lf 1 "' W'.'rt~:r ,

M 

,_ 1.\ /.\ ~-'lM l"' .. 

~ l

~( • ( ( ~ r~r ( ( ~ ( ~~ l (

{ 1C ( •• 0 CY.)4 UXIO.'\T\ \.XICI\ I '> n; Tc.fI C.l'!I G(t.).IO'I I O'I •• \~Jet " ;.u:._~ ,.f,. g~1,.},l,f ( I o((l({r (r (~ (~YJQ.'Sf~h (.{t.l)W'/ " (N~ ' ., \ \'(; UC.! 1 f\PII! l TCQII llllll tG(fVt \1JooHl ~~r?IS\ tU ~i(('o.'l ,,. ~'o"lS(; Iff\'91J -. \f1Ck." l.._ l .. (j,ol.f¥t.\"tH1ll f ;;1 ~SliVo T C)U ~c;ct " Wt>lM.H t\P -. ~f-l';; ......... lPII ot Vl '1»"ll ,., ~J:li\'01U.1>1) ltt'!o'f "' Q-.ltt 'I "' ~ ¥\ ._5C~l ~~ l Mitl"'.'loC I UG f\110 15>... ... '\o'IM'OG-! 1 ~! •. •ll f IU T .. 

Sc:ct Ill "' lG'o'II•O ~ U..C:U"\1ooCJ(~TSP(I SG\._..""""' t l)! P~ &~ 11' 

(G\"1.0-.Gt ~{ ll.oCilf""....C: l[!<ni>O S......_.~[ t ltf('P !(ll'l t ! KA T II' ,., 

(;\lo ft)' "' ~ f J~ : "»I 'Sv.l.£.l Y!M.ISA l iii'M"IIV'GI I(AA!l tO IS t.A\V A,.f ,v[·~lf 1'0'-l\ , .. 

19) 

\ 111 "'~ \,..Ut ,lV!AUM tl......,l"(; I ( .... ~ IO I (t.ltV.tl ;!h•(·.y;l t PQ~ U 

~c;ct S r 1!: : ".U.f S~(AYlJ..c IIW.t l fh~fh'G I• 1\,-.l ~IS t,_l~\'ll ~1\'(' '\o'St. PO~.l~ :'\1 

q .. u, '" fa-"\Mtii.M'OII~VTCK)G<;.\"i.~( t ' 'I 10-ltvt:r ...... ~l.Git•~ :': 

2-H 

I 4U-I"' ~· -•tl't(o:W'!IN<ll \1 I ""h'trl ..... ~l.~~li St-)\~ {l.t • Uw\lii..'TSI'-"f~S._,'.Ilrf '''I."'P•'f D""'~\ll&'ll~ ... • Source: NCBI a~t. hup://blolst.ncbi.nlm,nlh.gov' 

14 

1oem AcceSSIOn 
Figure 4: M3J! Qf VCC!Or p8112fs 

Pstl - 1196. 

BgiU . 8!57 

Bg/IJ - 234-4 

Spill - 2371 

r~1-).~v Pmt'l - 2492 

Right 

ec..a. Pst l - 3019 

pBI121fs Spill-3371 

13867 bp 

HindDl - -1950 

Spill - -1960 

Pstl - -1966 

Xmn l - 5821 

Smn I - ~823 

Ero Rl - 7091 Spil I - 63~-1 

Pstl - 6569 

IS 
Coleman. ~ Br.t\'Crm:l!1, M.: 03Jl1Cy, W. (201 .;) Group A·PcoJuct Analysis Tes:1 

Guidelines: X 14·2 Line orTrnn.11gcnic P:apaya Ri n~lJOl·Rdi~rnnr Papayn. Projtcl Number: 

05378. Unpublished study prcporcd by lnt<m:gioMI Research l'rojcct No. 4. 726p. 

Davis. 1. J. and Z. Yint;. 200·-1. lX\'<:IOJmtcnr of pap3yn bccct.Jing li ne~ with transgenic 

resistance to P(apnyn ring.11pot virus. Plunt Dis. 88:352-358. 

EPA. 2007. Cootl'rotcin ofl'ap.1yu Ring.SpOI Virus OJld the Genetic- fntcrial Necessary for 

its Pcoduction: E.xcmption from the Requirement of ::a Tokroncc. 72 FR 20434.20435. 

EPA 2013. RNAi Technology :u • Pe>ti<ide: Problem Fonnulation for llunun HC3lth :uxl 

Ecological Risk Assessment Submincd to 1hc FIFRA Scic~"C i\d\·isory Pane129 October 

2013. Docket 10: EPA-110·01'1'·2013·().185. 

l'cdcr•l Register. 40 CFR rnrt ISO (OPP-300538: FRL-5739·41 Co:tt protein ot"Papayn 

Ring.spol Virusund the Cr\.'nctic otc ri l Ncccssnry for ~ l'coduclion; Exemption from th~ 

Requirement ofu Tolcmnc\.'. Augus:t 22. 1997. 

Gonsalves. D .. Sw.uki, J., 'frip:uhi. S .. & Ferreira. S. (2009). 1•np:1ya. Comprndium of 

Transgtnic Crop Plams. 

Linbo. J./\. :uxl Doug.heny. W.O. 1992. Pothogen-derh·ed resisllln<c too potyvirus: 

Immune and ~istoncc ph<not)pt":S in l.r..lnSgt:nic tolxtcco c-xpn:ssing. altered fomts of D 

pot)"\'irus coat protein nudroli<k scqurncc:. Moleculnr Plnnt-~ticrobc Internet. 5: 14+ 153. 

Lin. Hsin-Tang: Yen. GO\\·Chin: !luang. Tins·Tzu: Chan, Lit·l' u: Cheng. Ying-Huey: Wu. 

Jhooi-Huci: Ych. yi·Don~: Wung. Sheng· Ynng: t.ioo. Jiunn· Wnn~:o (20 13). Toxicity 

1\ss\.'SSment ofTr.tnsgcnic f'npaya Ringspot Virus of 823-2210 l.inc l'apnyn fruits. Journal 

of AJ[riculluml & Foml Cht•mi(,Jry. 61(7). 1585 -1596. 

NCHI: llt.AST. 2015. hup; 11Na, ps~j, nlm.ni &o wl li"i· Rill: JWK58VEX113. (Accessed 

4115120 15). 

USDA-APHIS. 2009 ... P<tition> of :-lonn.-gul>ted Status Gr:111tcd or l'cnding by APHIS."" 

From hur:tJ\\"\\"W arhi~ uyl;a 1,!0\ ro 001 rcg.btml (nccc$.St.-d NO\'Cmbcr 2014). 

Ying. Z .. Yu.. X .. and D:nis. M. J. 1999. A n<-W m~lhod for obtaining tmnsgc:nic papayn 

plants b}' Agrobuctt·rlum·mc<li.:ucd tran.sfcnnation of sommic t:lnbryos. f'roc. Florida Stau: 

llort. Se<:. 112:201-205. 

Tnrnntino. L. !vi. (2008. Dcc..:mbec). Consuhntion lcncc by FDA on X 17-2 Pup::ayu. Food 

rand Drug Administration. 

16 
17 
•