MEMORANDUM

SUBJECT:

TO:

FROM: UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHI N GTON, D

. C. 20460

OFF I CE OF CHEM I CAL SAFETY AND POLLUTION PREVENTION 

## NOV 0 5 2015

Decision: 497658 DP Barcode: 424881 BPPD Environmental Risk Assessment for a Section (3) Registration Application for the Plant Incorporated Protectant, X l 7-2 Papaya 

### (Carica papaya L.) Ringspot Viral Coat Protein (EPA Reg. Nos.84427 -R). OECD Unique Identifier UFL-Xl7CP-6. MRIDs 494932-00,494932 -01 , and 494932-03. PC Code: 006701 Mike Mendelsohn, Senior Regulatory Action Leader Microb i al Pesticides Branch Biopesticides and Pollution Prevention Division (7511 P) Gail Tomimatsu,

# Ph**~** Microbial Pesticides Branch Biopesticides and Pollution Prevention Division (7511 P) Jeannette Martinez, PhD py

### ~ Microbial Pesticides Branch Biopesticides and Pollution Prevention Di PEER REVIEW: Chris Wozniak, PhD 

# /1/


# - C

- Biopesticides and

## Pollutb<~ention Di · ·on (7511P) Shannon Borges "" \. "'- Microb ia l Pesticides Branch

# ~**~** Biopesticides and Pollution Prevention Division (7511 P)
ENVIRONMENTAL ASSESSMENT 

### A. Introduction On behalf of the app l icant (Un i versity of Fl orida , Institute of food and Agricultural Sciences (UF / IF AS)) the USDA/IR-4 has app li ed for a FIFRA section (3) registration of the Plant Incorporated Protectant (PIP) , Event Xl7

- 2; a coat protein of the plant virus , Papaya Ringspot (PRSVcp) for transformation ofpapaya to improve plant resistance to Papaya Ringspot Virus (PRSV) , through biotechnological and molecular methodologies. This PIP has been designatedas OECD U niqu e Identifier UFL-X17CP-6. The applicant submitted scie ntifi c rationale, based on qualitative information fr om peer-reviewed literatu re and regulatory decision documents to s ati sfy the microbial p esticide test data requirements as publi s hed in 40 CFR 

### § 158.2150. The provided infonnation in addition to previous tolerance exempt ions fo r other vira l coat proteins (promoting RNAi protection) and minutes fr om FIFRA Scientific Adv i sory Pan el meetings concerning plant vi ral coat proteins are sufficient to satisfy all Tier I nontarget organism data requirement s and to suppo rt an environmental risk assessment for this new act ive ingredient. B. BACKGROUND In 1997, EPA gra nted an exemption from the requir ement of a tolerance for the coat protein produced by ge ne tically modified papaya VCP (55-I pap aya, "S un Up" variety) developed for Hawaii (U.S. EPA, 1997). T he rationale for the to le ra nc e wa s the documented occurrence of the PRSV in f ood, lack of any indi cation of mammalian tox ic ity with it s current consumption and the publi s h ed sc ientific literature indicating lack of mammalian pathogenicity for plant v iru ses. The UF / IFAS (app li cant for papaya Event X 17-2) reported that th e current papaya VCP in se rt for the Florida hybrid had 96% amino acid homol ogy t o the H awa ii an papaya ringspot i solate (MRID 494932-01 ). Resistance to the ringspot virus i s conferred through posttran sc riptional gene silencing, a type of RNA-mediated protection (Gonsalves 1998). The amino acid homo logy between Xl7-2 and it s original so urce ( HI K strain of PRSV-P , MRID 494932-0 1) wa s reported to be 99.7%. In 2004 and 2005, US EPA held FIFRA Scientific Advisory Panel (SAP) meetings to discuss

" i ss ue s associated with deployment of a type of Plant-Incorporated Protectant (P IP ), specifically tho se ba se d on Plant Viral Coat Protein s (PVCP- PIP) " and to provide recommendations to the Agency on gene flow , weediness, viral interaction s, dietary expos ure to human s, and risk to wi ldlif e.

• Addressing concerns regarding gene flow and weediness , th e SAP concluded that primarily fruit and vegetable crops spec ie s were not expected to form viable hybrid s with wild relatives in the US and it s territorie s . Even thou g h introgre ss ion s hould happen infreque ntly , and the consequences of in trogression co uld be controlled overridingly by genes linked with th e transgene , there was a s light c hance that a boost in tolerance to a wild plant co uld be eco logicall y significant. But based on knowledge obtained from observation of cult ivated crops in the agroecosy s tem , th e maj o rity of the Panel concluded that it wou ld be unlikely that a plant p op ul at ion freed from viral pressure would give a plant s p ec ie s a competitive advantage. The Panel was not aware of any study that had purpo se fully freed a weed species of a known virus and determined it s competitive ability or looked at the population dynamics of virus inf ected versus uninfected plants in multiple s pecie s ecosystems. • With re s pect to dietary concerns for human s, the Panel agreed that because of the human hi s tory of consuming virus infected food , unaltered PV C P s do not present new dietary exposures. There was no clear consensus on how much c h ange would be necessary to invalidate thi s assumption , although there wa s general agreement that the appropriate compari son was to the rang e of natural variati on in th e virus population. • Conce rn s for non-t a rget s pecie s were addressed as follows: the Panel agreed that virusprotected crops s hould not be as attractive to aphids as di seased crops and thu s m ay 2reduce the numbers and types of organisms that spend at least part of their li fe cycles in spcc ifie agroecosystems. Becau se of the transie nt nature of agroecosystems, a reduc t ion of aphids over l arger scales s hou ld be negligible. The Panel was not aware of any risk po sed by PVCP-PIP s crops to w ildlif e. On September I , 2009, the Animal Plant Hea l th and In spectio n Service (= APH IS, U.S. Department of Agricu lture) pub li shed a n ot ice in the Federal Re g ister to inform the public that transformation Event Xl7-2 papaya was no lo nger considered a regulated article under it s regul at ions. This determination was based on eva luation of data subm itted by th e IF AS of the Uni ve rsity of Fl orida, analy s i s of sc ientific data from the open literature, APH IS' re sponse to comments received from the public on the petition for deregulated statu s for this new event and associated environ mental assessment for a Find in g ofNo Significant Impact (USDA, 2009). EPA ha s granted tol e ranc e exemptio n s for o ther viral coat proteins wh ich promote RNAi (posttranscriptional gene-si lencing) in plants; the se include Zucchini Ye ll ow Mosaic , Watermelon Mosaic-2 , and Cucumber Mosaic Viruse s as expressed in squash 

### (Cucurbita pepo) plants , line ZW -20 and vario u s me lons. Th ese vira l coat proteins were granted toleran ce exemptions based on rat io n a le of the ub iq uity of plant viruses in the agricultura l env ironment, lack of any indication of mammalian toxicity wi th cu rrent dietary consumpt ion of th e entire v iru s in clud in g the plant v ir a l coat protein , and lack of mammalian patho gen ic ity for plant v iru ses [ 40 CFR 147.514 and 40 CFR 174.515 , respectively]. Additionally , the presence of plant viruses, and therefore viral coat protein s, is higher in naturally infected crops than in the transgenic lines of papaya. 

### C. CONCLUS IONS AND RECOMM EN DATIONS BPPD conc l udes that the PIP a.i., PRSV cp produced by papaya that have been transformed with Event X l 7-2 does not produce any new, or inc reme ntal environme n tal exposures of concern beyond tho se of papaya tree s or fruit naturally infected by Papaya Ring spot virus (PRSV) population s in Florida. The evide nce from the regulatory records (EPA , 1997; EPA , 2007) , documented minutes of F IFRA Scient ific Advisory Panel s ( SAP reports of 2000, 2004, and 2006) , information from the cited li terature and from submitted sc ien t ific rationa l e justi fy no additional nontarget organism testing as recommended in th e 40 CF R 158. 2 150 series of microbial pe s ticide d ata requirements. A detai led data evaluation record (DER) of th e submitted information i s appended to this memorandum. 

### D. NON-TA RG ET ORGANISM RI SK ASSESSMENT PROCESS FOR PLANTINCORPORATED PROTECT AN T S ( PIP s) The Ser ies 885 guidelines app ly to microbes and m icrobia l toxins when used as pesticides (Table I; 40 CFR 158.2150 including those that are naturally occurring , and those that are strai nimproved , either by natural se lection or by deliberate genetic manipulation. Therefore PIP s that have microbial genetic material deliberately introduced into plants are also covered by these testing guide lin es . The EPA be l ieves that thi s tiered paradigm to testing of non -targe t organisms ensures, to the greatest extent possible , that a minimum amount of data would be required to make scie ntifi ca ll y sound regulatory decisions related to the use of pl ant incorporated p esticidal substances and for


### 3id e ntifying negati ve re s ult s with a reaso n ab le degr ee of confidence. Th e Agency expects that scie ntific consideration of Ti e r 1 data requirem e nt s is s ufficient to s upport non-target organism ri s k assessment for PIP regi s trations (EPA-SAP , 20 00). T he fate of pe s ti c id a l s ub s ta nce s in the envi ronment is a critical factor in the Agency's ri s k assess m e nt for all PIP s. The Agency u ses env ir onme nta l fate data to d ete rmin e pot e ntial fo r adver se impact s of a PIP , s uc h as PRSV cp entering and pers is ting in th e e nv i ronment. Therefore, EPA co n s id e rs gene flow , p ote nti a l invasiveness of the pl a nt ex pr essi ng th e PIP , and the potential environmental imp acts when evalu at in g PIP s . Tab l e I. Current Non-Target Organism s and Enviro nmental Fate Tier I Data Requirements for Evalu ation of the Papaya active ingr edient, PR S Vcp Avian Oral Toxicity Av ian Inh alation Toxi c it y

/ Pathogenicity Wild Mammal To x ic ity / Pathogenicity Freshwa te r Fi s h Toxicity/Pathogenicity Freshwater In vertebrate Toxicity/Pathogenicity Estuarine and M ari ne A nim al Toxicit y and Path oge ni ci ty No n- Target Plant Testing Non-Target In sec t Testing OSCPP 885.4050 OSCPP 885.4100 OSCPP 885.4 150 OSCPP 885.4200 OS C PP 885.4240 OSCPP 885.4280 OSCPP 885 .4 300 OSCPP 885.4340 OSCPP 885.4380 Honey Bee Testing Gene Flow Weediness Potential No pub li s he d guideline; EPA-SAP 2004, and 2006 No publi s he d g uid e line ; EPA-SAP 2004 , and 2006



### 1. 

### Rationale and Justification The applicant ha s s ubmitt ed sc ientific rati o n al e and justifi cat ions th at th ese data requirement s co uld be addressed by a qualitative summary of infonnation cited from th e ope n li terature and other regulatory deci s io n documents to support regis tration of the PIP a.i., PRSVcp (and it s associati ve genetic mat e rial) for Event X 17-2 papaya. ln ad dition , EPA rev iewe rs h ave c ited previous docum e nt a tion from propo sed rul es a nd m ee ting s w ith sc ie ntifi c adv isory panels (SAP) as re levant to assessing ri sks o f a PIP that is developed w ith pl ant v iral coat protein

. Furthermor e BPPD believes that data from guideline te s t s wo uld not provide additional information for regu l atory assessment of the active ingredient , a v iral coat protein of pap aya rin gs pot v iru s Strain HI K (US DA , 2009). He r eaft e r the active in g red i e nt w ill be referred to as PR SVcp; the pe s ticidal active ingredient includ es other genetic m ate rial ( CaMV promoter genes fr o m cauliflower mo saic virus and 

### npt m ark e r genes) (40 CF R 174. 3 )



### a. Non- Target P l ants ln seven years of eva lu atio n of th e transgenic p a paya ex pre ss ing the PIP , PRSVcp , th e re were no differences of d i sease or in sect s u sc eptibility and resistanc e between the transge ni c papaya and non-transgenic p ap aya in Fl or ida , and there was no ev iden ce that th e tran sfo rmati on trait wo uld have any impact on th e p ote ntial as a weed , if th e PR SVcp escaped int o " [w]ild" papaya.
4The PR SVcp is produced by tran sge ni c papaya Event X l 7

- 2, whi ch re li es on a m ec h anism of RNA ge ne s il enci ng for ex pr essi ng resis tan ce to PRSV. Papaya Rin gspot V iru s cp production from papaya tran sfo nn ed by Eve nt X17-2 does no t produce a ny n ew environmental ex po s ures co mpar ed w ith PR SV- in fecte d fru i t (USDA , 2009). b. Other Non- Ta r ge t Organisms A vi an Oral Tox icity and Avian Inha lation Tox icity / Pathogenicity There is a lo n g history of consu mption o f the plant virus p a rti cles (i.e., p apaya ringspot) in food , w ith out causing deleterious effects o r ev id e nce of toxici ty; v iru s -i nfected plants are part of th e avia n diet, and there h ave been no findin gs which indicate that pl a nt v iru ses are capa bl e of rep li cation in man1m a ls o r o th e r ve 11ebrate s, thus e li minating the po ss ibility o f av ia n inf ecti on Furth e nnore, inf ect io u s particles are not f o nn ed from the transgenic pap aya, w hi c h ha s been modified with the p o rti on of the geno m e coding for r es istanc e to P apay a Rin gspo t v iru s and subcom p o nents of the resistance gene. The PR SVcp is produced by transgenic p ap aya Event X 17-2 , w hi c h re li es on a m ec h anism o f RNA gene silenc ing for expressing res i stance to PR SV. Since this gene is expressed only within plant tissues, exposures are expected t o be minimal , and are not like l y to i mpact bi rd s o r in sects that serve as a f ood sou r ce for birds; as the gene tic material is incorporated int o papaya pl ant tissue to overco me PRSV di sease, a nd it is not atomized to create inh a lab le particl es to birds. Wild Mammal T ox ic it y / Path oge ni c i ty There is a long hi story of consumpt ion of the plant v iru s p art icles (i.e. , papaya ringspot) in food, wi th out cau sing deleterious effec t s or evidence of tox ic ity; virus-i n fec ted pl ants are part of the mamma li an diet , and there have been no findings wh i ch indicate that plant viruses are capable of rep li cation in mamm als or other verteb rates , thus elim in atin g the p oss ibili ty of wi ld mamm al in fect io n (US D A, 2009). Transge nic Event X 17-2 papaya does not re pr ese n t a source of n ove l p oten ti al allergeni c o r a nti -nutr i e nt pro te in s. Inf ect io u s p articl es are not fo 1med from th e transgen i c pap aya, which h as been m od ified w ith the portion of th e ge n o m e cod in g for re sis tan ce to Pap aya Ring spot vi ru s and s ub comp o nents of the resis ta nce gene. T h e PR SV coat protein is produced by pap aya that h as bee n tra n sformed with Event X 17

- 2 w hi c h relies on a mechanism of R NA gene s il e nc in g f o r exp ressing res istance to PRSV. No deaths or abnonnalities we re reported i n five wee k- o ld a lbino r ats (I 0 m a les a nd 10 f e m a les p er treatm e nt group) when fed pap aya fruit p owde r from tran sge ni c (3 stra in s) a nd one no n tr a n sge nic contro l a t a d a il y do se of2 g frui t powde r /kg body weight/day f or 28 d ays (L in , e t al. , 2013). In addition, th e re were no s ignifi ca nt di ffe re nces of foo d consumption , feed e ffici e ncy and body weights a m ong a ll treated groups. Freshwater Fis h , Invertebrate and Estuarine / M arine A nim a l T ox icity / Pathogenicity The applicant asse rt s that data are not needed because th e re is no expected expos ure to fi sh, estuari ne a nd / or marine an im a ls in water. Si n ce the gene is only ex pr essed w ithin plant tissues, 

### 5the occu rrence would be minimal to non-existent. PRSY cp production from Event 17-2 papaya doe s not produce any new environmental ex po sures com p ared with PR SY -in fected fruit. Off- s ite move ment o f tra n s genic papaya tissu es (e.g., primaril y seed or fruit) int o aquatic ecosystems cou ld re s ult from agriculturaVhorticultural operations (e.g. , planting) and wea ther activity (e.g., hurricane s, tropical storms uprooting th e papay a tree) and an im al dispersal of partiall y -con s umed fruit; however there is uncertainty in re gard s to the s ignificance of these exposures. Neve rthele ss, no adverse effects to aquatic o rga ni s m s are anticipated from the viral coat prot e in of papaya rin gspot virus u sed for the PIP , becau se no inf ectiou s particles are formed from the tran s genic papaya and the mode of action i s spec ifi cally for expressing re s i sta nce through a ge ne s il e ncing mechanism to PRSV , a plant virus s p eci fic to papaya. Non-Target In sect Toxicity and Pathogenicity The applicant asse rt s that there is no expected exposure to no n-target in sec t s, because the gene i s only exp ressed within plant ti ss ue s, occurrence would be minimal to non-exi ste nt. "[L]eve ls of detec tab le PRSY

### cp do not increa se the iss ue of potential imp acts to no n-targe t organisms (i.e., b e ne fi c i a l in sec t s s uch as the ladybird beetle , green lacewing , para s iti c wa s p) as the PRSV coat protein i s not known to ha ve any toxic prop ert ies and is ex pre ss ed at a very low leve l when compared to papaya s in fecte d with PRSV " (USDA , 2009). As no ted above, BPPD belie ves that nont arget in sects co uld b e exposed to tran sgenic p apaya ti ss ues which co ntain PR SYcp

. Di spersal and moveme nt of tran sge ni c p apaya tissues (e.g., primaril y seed o r fruit) could result from agricultural /ho rti c ultural ope rations (e.g., plantin g) and weather a c tiv i ty (e.g., hurricanes , tropical s torm s uprooting th e papaya tree) and animal disper s al of partially-con s um ed fruit ; howeve r there i s un certa int y in regards to the significa nce of these expos ures to no ntarget in sects. V iral coat prote in s are ro utin e ly inge s ted by vi rtually all organ is m s, i ncluding human s, when virus-infected fruits and vegetab l es are consumed. Thu s, b ecau se of th e ubiquit o u s nature of plant viruses and th e li ke lih oo d of expos ures, impact(s) to non-t ar get organi s m s, including beneficial in sects, is v irtu all y non-ex iste nt (EPA , 1997 ; EPA, 2007; USDA / APH IS , 2009). BPPD re viewers not e that the virus is specific to only the papaya plant , and nontarget insect pe s t s s uch as aphid s, two-spotted spider mites and pap aya fruit fly are no t adversely affected b y th e ex press io n of th e PR SVcp (USDA/AP HI S , 2009). Becau se no inf ec tiou s particles are form e d from th e tran sge nic pap aya and the mode of action is s p ec ifi ca ll y for expressing r es is ta nce through a gene s ilencing mechani s m to PR SY, no adverse effects are anticipated to nontarge t in sect s, regard less of the exp osure. H oney B ee The a ppli cant asserts that there is no expected exposure to honeybee, becau se the gene is only expressed within pl ant ti ss ues; theref ore, occurrence of ex p os ure wou ld be minim a l to nonex i s te nt. Leve l s of detectable PRSV

### cp do not in c rease th e iss ue o f potential impacts to n o n-targ et organisms , s uch as the h oneybee as the PRSV

### c p is not known to have any tox ic properties and i s exp ressed at very low l eve ls when compared to pap ayas inf ected with PRSV " [Braverman , et al. , 20 14, MRID 494932-03]. 

### 6In addition, viral coat proteins are routinely inge s ted by virtually all organisms, including humans , when v iru s- inf ected fruits and vege tabl es are consumed. Thu s, b ecau se of th e ubiquitous nature of pl ant v iru ses and the li ke lih ood of previou s exposure , the like lihood of impac t to non-target organ i s m s, including the ho neybee, i s v irtu a ll y n on-ex i ste nt (EPA , 1 997; EPA, 2007; USDA/APH IS, 2009)

. BPPD re viewers note th at the virus is specific to only the papaya plant , and nontarget insect pests such as aphids , two- s potted spider mite s and papaya fruit fly are n ot adverse l y affected by th e expression ofthe PR SVcp (US D A/A PHI S , 2009). Because no inf ectious particles are formed from the transgenic papaya and the mode of action i s specifically for ex pr essing resistance through a ge ne si len c in g mech anism t o PR SV, no adverse effects are anticipated to honeybees, regard less of th e exposure. E. Ec o l ogical Ex po s ure and Environmenta l Ri s k Characterization l. T e rrestria l Animals and Plants 

### Birds and Mammals Birds and mamma ls wou ld be exposed to PR SVcp primarily through inge s tion of tissues , seed or fruits of transgenic papaya X17-2, or from prey that had inge sted transgenic papaya. Contact with these surfaces i s not expected to re s ult in s ig nifi cant exposure, though ora l exposure may occur incidentally thro u g h grooming

. T he s ubmitt ed data and information are s ufficient to conc lud e that PRSV cp is unlikely to infect bird s or w il d mammal s and cause adverse effec t s . 

### Nontarget In sec t s and Honev Bees Nontarget insects and honeybees would be ex p osed to PRSVcp primarily through i ngestion of tissues , fruits o r pollen of transgenic papaya X 17-2, or from prey that had inge s ted transgenic papaya. Contact with the se su r faces i s not expected to result in sign i ficant exposure, though oral exposure may occu r in ciden t ally through grooming. The submitted data a nd inf o rmation are s ufficient to conclude that PRSV cp is unlik e ly to i nfect nontarget i n sects and ho neybees and ca u se adverse effects. 

### Nontarget Plants For hazard evaluation of a plant incorporated pesticidal s ub stance that i s produced from a plant pathogenic mic roorganism , the Agency i s concerned that it s use(s) wou ld not predi spose other nontarget plants, and n o n-transgenic papaya to other diseases or pests that might trigger adverse effects. I n addition , cont inu ed express ion of the pe s ticidal s ub s tan ce in tran sge nic papaya may trigger potential for off-site m ovement (gene flow) and introgr ession i nto non-transgenic, w ild or "feral" papaya populations. Ri sk considerations for ge ne flow and invasiveness are d i sc us se d further be low in Section 3. Environmental Fate , Pe rs i s tence and Ri sk Assessment. The Agency does n ot an ti cipate adverse effects to n o ntarg e t plants outs id e of th e papaya species 

### (Carica papaya); and a nti ci pat es minimal risks t o wild or "feral " papaya that may be present in the subtro pical U.S. Informat io n from the open literature was evaluated to determine that it is un l ikely that PRSVcp when incorpo rated into papaya (i.e., Eve nt Xl7-2 transgen i c papaya) w ill produce any new , or incremental e nviro nment a l exposu res of concern beyond those of papaya trees or fruit infected by the natural populations of papaya ring spot vir u s (PRSV) popu lations.
7

### 2. Aquatic Anima ls and Plant s Freshwater Fish and Invertebrates B ased on the inf o nnati on c ited from the li t e r ature, which i s discussed above, ri s k s to freshwater fish and invertebrates are not ant ic i pated as a result of the proposed u ses of PRSYcp in papaya Event X 1 7-2. 

### Marine /

Estua rin e Fish and I nvertebrates B ased o n th e inf ormatio n c it ed from the literature, ri s k s t o marin e/ es tuarine fish and in vertebrates are n ot a nti c ip a t e d as a r esu lt of th e propo se d u ses of PRSY

### cp in p apaya E ve nt X 1 7-2, as th e produc t i s not inte nded for direct app li cat io n in t o estu ar in e o r m ari n e e n vi r onme nt s a nd the pe s t ic id a l m ode of act io n i s s p ecificall y for ex pr ess in g re s i sta nce through a ge ne s ilencin g m ec h an i s m to PR SY, a plant v iru s spec ifi c to p apaya. 

### Aquatic Plants As discussed above , th e proposed u se ofPRSVcp in pap aya Eve nt X l 7-2 i s s p ec ific for transgenic papaya to contro l a viral disease. Also, the product i s n o t inte nd e d fo r direct applicat io n into estuar i ne or marine environments , and the p es ti c id al mode of action is spec ifi ca ll y for express i ng resistance through a ge n e s il e n c i ng mechanism to PR SY , a plant v irus specific to papaya. Th e refore , adverse effects to freshwate r and marine or estuarine plant s are not anticipa t ed. 

### 3. E n v ironm e ntal Fate , Persistence and Ri sk Assessment A. Gene Flow F or PI P regi st ration act ions, th e US EPA a l so assesses th e l ike lih ood and effects of gene flow and introgress io n from the PIP plant to sexuall y compat ib l e wild r e l at i ves (SCWR) in the US and i ts territories. Re g i s trant s a re, therefore , asked to s upply a ge n e fl ow ri sk assessme nt th at di sc u sses th e p ossibi lity of po ll en fl ow to com pat ib le pl ants a nd h ybrid iza tion , in trogression , and stab l e inh e rit ance. If E P A's ri s k ana l ys i s concludes that a proposed PIP trait ha s th e potential to confer a select ive advantage to wi ld and fera l re latives b y po ll e n transfer lead in g t o h y bridi zat ion , introgre ss ion , and stab le inh e ritance , then proactive mea s ures mu st b e tak en t o mitigate id e ntified imp acts upon registr atio n of suc h a PIP
. The a ppli cant (UFIIFAS) o f the propo sed P IP (a coat protei n of papaya rin gspot virus produ ced from transgenic papaya Event X17

- 2) intend s to re lease this geneti cally modified papaya h y brid for commercial ization exclusively in Florida. No closely related wild and feral rel atives coexist in this geographi c regi o n (US DA 2009) , and ri sk from ge n e fl ow a nd i n trogression i s , theref ore , highly unlikel y. T h e closest related relati ve i s th e hi g hl and papaya ( 

### Vasconcellea s pp.)

. which i s n at ive to Central and South America (US D A 2009). If 

### Carica papaya L. were commercialized in an area w h e re 

### Vasconella s pecie s co - occurred, the risk of h y bridi zat io n from gene fl ow would be low. T hi s s t at e ment i s s upp orted b y h ybrid iza tion attempts betwee n 

### Carica and 

### Vasconelfa spec ies th a t have fa il ed to produce intergen eric hybrid s (OEC D 2005). In addition, th e FIFRA
8SAP (2004, 2005) concluded that papaya would not form a viab l e hybrid with w ild re latives in the U.S. and it s territories. BPPD co nclude s that the risk of gene flow via pollen transfer from the P IP X 17-2 plants resulting in hybridization of a SCW R is very unlike ly in the United States and its territories but especia ll y in the propo se d geograp hic regi o n of commercializat ion (F lorida). 

### B
. Inva s i ve n ess A PIP plant expressing a protein conferring disease or pest re s istance has the potential to incur a competitive advantage over other plant species with which it i nteracts and shares an environment (SAP 2004). Therefore, as part ofthe re gistration re que s t , EPA requires applicants to di sc u ss the potentia l for a PIP p lant to become invasiv e in th e environment. Typically , appl i cants are also asked to subm it information addressing the li kelihood th at a SCWR can be co m e a weed if the transgene introgresses . PIP po ll en flow to SCWR h as th e potential to confer a selec ti ve advantage to wild and feral relatives if hybridi za ti on, introgre ss ion , and s t able inh eritance are possible. Th e Univers ity of Florida demonstrated that there were no s ignificant difference s in pheno logy between ge netically engineered X l 7-2 papaya and non-GMO 

### C. papaya plants (USDA, 2009). The la ck of detectable s ignificance in plant vigor did not support th at Xl7-2 papaya would b e weedier in nature than it s non-GMO counterpart. Therefore , BPPD concludes that the like l ihood for X 17-2 papaya to become invasive is low in the U nited States where its comme r cializati on would be l imited to Flor id a. BPPD further concludes s inc e th e re i s no ev idence that 

### C. papaya ha s 'Yild or feral re latives in the United State s and its territory with which hybridization could occur, that at the present time the concern for the VCP tran sge ne to confer a selective advantage of invasivene ss to SCWR in the United States i s not substantiated.


### 4 . T hr eatene d a nd E nd a n ge r ed S peci es Assess m ent EPA has determined that no effec t s are anticipated fo r nontarget spec ies exposed to PRSVcp in papaya X 17-2 including mammals, birds , terrestrial and aquatic plants, freshwater and marin e/ es tuarine animals and in sec ts. Therefore, a ''No Effect" detennination i s made for dire ct and in di rect effects to federa ll y listed endangered and threat e n ed (" li s ted") spec ies and for their de s ignate d c ritical habitat. 

### BIBLIOGRAPHY I. 

### Data S ubmi ss io n Braverman, M

. , W. P. Barney , K. D. Co leman. 2014. Nontarget waivers for Xl7-2 Lin e oftra n sge nic Papa ya Ring spo t-Re s i sta nt Papaya. Non-p ub l ished report s ubmitt ed to th e Agency, MRID 494932-03.



### 9Braverman , M. , W. P. Barney, K. D. Coleman. 2014

. Group A -product analysis test gui d elines. Non-published rep ort submitted to the Agency, MRID 494932-01 Lin , H-T., G-C.Yen, T-T.Huang, L-F.Chan, Y-H, C heng , J-H. Wu, S-D.Yeh, S -Y .Wan g, and J-W .Lia o. 2013. Toxicity a ssess ment of transgenic papaya ringspot v iru s of 823-22 10 Lin e P apaya fruits. J. of Agri cu ltural and Food Chemistry 6 1: 15 85-1596. USDA. 2009. In response to University of Florida Petition 04-337-0 I P seeking a determination ofNonregulated Status for X 17-2 Papaya Resistant to Papaya Ringspot Virus. OECD Unique Identifier UFL-X 17CP-6. Animal and Plant Health Inspection Service, Biotechnology Regulatory Services. 

### II
. Other References EPA-SAP. February 4, 2000. Characterization and non-target organism data requirements for protein plant-p es ticides. SAP Report No. 99-06A for FIFRA Scientific Advisory Panel Meeting held December 8, 1999 , held at the Sheraton Crys tal City H ote l , Arlington , VA EPA-SA P. December 27, 2004. Issues associated with deployment of a type of plantincorporated protectant (PIP), specificall y tho se ba se d on plant vira l coat proteins (PVCP-PIP s). SAP R eport No. 2004-09 for F IFRA Sc ientifi c Advisory Panel Meeting held on October 1 3- 1 5 2004 at the H o lid ay Inn

- Nati o nal Airport , Arlington , VA. U.S. EPA-SA P
. February 28,2006. A set of sc ientific issues being considered by the Environmental Protecti on Agency regarding: Plant-incorporated prot ectants based on virus coat protein genes: Science i ss ue s as soc iated with the proposed rule. SAP Rep o rt No. 2006-1 for FIFRA Scientific Advisory P anel Meeting held on December 6-8 , 2005 at the Holiday Inn
- National A irp ort, Arlington, VA. U.S

. EPA 1997. Coat Protein of Papaya Ring s pot Virus and the Genetic Material Necessary for it s Production; Exemption From th e Requirem e nt of a Tolerance. 62 FR 44572-44575. Gonsalves, D. 1998. Control of Papaya ring spo t virus in papaya. Ann. Rev. Phytopath., Vo1.36: 415-437. OECD. 2005. Consensus D oc ument on the Bi o logy of Papaya 

### (Carica papaya).

10No ntarget Organi s m Tes ting- Review of Literature To S uppor t Data Requirements (40 CFR 158.2150 ; OSCPP G uid e lin es 885.4000 seri es Vira l Coat Protein from Papaya Ringspot V iru s; X 1 7-2 Transge nic Line of Papaya ( P C Code 00670 1)/EPA File Symboi84427-R-Xl7

- 2 S ubmi ss ion No. 960423/ Decision No. 497668 / DP Barcode: DP 424881 Primary Reviewer: Gail Tomimatsu ,



### Ph.D. **~** Secondary R eviewer: S h a nn o n B o r ges, LeaJ



### Biologi **~** 

## NOV 0 5 

## 2015
EXECUTIVE SUMMARY: On behalf of the ap pli cant , the U ni versity o f FL/ l nstitute of Food and Agricu ltural Sc ie nces (Company No. 84427), USDA-IR-4 submitted qualitative s umm ary rationale s upp orted by in format io n from peer- reviewed sc ientifi c li terature and th e USDA/A PHI S P etit io n for No n-Regulated S tatus a nd Finding ofNo S ignifi cant Impact [I] to s upp o rt the nontarget o rg a ni s m s data requir e m e nt s f o r th e eco logical ri s k assessment of the plant in co rporated prote c tant (P I P) , a v iral coat prote in ( c p)from papaya ring s p o t virus (PRSV) produced b y Eve nt X17-2 transgenic lin e of papaya (OECD Unique Id e n tifier U FL-Xl7CP-6)

. The we i ght

- of

- evidence in the s ubmitt ed rationale an d the ci ted li terature do not justify add itional nont arget orga ni s m testing as recommended in the O CS PP T es t Guidelines 885.4000 se rie s of mi crobial pe s ticide data requirement s . T he US D A- IR-4 submi tt ed ratio n ale supported by relevant citation s from the open scientifi c lite rature to concl ud e that the PR SV coat pro te in that is produ ced b y X 17-2 transgen ic papaya d oes not produce any n ew, or environmental ex p os ure s of conce rn beyo nd tho se of papaya trees or fruit naturally infecte d b y the Papaya Rin gs pot virus (PRSV) populations in FL. No tabl y, th e PR SY gene u se d to e ng ineer X 17-2 is f rom a F lorida iso late of th e v iru s (H l K , 

### ll J) a nd repo rt s h ave s ho wn that s imil ar ge ne c o n stru cts give resistance to th e di sease only from hi g hl y s imilar vira l stra in s (OECD 2005). In additio n , th ere are no known adve rse ef f ects for th e use of a PR SV -r es istant transgenic p ap aya in HI , which is a lso ba se d on a PR SV coat protein , a nd genetically e ngi nee red from a Ha wa ii an PR SY stra in [1]

. CLASS IFI CAT IO N: S ubmitted Rationale and C la ssification of Indi v idual Rationale: I. Avian Oral (O PP TS 885.4050) A v ian Inhalation Toxicity/Pathogenicity , Ti er I ( OPPT S 885.41 00) Th e appli cant claims that data are not needed becau se th ere is a lo ng hi story of co n s umption o f th e pl a nt v iru s parti c les (i.e., pap aya rin gs p ot ) in f ood, without caus in g de l e te ri o u s e ffe c t s or ev id e nce of to x icity ; v iru s- infe cted pl ants a re part of th e av i a n diet , a nd th e re h ave b een no findin gs wh ich indi cate th a t plant viruses are cap able of r e plication in mamma ls or other vertebrates, thus e limin atin g the p ossi bility of avia n inf ect ion [ 

### 11. Furth ermo re, infectiou s part i cles are no t formed f r o m th e transgen i c papaya , w hi c h has be e n modified with the porti on of the genome codi ng for re s istance t o Papaya Ring s p ot virus and s ub comp onen t s of the resistance gene. The PR SY coat protein i s prod uced b y X 17-2 papaya, w hich relie s on a mechanism of RNA gene s ilen c i ng for exp ress ing re s istan ce to PRSV. S inc e thi s gene is ex pr essed o nl y w ithin plant tissues, exposure s are ex p ected to be minimal , a nd a r e not like l y to impact bird s o r in sectsN ont arget Org ani s m T es tin g - R ev iew of Lite ratur e To S upport D a ta Requir e men t s (40 CF R 158.2150 ; O SCP P Guid e line s 885.4000 se r ies V ir a l C o a t Pr o t e in from Papay a Rin gs pot Viru s; X l7-2 Tran sge nic Lin e of P a pay a (PC Co de 00670 1)/E P A F il e Sy mboi8442 7- R-Xl 7

- 2 S ubmi ss ion No. 9 60 42 3 / D e ci s ion No. 49 7668/ DP B a r c o de: D P 424881 that :S erve as a f oo d so urc e for birds ; as the genetic mate ri al is incorp o rated int o papaya plant ti ssue to ove rcome PRSV d isease , and it is not atomized to create inha lable particle s to bird s . C la ss ification of Submitted Rationa l e for Avian Oral and Avian I nh alation Data Requ i rement s : Acceptab l e. 2

. W i ld Mam m a l Te s t ing (OPPTS 885.4 1 50) T he app li cant claim s that data are no t n eeded becau se th e re i s a l o ng hi story of consump ti o n of the plant v iru s particles ( i .e. , papaya r i ngspot) in food , without causing de l eterious effects or evidence of tox ic ity ; v i ru s - i nfected plants are part of th e mammal ian diet , and there have bee n no findin gs which indicate that plant viruses are capab l e of rep li cation in mammal s or other ve rt e brate s, thu s e l im i nating the po ss ibility of wild m amm a l i nfection 11)

. The X 1 7

- 2 papaya d oes not re present a so urce of novel potential allergenic or anti

- nutrient protein s. No deaths or abnorma l ities were reported i n fi ve week-old al bin o rat s ( I 0 male s and l 0 fema les per treatme nt gro up ) whe n fed papaya fruit po w der from 3 tran sgen ic papaya s train s (other than X 17

- 2) and one non-transgenic control at a dail y do se o f 2 g fru it powd e r /kg bod y weight/day for 28 d ays 12). In addition , the re were n o sign i ficant differences of food consum p t ion, feed e ffi c ie ncy a nd body weights among all treated gro up s. Infecti o u s particle s are not formed from the tran sge ni c p apaya, w hi ch ha s been modified with th e port ion of the genome c o ding for resistance to Papaya Rin gs p o t virus and subcomponents of the re s is tan ce gene. T h e PR SV coat protein is produced by X 17-2 papaya , w hi ch re li es on a mechani s m of RNA gene s i lencing for expre ss i ng res istance to PR SY. C l assification of Subm i tted R ationa le fo r W il d Mammal Data Req u i rement: Acce ptable.

3. Freshwater Fis h T es ti ng, T ier 1 (OPPTS 885.4200) Es tuarin e and Marine Anima l Testing , Tier 1 (OP PTS 885 .4 28 0) The app li cant claims that data are not needed bec a u se th e re is n o ex p ec t e d ex po s ure to fi s h , estuarin e and/o r marine animal s in water. Since the ge ne is only exp re ssed with in plant tissue s, the occ urre nce would b e minimal to non- ex is tent. PRSY cp produ c ti on from transgenic papaya X 17-2 does no t pr o du ce an y ne w e nv i ronmental exposures compared with PRSV-infected fruit. BPPD rev iewe rs disagree th at fre s hwater , estuarine and m arine anima ls wou l d no t be exposed to the P I P a.i. , (P R SV cp -tran s formed X17-2 ti ss ue s) even minim all y . Off-site movement of tran sge nic papaya t i ss ue s (e.g. , primari l y see d or fruit) int o aquatic ecosystems could result from agricu l tura l /horticultura l operati ons (e.g. , planting) , weather activity (e.g. , hurricanes , tropical s torm s uprooting the papaya tree) and anima l disper sa l of partia ll y-consumed fru i t ; however there i s unee 1 ta i nty in regards to the signi ficance of th ese ex po s ure s. Nevertheless , B PP D does



### . 2

### Non t a r get O rga ni s m Test in g- Rev i ew o f Li te ratur e T o S uppor t D a t a R e qui re m e nts ( 4 0 CF R 1 58

. 2 1 5 0 ; O SC PP G uid elin es 885.4000 se ri es Vira l C o at P ro te in fr o m P a p aya Rin gs p ot V iru s; X17- 2 Tra n sge ni c Li n e o f Pa p aya ( PC Code 

### 006701)/E PA F il e Sy mb oi84 427 -R- X 1 7-2 S ubmi ss i o n No
. 960423/ Dec is i o n N o . 4 97668/ D P B a r co d e: DP 4 24 881
not a ntic i pate adve r se e f fects to a qu at ic o rga ni s m s from expos ur es to th e v ira l coat protein ( cp) of p apaya rin gsp ot vir u s and other gene ti c material (CaMV promot e r genes from caulifl ower mosaic v iru s and npt marker genes) u sed fo r the PIP , because no infectiou s particles are formed from the transgenic papaya and th e mode of act io n i s s pecifically for express in g resistance through a gene si le n ci n g mechanism t o PR SV, a plant virus s p ecifi c to papaya. C lass ification of Subm i tted Rationale for Freshwater, Estuarine and Mar i n e Animal Data R e quire m e nt s : Supp le m e ntal. Some text i n th e s ubm i tted rational e was n o t s upp o rt ed by re le va nt ref e re n ce citations

. 4

. Freshwater In vertebrate Test in g, Tier 1 (OPPTS 

### 885.4240) The applicant claims th at data a re not needed b eca u se th e re are n o expected exposures to fre s hwater in verteb ra tes. S in ce the gene i s on ly expressed wit hin plant ti ss u es, th e "[o]ccurrence wou ld b e minimal to n o n- ex i ste nt "

. Exposure s to plant ti ss ue s that exp ress th e p esti c idal gene are a nti c ip ated (see t ex t b e low), h owever th e re i s un certa inty with re spect to th e ex te nt and persistence of the ex pre ssed genes in planta. B eca u se i nfect i ou s particl es are not fonned from th e tra n sge nic papay a, which has been modified with the portion of the genome coding for res i stance to PR S V a nd s ub co mp o n e nt s of th e res i stance gene, adverse effects to aqua tic in ve rtebrates a re not ex p ected. The PR SV coat protein i s produced b y tran sge nic papaya X 

### 17-2 whic h re l ies on a mechanism of RNA ge n e sile n cing for exp ressi n g resistance to PRSV. P a paya R ing s pot V iru s cp pr oducti on from transgen ic papaya doe s n ot produce any n ew env ironme ntal expos ures co mp a red with PRSV-infected fruit. BPPD re v i ewers di sagree th a t fre s hwat e r in ve rt e bra t es would no t be ex p osed to the PIP a.i. PR S V cp- tran s form e d X 

### 17-2 ti ss ue s ) , even min i m a ll y. Off- s ite movem e nt of tran sge nic papaya t i ss u es (e

. g . , prim a ri ly see d or fru i t) i nto aquatic ecosys tem s cou ld re s ult fro m agricu ltural /hortic ultura l ope ra tion s (e.g. , planting) , o r weather act ivity ( e,g, hu rri ca ne s, tropi ca l s torms up rooti n g the papaya tree), and a nim a l di spe r sa l of raw o r co n su m ed fruit ; however th e re i s uncertainty t o the s ign ifi ca n ce of the se ex p osu res. Neve rth e l ess, BPPD doe s n ot anticipatead ve r se effects to freshwater invertebrates from exposures to th e v iral coat protein (cp) of papaya ringspot v iru s and ot h er ge n etic material ( Ca M V promoter genes from caul iflo wer mosaic virus and npt marker ge n es) u sed f or the PIP , because n o in fect io u s particles are formed from the tran sge ni c p a pa ya and th e mod e of action i s s p ec ifically for expre ss in g re s i sta nce through a gene s i le ncin g m ec hani s m to PR SV , a pl a nt virus spec i fic to papaya. C lassificat ion of S ubmi tte d Rationale for Freshwater, Estuarine and M a rin e An im a l D a t a R equiremen ts: Supp l emen t al. Some text in th e sub mi tted rationale was not s upported by relevant refere n ce citat ions .

3No ntarget Organism Testing- R ev iew of Literature To Support Data R eq uirements (40 CFR 158.2150; OSCPP G uid e line s 885.4000 series V ir a l Coat Protein from Papaya Rin gs pot Vi ru s; X17-2 Tra n sgenic Line of Papaya (PC Code 006701)/E PA File Symbo l84427-R-X1 7-2 Submiss io n No. 960423 / Deci sio n No. 497668/ DP Barcode: DP 424881 5. Nontarget Plant Stud ies, Tier I (OPPTS 885.4300) The applicant claims that data are not ne eded based on several l ine s of evidence: a) "(N]o differences in susceptibility or resistance to othe r in sec t pest (two-spotted spider mites and papaya fru it fly) or pathogen (anthracnose and powdery mildew) populations compared to control non-transgenic plants in these trials cond u cted on X 1 7-2 papaya for 7 years in FL [1]. The appl ica nt did n ot address the rationa l e for why the c ited text i s re levant. Based on these obse r vat io n s, th e reviewer opines that th e re wou ld lik e l y be no signifi cant effect on predispo s iti on of nontransgenic papaya to the organisms ; and minimal synergy of the PRSV cp and the cited organ i sms. b) Th e cp of PR SV in transgenic papayas may have "[ l] ittle influ e nce on soi l microorganisms, due to low level s or has not been secreted from roots in to so i l ", and therefore , plants obtaining nutrient s from the surroundi n g so il will not be affected [31

. The applicant did n ot address the rationale for h ow the cited text would be re levant towards assessing potent ial h azards of the v iral coat protein to nontarge t papaya (e.g., organic papaya) or t o other nontarget plants 

### in the vici nity of plantings of FL X 17-2. The re v iewer noted that re su lt s of this limited study (conducted on tran sge ni c Taiwan papaya s) indicated that there was an overall s imilarity (> 80%) of soil micro o rgani s m s in Taiwan soi l profiles of transgenic papaya (wi th the PRSV cp) and nontran sge nic papaya. Because these results were ex trapolated from results of molecular methodologie s used to determine microbial " profiles ", and that additiona l experimentation i s nece ssa ry to confirm the reported observations , this part of the rationale is not rele vant to assessing ha zard s of the v iral coat protein to nont arget plant s, including non-transformed papaya. c) There i s no evidence that the Xl7-2 trait wou ld ha ve any imp act on the potential as a weed if th e PRSV c p escaped into "(w] ild " papaya . Th e app li ca nt did not provide text from a reference c it ation to support this sta teme nt. The reviewer n otes that n o close l y related wild and feral re latives coexist in this geographic region [1 ] , and ri sk from gene fl ow and introgression i s, therefore , highly unlik e ly. BPPD concludes th at the risk of ge ne t1ow via pollen tran sfe r from the PIP X 17-2 plants resulting in hybridization of a sexua ll y compat ibl e wild re lat i ve (SCWR) is very unlik e ly in the United States and its territories but espec ia ll y in the proposed geographic region of commercializat ion (Florida). d) US DA-APHIS was unable to identify any characteristics of X 17-2 th at might indicate an increased propensity to become weedy or in vasive. Feral p o pulation s of papaya may exist in so me locati o n s, however hybridizations with X 17-2 will not I ikel y re s ult in production of plants with weedy o r invas ive characteristics [1]

. e) The PRSV coat protein is produced by Xl7-2 papaya , which re lie s o n a mechanism ofRNA gene s ilencing for expressing resistance to PR SV. Papaya Ring s pot Virus cp production from X 17-2 papaya does n ot produce any new env ironm ental ex po s ures compared with PRSVinfected fruit 111

. 4No nt a r ge t Org ani s m T es tin g - R ev i ew of L it e r a tu re T o S upport Data R e quir e m e nt s ( 4 0 CF R 158.2150 ; O SCP P G uid e lin es 8 85 .4 00 0 seri es V ir a l C o a t Pro te in from P a p ay a Rin gs po t V iru s; X 1 7

- 2 Tra n sge ni c L in e o f P a p aya (PC Code 00670 1)/E P A Fil e Sy mbol8442 7 -R-X17-2 S ubmi ss ion No. 96 042 3/ D e ci s ion N o . 497 668/ 

### DP Ba rc od e: DP 424881

C lassificat io n of Subm itte d R ational e for No nt arg e t Plant Dat a Re quirem ents: Acceptable. Although th e re was no ex pl a n at io n of why some o f the submitted text were re l ev ant for th e d ata r eq uire m e nt , a nd some text in the su bmitted rati o n a le was not supp orted by re levan t r e f e rence c i tatio n s, the inf ormation provided in the study d ocu m en t is sufficie nt to conclude that no unreaso n able , in c re m e nt a l ri sks a re anti ci p ated fo r no nt arget pl a nt s, including papaya f o r o rgan ic production

. 6. Nontarget In sec t Studies , T i er I (O PP T S 885.43 40) Th e app li cant claims th at data are not nee d e d b ased on severa l line s of ev id e n ce: T he re is no expec ted exposu re to non-target in sec t s. S in ce the gene is on ly expressed within plant tissues , occurrence would be minim a l to no n- ex is tent. " Xl7-2 papaya does exp ress d etectabl e PR SV 

### [1) , but at very low le ve ls co mp ared to p ap ayas infe cted with PR SV. {These levels} d o not increa s e the iss ue of p ote nti a l imp acts to no n-target orga ni s m s (i.e. , b e ne fici a l in sect s s uch as th e la d yb ird b ee tl e, green l acew in g , p aras itic wasp and hon eybee) as the P RS V coat protein is n ot known to h ave any toxic p rop e r ties 

### [1) . The applicant did no t prov id e t ext fro m a re f e re nce c itation to s upp o rt th i s s tate ment. The rev i ewe r d oes not agree th at no ntarget insect s, e.g., ap hid s, o th er s uc k i ng in sec t s wo uld not be ex po sed to the PI P a. i

. , ( PR SV cp) even mi nima ll y. Aphids s pre ad PR SV in a non-persistent manner, the vi ru ses are rapidly acquired from infected pl a nt s and can be tra n sm itted to other ( h ea lth y) papaya w ithin minutes [1]. Furth e nnore , th e applicant noted that th e tra n sgenic P RSVresis tant c ulti v ar R a inb ow ( H I tra n sgenic p apa ya) is s u sce ptible to both th e lea fhopper and mit es, but did not pr ov id e ju s tifi ca ti o n why th e cited t ex t is r e l ev ant for the no nt arge t in sec t data re qui re m e nt. T he reviewer notes that the PR SV (a plant-pathogenic v iru s) i s path oge ni c t o o nly the pap aya plant , and in sec t s suc h a s aphid s are no t ad ve rse l y a f fecte d

. Compared to no n-tra n sge ni c plants , the re was n o c h an ge in s u sceptibili ty or resistance t o two-spotted s pid er mit es a nd th e p ap aya fruit fl y in the tran sge ni c papaya plants ( 1) . In add iti o n , viral coat proteins are rou t in e ly in ges ted b y v irtu all y all o r ga ni s m s, in c ludin g hum a n s, wh e n virus-infected fruits and vege tabl es a r e co n s umed. Thus, b eca u se of the ubiqu i to u s nature o fpla nt v i ru ses and the like l ihood of previous exposure, the like l ihood of impa ct to non-target organisms , includ i ng b e n e fi c ia l in sec t s, is vi rtua ll y non- ex i s tent [ 1 , 5)

. C lassificat ion of S ubmitt e d R ati o nal e for No ntarget Insect Data R equire m e nt s: S upplem ental. So m e text in the sub mitt ed ratio n a le was n ot s upported b y relevant re f e re nce citations

.



### 5N on ta r g e t Orga ni s m T est in g - R e vi ew of Lite r a tur e T o S upp o r t D a ta R e quir e ments (40 C FR 15 8

. 2150 ; O SC PP G ui de lin e s 885.4000 se ri es V ira l C o a t Prote in from P a p aya Rin gs po t V iru s; X l 7 -2 T r a n sge ni c Lin e o f P ap aya (P C Code 00670 1)/E PA F il e Sy mb o l8442 7 -R- X 1 7

- 2 S ubmi ss i o n No
. 960 4 23 / D e ci s ion N o. 4 97 6 6 8/ D P B a r co d e : D P 4 248 81 7. H oneybee S tudi es, Tier 1 ( OPPT S 885.4380) Th e applicant clai m s that data are not needed b ase d on severa l lin es of evidence as pre se nted above for It e m 6 , No ntarge t In sec t Studies, and cited te x t from th e ope n literature:

a ) Morandin and Win s ton , 2005 s tudy [ 6 ] : O n e i nt e rpre tat ion of th is st udy is that there was a s imil ar ' p o lli nato r force' i n all " fie ld type s " base d on observations of"relatively eq u al number of see d s p e r s il ique for op e n-poll i nate d fl ow e rs i n org an ic, convent ional, a nd GM fie l d s . Th e purp ose of th e cited s tudy wa s to observe d i fferences b e twe e n pote ntia l and actual po lli nation (i.e. , p o ll in ati o n defic it s) and bee abundance in or ga ni c, conventional and herbicide-re s istant , genetica ll y mo difi ed canota fields. The applicant did not ex plain why th e cited text i s relevant to ju st i fy why th e re would b e no adverse effects of PR SV cp to hon ey be es, if a properly d es igned guideline test were co nducted

. BPPD concludes th at th ese u ses a nd exposures are s ubstantiall y different th an th ose of a p lant v iru s coat pr o te in ; the r efo re, th e c itat io n is irrel evant and thi s portion of th e rationale i s Unacceptable. b) "X 17-2 p apaya does express detectable PR SY 

### Ill, but at very low leve l s co mp ared to p ap ayas infected with PRSY ''

. {These le vel s} do not in c rease th e i ss ue of p oten ti al imp act s to non-t arget organ i s m s (i.e., beneficia l in sec t s s uch as th e ladybird b eet le, g ree n la cew in g, p aras it ic wasp and honeybee) as th e PRSV coa t protein i s n ot kno wn to have any tox ic prope rt ies [1]

. PRSV cp production fro m X 1 7

- 2 papaya doe s not pro duc e any n ew e nvi ro nm e ntal expos ur es compared w ith P RSY- i nfec te d fruit , which is not known to have adve rse (e)ffect s on animals or plant s to w hi c h th ey are exposed. In addition, v iral coat protein s are ro utin e l y in ges ted by virtua ll y all o rga ni s m s, including humans , when vi ru s - infecte d fruits and vegetab l es are con s umed. Thu s, be ca u se of th e ub iqu i tou s natu re of p lant v i ruse s and the li ke liho o d of pre viou s exposure, th e l ike l ihood of impac t to non - target organisms , incl udin g the honeybee , i s v i1 tua ll y no n -ex iste nt 

### [1 , 5, 

### 61 C lass ifi ca ti o n of S ubmitted R ationale for Nontarget In sec t Data Re qu ire me nt s: Sup p lemental. Some text i n th e s ubmitted rationale wa s not supported by re l eva nt refe rence citations or co n s id e red irre leva nt. The reviewer note s that the PR S V (a p lant-pathogenic v iru s ) i s pathoge nic to on ly th e p a p aya plant , and ho ne ybees are no t anti c ip ated to be adversely affected. R e f e r e nc es a nd Li te ratur e C ited : I. US D A-A PHI S. 2009. Draft Env ironmental Assessment , Finding o f No S ignifi c ant Impa c t. In re s pon se to U ni v. FL Petition 04

- 337

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6No nt a rget Orga ni s m Testing- Rev iew of Lite ratur e To S upport Data R eq uirements (40 CF R 158.2150 ; O SCPP G uid e lin es 885.4000 seri es Vira l Co at Prote in from Papay a Ringspot V iru s; X17-2 Transgen i c Line of Papaya (PC Code 006701)/E PA F il e Sy mboi84427-R-X17 -2 S ubmis s ion No. 960423 / D ecis ion No. 497668 / DP Barcode: DP 424881 3. Hsieh , Y.T. and T.M.Pan. 2006. Influ ence of planting papaya rin gspot v iru s re s istant tran sge nic papaya on so il microbial biodivers ity. J .Agric. Food C hem.54: 130-137. 4

. OECD. 2005. Co n sensus Document on the Bio logy of P apaya (Carica papaya). 5. E PA 1997. Coat Protein of P apaya Rin gspot Virus and the G enetic Material Necessary for it s Prod uct io n ; Exempt io n F ro m th e R equi r ement of a Tolerance. 62 FR 44572. 6. Morand in , L. A. a nd M.L.W in s ton. 2005

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