MEMORANDUM 

SUBJECT: 

TO: 

FROM: 

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEM ICAL 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 

Microbial Pesticides Branch 

Biopesticides and Pollution Prevention Division (7511 P) 

Gail 

Microbial 

Tomimatsu, 

Pesticides 

Ph~ Branch 

Biopesticides and Pollution Prevention Division (7511 P) 

Jeannette 

Microbial 

Martinez, 

Pesticides 

PhD

Branch 

py ~ 

Biopesticides and Pollution Prevention Di 

PEER REVIEW: Chris Wozniak, PhD /1/ -C - Biopesticides and Pollutb<~ention Di · ·on (7511P) 

Shannon Borges "" \. "'-

Microbial Pesticides Branch ~ ~ Biopesticides and Pollution Prevention Division (7511 P) 

ENVIRONMENTAL ASSESSMENT 

A. Introduction 

On behalf of the applicant (University of Florida, Institute of food and Agricultural Sciences 

(UF/IFAS)) the USDA/IR-4 has applied 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 of papaya to improve plant resistance to Papaya Ringspot Virus 

(PRSV), through biotechnological and molecular methodologies. This PIP has been designated 
as OECD Unique Identifier UFL-X17CP-6. The applicant submitted scientific rationale, based 

on qualitative information from peer-reviewed literature and regulatory decision documents to 

satisfy the microbial pesticide test data requirements as published in 40 CFR § 158.2150. The 

provided infonnation in addition to previous tolerance exemptions for other viral coat proteins 

(promoting RNAi protection) and minutes from FIFRA Scientific Advisory Panel meetings 

concerning plant viral coat proteins are sufficient to satisfy all Tier I nontarget organism data 

requirements and to support an environmental risk assessment for this new active ingredient. 

B. BACKGROUND 

In 1997, EPA granted an exemption from the requirement of a tolerance for the coat protein 

produced by genetically modified papaya VCP (55-I papaya, "Sun Up" variety) developed for 

Hawaii (U.S. EPA, 1997). The rationale for the tolerance was the documented occurrence of the 

PRSV in food, lack of any indication of mammalian toxicity with its current consumption and 

the published scientific literature indicating lack of mammalian pathogenicity for plant viruses. 

The UF/IFAS (applicant for papaya Event X 17-2) reported that the current papaya VCP insert 

for the Florida hybrid had 96% amino acid homology to the Hawaiian papaya ringspot isolate 

(MRID 494932-01 ). 

Resistance to the ringspot virus is conferred through posttranscriptional gene silencing, a type of 

RNA-mediated protection (Gonsalves 1998). The amino acid homology between Xl7-2 and its 

original source (HI K strain of PRSV-P, MRID 494932-01) was reported to be 99.7%. 

In 2004 and 2005, US EPA held FIFRA Scientific Advisory Panel (SAP) meetings to discuss 

" issues associated with deployment of a type of Plant-Incorporated Protectant (PIP), specifically 

those based on Plant Viral Coat Proteins (PVCP-PIP)" and to provide recommendations to the 

Agency on gene flow, weediness, viral interactions, dietary exposure to humans, and risk to 

wildlife. 

• Addressing concerns regarding gene flow and weediness, the SAP concluded that 

primarily fruit and vegetable crops species were not expected to form viable hybrids with 

wild relatives in the US and its territories. Even though introgression should happen 

infrequently, and the consequences of introgression could be controlled overridingly by 

genes linked with the transgene, there was a slight chance that a boost in tolerance to a 

wild plant could be ecologically significant. But based on knowledge obtained from 

observation of cultivated crops in the agroecosystem, the majority of the Panel concluded 

that it would be unlikely that a plant population freed from viral pressure would give a 

plant species a competitive advantage. The Panel was not aware of any study that had 

purposefully freed a weed species of a known virus and determined its competitive ability 

or looked at the population dynamics of virus infected versus uninfected plants in 

multiple species ecosystems. 

• With respect to dietary concerns for humans, the Panel agreed that because of the human 

history of consuming virus infected food, unaltered PVCPs do not present new dietary 

exposures. There was no clear consensus on how much change would be necessary to 

invalidate this assumption, although there was general agreement that the appropriate 

comparison was to the range of natural variation in the virus population. 

• Concerns for non-target species were addressed as follows: the Panel agreed that virusprotected crops should not be as attractive to aphids as diseased crops and thus may 

2 
reduce the numbers and types of organisms that spend at least part of their life cycles in 

spcci fie agroecosystems. Because of the transient nature of agroecosystems, a reduction 

of aphids over larger scales should be negligible. The Panel was not aware of any risk 

posed by PVCP-PIPs crops to wildlife. 

On September I, 2009, the Animal Plant Health and Inspection Service (=APH IS, U.S. 

Department of Agriculture) published a notice in the Federal Register to inform the public that 

transformation Event Xl7-2 papaya was no longer considered a regulated article under its 

regulations. This determination was based on evaluation of data submitted by the IF AS of the 

University of Florida, analysis of scientific data from the open literature, APH IS' response to 

comments received from the public on the petition for deregulated status for this new event and 

associated environmental assessment for a Finding ofNo Significant Impact (USDA, 2009). 

EPA has granted tolerance exemptions for other viral coat proteins which promote RNAi (posttranscriptional gene-silencing) in plants; these include Zucchini Yellow Mosaic, Watermelon 

Mosaic-2, and Cucumber Mosaic Viruses as expressed in squash (Cucurbita pepo) plants, line 

ZW -20 and various melons. These viral coat proteins were granted tolerance exemptions based 

on rationale of the ubiquity of plant viruses in the agricultural environment, lack of any 

indication of mammalian toxicity with current dietary consumption of the entire virus including 

the plant viral coat protein, and lack of mammalian pathogenicity for plant viruses [ 40 CFR 

147.514 and 40 CFR 174.515, respectively]. Additionally, the presence of plant viruses, and 

therefore viral coat proteins, is higher in naturally infected crops than in the transgenic lines of 

papaya. 

C. CONCLUSIONS AND RECOMMENDATIONS 

BPPD concludes that the PIP a.i., PRSVcp produced by papaya that have been transformed with 

Event X l 7-2 does not produce any new, or incremental environmental exposures of concern 

beyond those of papaya trees or fruit naturally infected by Papaya Ringspot virus (PRSV) 

populations in Florida. The evidence from the regulatory records (EPA, 1997; EPA, 2007), 

documented minutes of FIFRA Scientific Advisory Panels (SAP reports of 2000, 2004, and 

2006), information from the cited literature and from submitted scientific rationale justify no 

additional nontarget organism testing as recommended in the 40 CFR 158.2 150 series of 

microbial pesticide data requirements. A detailed data evaluation record (DER) of the submitted 

information is appended to this memorandum. 

D. NON-TARGET ORGANISM RISK ASSESSMENT PROCESS FOR PLANTINCORPORATED PROTECT ANTS (PIPs) 

The Series 885 guidelines apply to microbes and microbial toxins when used as pesticides (Table 

I; 40 CFR 158.2150 including those that are naturally occurring, and those that are strainimproved, either by natural selection or by deliberate genetic manipulation. Therefore PIPs that 

have microbial genetic material deliberately introduced into plants are also covered by these 

testing guidelines. 

The EPA believes that this tiered paradigm to testing of non-target organisms ensures, to the 

greatest extent possible, that a minimum amount of data would be required to make scientifically 

sound regulatory decisions related to the use of plant incorporated pesticidal substances and for 

3 
identifying negative results with a reasonable degree of confidence. The Agency expects that 

scientific consideration of Tier 1 data requirements is sufficient to support non-target organism 

risk assessment for PIP registrations (EPA-SAP, 2000). 

The fate of pesticidal substances in the environment is a critical factor in the Agency's risk 

assessment for all PIPs. The Agency uses environmental fate data to determine potential for 

adverse impacts of a PIP, such as PRSVcp entering and persisting in the environment. 

Therefore, EPA considers gene flow, potential invasiveness of the plant expressing the PIP, and 

the potential environmental impacts when evaluating PIPs. 

Table I. Current Non-Target Organisms and Environmental Fate Tier I Data Requirements for 

Evaluation of the Papaya active ingredient, PRSVcp 

Avian Oral Toxicity 

Avian Inhalation Toxicity/ Pathogenicity 

Wild Mammal Toxicity/Pathogenicity 

Freshwater Fish Toxicity/Pathogenicity 

Freshwater Invertebrate Toxicity/Pathogenicity 

Estuarine and Marine Animal Toxicity and Pathogenicity 

Non-Target Plant Testing 

Non-Target Insect Testing 

OSCPP 885.4050 

OSCPP 885.4100 

OSCPP 885.4 150 

OSCPP 885.4200 

OSCPP 885.4240 

OSCPP 885.4280 

OSCPP 885.4300 

OSCPP 885.4340 

Honey Bee Testing OSCPP 885.4380 

Gene Flow 

Weediness Potential 

No published guideline; EPA-SAP 2004, and 2006 

No published guideline; EPA-SAP 2004, and 2006 

1. Rationale and Justification 

The applicant has submitted scientific rationale and justifications that these data requirements 

could be addressed by a qualitative summary of infonnation cited from the open literature and 

other regulatory decision documents to support registration of the PIP a.i., PRSVcp (and its 

associative genetic material) for Event X 17-2 papaya. ln addition, EPA reviewers have cited 

previous documentation from proposed rules and meetings with scientific advisory panels (SAP) 

as relevant to assessing risks of a PIP that is developed with plant viral coat protein. Furthermore 

BPPD believes that data from guideline tests would not provide additional information for 

regulatory assessment of the active ingredient, a viral coat protein of papaya ringspot virus Strain 

HI K (USDA, 2009). Hereafter the active ingredient will be referred to as PRSVcp; the pesticidal 

active ingredient includes other genetic material (CaMV promoter genes from cauliflower 

mosaic virus and npt marker genes) (40 CFR 174.3) 

a. Non-Target Plants 

ln seven years of evaluation of the transgenic papaya expressing the PIP, PRSVcp, there were no 

differences of disease or insect susceptibility and resistance between the transgenic papaya and 

non-transgenic papaya in Florida, and there was no evidence that the transformation trait would 

have any impact on the potential as a weed, if the PRSVcp escaped into "[w]ild" papaya. 

4 
The PRSVcp is produced by transgenic papaya Event X l7-2, which relies on a mechanism of 

RNA gene silencing for expressing resistance to PRSV. Papaya Ringspot Virus cp production 

from papaya transfonned by Event X17-2 does not produce any new environmental exposures 

compared with PRSV-infected fruit (USDA, 2009). 

b. Other Non-Target Organisms 

A vi an Oral Toxicity and Avian Inhalation Toxicity/Pathogenicity 

There is a long history of consumption of the plant virus particles (i.e., papaya ringspot) in food, 

without causing deleterious effects or evidence of toxicity; virus-infected plants are part of the 

avian diet, and there have been no findings which indicate that plant viruses are capable of 

replication in man1mals or other ve11ebrates, thus eliminating the possibility of avian infection 

Furthennore, infectious particles are not fonned from the transgenic papaya, whi ch has been 

modified with the portion of the genome coding for resistance to Papaya Ringspot virus and 

subcomponents of the resistance gene. The PRSVcp is produced by transgenic papaya Event 

X 17-2, which relies on a mechanism of RNA gene silencing for expressing resistance to PRSV. 

Since this gene is expressed only within plant tissues, exposures are expected to be minimal, and 

are not likely to impact birds or insects that serve as a food source for birds; as the genetic 

material is incorporated into papaya plant tissue to overcome PRSV disease, and it is not 

atomized to create inhalable particles to birds. 

Wild Mammal Toxicity/Pathogenicity 

There is a long history of consumption of the plant virus particles (i.e., papaya ringspot) in food, 

without causing deleterious effects or evidence of toxicity; virus-infected plants are part of the 

mammalian diet, and there have been no findings which indicate that plant viruses are capable of 

repli cation in mammals or other vertebrates, thus eliminating the possibility of wild mammal 

infection (USDA, 2009). Transgenic Event X 17-2 papaya does not represent a source of novel 

potenti al allergenic or anti-nutrient proteins. 

Infectious particles are not fo1med from the transgenic papaya, which has been modified with the 

portion of the genome coding for resistance to Papaya Ringspot virus and subcomponents of the 

resistance gene. The PRSV coat protein is produced by papaya that has been transformed with 

Event X 17-2 whi ch relies on a mechanism of RNA gene silencing for expressing resistance to 

PRSV. 

No deaths or abnonnalities were reported in five week-old albino rats (I 0 males and 10 females 

per treatment group) when fed papaya fruit powder from transgeni c (3 strains) and one nontransgenic control at a daily dose of2 g frui t powder/kg body weight/day for 28 days (Lin, et al., 

2013). In addition, there were no significant differences of food consumption, feed efficiency 

and body weights among all treated groups. 

Freshwater Fish, Invertebrate and Estuarine/Marine Animal Toxicity/Pathogenicity 

The applicant asserts that data are not needed because there is no expected exposure to fish, 

estuarine and/or marine animals in water. Since the gene is only expressed within plant tissues, 

5 
the occurrence would be minimal to non-existent. PRSYcp production from Event 17-2 papaya 

does not produce any new environmental exposures compared with PRSY-infected fruit. 

Off-site movement of transgenic papaya tissues (e.g., primarily seed or fruit) into aquatic 

ecosystems could result from agriculturaVhorticultural operations (e.g., planting) and weather 

activity (e.g., hurricanes, tropical storms uprooting the papaya tree) and animal dispersal of 

partially-consumed fruit; however there is uncertainty in regards to the significance of these 

exposures. Nevertheless, no adverse effects to aquatic organisms are anticipated from the viral 

coat protein of papaya ringspot virus used for the PIP, because no infectious particles are formed 

from the transgenic papaya and the mode of action is specifically for expressing resistance 

through a gene sil encing mechanism to PRSV, a plant virus specific to papaya. 

Non-Target Insect Toxicity and Pathogenicity 

The applicant asserts that there is no expected exposure to non-target insects, because the gene is 

only expressed within plant tissues, occurrence would be minimal to non-existent. "[L]evels of 

detectable PRSY cp do not increase the issue of potential impacts to non-target organisms (i.e., 

benefi cial insects such as the ladybird beetle, green lacewing, parasitic wasp) as the PRSV coat 

protein is not known to have any toxic properties and is expressed at a very low level when 

compared to papayas infected with PRSV" (USDA, 2009). 

As noted above, BPPD believes that nontarget insects could be exposed to transgenic papaya 

tissues which contain PRSYcp. Dispersal and movement of transgeni c papaya tissues (e.g., 

primarily seed or fruit) could result from agricultural/horti cultural operations (e.g., planting) and 

weather activity (e.g., hurricanes, tropical storms uprooting the papaya tree) and animal dispersal 

of partially-consumed fruit; however there is uncertainty in regards to the significance of these 

exposures to nontarget insects. Viral coat proteins are routinely ingested by virtually all 

organisms, including humans, when virus-infected fruits and vegetables are consumed. Thus, 

because of the ubiquitous nature of plant viruses and the likelihood of exposures, impact(s) to 

non-target organisms, including beneficial insects, is virtually non-existent (EPA, 1997; EPA, 

2007; USDA/APH IS, 2009). 

BPPD reviewers note that the virus is specific to only the papaya plant, and nontarget insect 

pests such as aphids, two-spotted spider mites and papaya fruit fly are not adversely affected by 

the expression of the PRSVcp (USDA/APHIS, 2009). Because no infectious particles are 

formed from the transgenic papaya and the mode of action is specifically for expressing 

resistance through a gene silencing mechanism to PRSY, no adverse effects are anticipated to 

nontarget insects, regardless of the exposure. 

Honey Bee 

The applicant asserts that there is no expected exposure to honeybee, because the gene is only 

expressed within plant tissues; therefore, occurrence of exposure would be minimal to nonexistent. Levels of detectable PRSV cp do not increase the issue of potential impacts to non-target 

organisms, such as the honeybee as the PRSV cp is not known to have any toxic properties and is 

expressed at very low levels when compared to papayas infected with PRSV" [Braverman, et al., 

20 14, MRID 494932-03]. 

6 
In addition, viral coat proteins are routinely ingested by virtually all organisms, including 

humans, when virus-infected fruits and vegetabl es are consumed. Thus, because of the 

ubiquitous nature of pl ant viruses and the likelihood of previous exposure, the likelihood of 

impact to non-target organisms, including the honeybee, is virtually non-existent (EPA, 1997; 

EPA, 2007; USDA/APH IS, 2009). 

BPPD reviewers note that the virus is specific to only the papaya plant, and nontarget insect 

pests such as aphids, two-spotted spider mites and papaya fruit fly are not adversely affected by 

the expression ofthe PRSVcp (USDA/APHIS, 2009). Because no infectious particles are 

formed from the transgenic papaya and the mode of action is specifically for expressing 

resistance through a gene silencing mechanism to PRSV, no adverse effects are anticipated to 

honeybees, regardless of the exposure. 

E. Ecological Exposure and Environmental Risk Characterization 

l. Terrestrial Animals and Plants 

Birds and Mammals 

Birds and mammals would be exposed to PRSVcp primarily through ingestion of tissues, seed or 

fruits of transgenic papaya X17-2, or from prey that had ingested transgenic papaya. Contact 

with these surfaces is not expected to result in signifi cant exposure, though oral exposure may 

occur incidentally through grooming. The submitted data and information are sufficient to 

conclude that PRSVcp is unlikely to infect birds or wild mammals and cause adverse effects. 

Nontarget Insects and Honev Bees 

Nontarget insects and honeybees would be exposed to PRSVcp primarily through ingestion of 

tissues, fruits or pollen of transgenic papaya X 17-2, or from prey that had ingested transgenic 

papaya. Contact with these surfaces is not expected to result in significant exposure, though oral 

exposure may occur incidentally through grooming. The submitted data and information are 

sufficient to conclude that PRSVcp is unlikely to infect nontarget insects and honeybees and 

cause adverse effects. 

Nontarget Plants 

For hazard evaluation of a plant incorporated pesticidal substance that is produced from a plant 

pathogenic microorganism, the Agency is concerned that its use(s) would not predispose other 

nontarget plants, and non-transgenic papaya to other diseases or pests that might trigger adverse 

effects. In addition, continued expression of the pesticidal substance in transgenic papaya may 

trigger potential for off-site movement (gene flow) and introgression into non-transgenic, wild or 

"feral" papaya populations. Risk considerations for gene flow and invasiveness are discussed 

further below in Section 3. Environmental Fate, Persistence and Risk Assessment. 

The Agency does not anticipate adverse effects to nontarget plants outside of the papaya species 

(Carica papaya); and anti cipates minimal risks to wild or "feral" papaya that may be present in 

the subtropical U.S. Information from the open literature was evaluated to determine that it is 

unlikely that PRSVcp when incorporated into papaya (i.e., Event Xl7-2 transgenic papaya) will 

produce any new, or incremental environmental exposures of concern beyond those of papaya 

trees or fruit infected by the natural populations of papaya ringspot virus (PRSV) populations. 

7 
2. Aquatic Animals and Plants 

Freshwater Fish and Invertebrates 

Based on the infonnation cited from the literature, which is discussed above, risks to freshwater 

fish and invertebrates are not anticipated as a result of the proposed uses of PRSYcp in papaya 

Event X 17-2. 

Marine/Estuarine Fish and Invertebrates 

Based on the information cited from the literature, risks to marine/estuarine fish and 

invertebrates are not anticipated as a result of the proposed uses of PRSY cp in papaya Event 

X 17-2, as the product is not intended for direct application into estuarine or marine environments 

and the pesticidal mode of action is specificall y for expressing resistance through a gene 

silencing mechanism to PRSY, a plant virus specifi c to papaya. 

Aquatic Plants 

As discussed above, the proposed use ofPRSVcp in papaya Event X l7-2 is specific for 

transgenic papaya to control a viral disease. Also, the product is not intended for direct 

application into estuarine or marine environments, and the pesticidal mode of action is 

specificall y for expressing resistance through a gene silencing mechanism to PRSY, a plant virus 

specific to papaya. Therefore, adverse effects to freshwater and marine or estuarine plants are not 

anticipated. 

3. Environmental Fate, Persistence and Risk Assessment 

A. Gene Flow 

For PIP registration actions, the US EPA also assesses the likelihood and effects of gene flow 

and introgression from the PIP plant to sexually compatible wild relatives (SCWR) in the US and 

its territories. Registrants are, therefore, asked to supply a gene flow risk assessment that 

discusses the possibility of pollen flow to compatible pl ants and hybridization, introgression, and 

stable inheritance. 

If EPA's risk analysis concludes that a proposed PIP trait has the potential to confer a selective 

advantage to wild and feral relatives by pollen transfer leading to hybridization, introgression, 

and stable inheritance, then proactive measures must be taken to mitigate identified impacts upon 

registration of such a PIP. 

The applicant (UFIIFAS) of the proposed PIP (a coat protein of papaya ringspot virus produced 

from transgenic papaya Event X17-2) intends to release this genetically modified papaya hybrid 

for commercialization exclusively in Florida. No closely related wild and feral relatives coexist 

in this geographic region (USDA 2009), and risk from gene flow and introgression is, therefore, 

highly unlikely. The closest related relative is the highland papaya ( Vasconcellea spp.). which is 

native to Central and South America (USDA 2009). If Carica papaya L. were commercialized in 

an area where Vasconella species co-occurred, the risk of hybridization from gene flow would be 

low. This statement is supported by hybridization attempts between Carica and Vasconelfa 

species that have failed to produce intergeneric hybrids (OECD 2005). In addition, the FIFRA 

8 
SAP (2004, 2005) concluded that papaya would not form a viable hybrid with wild relatives in 

the U.S. and its territories. 

BPPD concludes that the risk of gene flow via pollen transfer from the PIP X 17-2 plants 

resulting in hybridization of a SCWR is very unlikely in the United States and its territories but 

especiall y in the proposed geographic region of commercialization (Florida). 

B. Invasiveness 

A PIP plant expressing a protein conferring disease or pest resistance has the potential to incur a 

competitive advantage over other plant species with which it interacts and shares an environment 

(SAP 2004). Therefore, as part ofthe registration request, EPA requires applicants to discuss the 

potential for a PIP plant to become invasive in the environment. Typically, applicants are also 

asked to submit information addressing the li kelihood that a SCWR can become a weed if the 

transgene introgresses. PIP pollen flow to SCWR has the potential to confer a selective 

advantage to wild and feral relatives if hybridization, introgression, and stable inheritance are 

possible. 

The University of Florida demonstrated that there were no significant differences in phenology 

between genetically engineered Xl 7-2 papaya and non-GMO C. papaya plants (USDA, 2009). 

The lack of detectable significance in plant vigor did not support that Xl7-2 papaya would be 

weedier in nature than its non-GMO counterpart. Therefore, BPPD concludes that the likelihood 

for X 17-2 papaya to become invasive is low in the United States where its commercialization 

would be limited to Florida. 

BPPD further concludes since there is no evidence that C. papaya has 'Yild or feral relatives in 

the United States and its territory with which hybridization could occur, that at the present time 

the concern for the VCP transgene to confer a selective advantage of invasiveness to SCWR in 

the United States is not substantiated. 

4. Threatened and Endanger ed Species Assessment 

EPA has determined that no effects are anticipated for nontarget species exposed to PRSV cp in 

papaya X 17-2 including mammals, birds, terrestrial and aquatic plants, freshwater and 

marine/estuarine animals and insects. Therefore, a ''No Effect" detennination is made for direct 

and indirect effects to federally listed endangered and threatened ("listed") species and for their 

designated critical habitat. 

BIBLIOGRAPHY 

I. Data Submission 

Braverman, M., W. P. Barney, K. D. Coleman. 2014. Nontarget waivers for Xl7-2 Line 

oftransgenic Papaya Ringspot-Resistant Papaya. Non-published report submitted to the 

Agency, MRID 494932-03. 

9 
Braverman, M., W. P. Barney, K. D. Coleman. 2014. Group A-product analysis test 

guidelines. Non-published report submitted to the Agency, MRID 494932-01 

Lin, H-T., G-C.Yen, T-T.Huang, L-F.Chan, Y-H, Cheng, J-H. Wu, S-D.Yeh, 

S-Y.Wang, and J-W.Liao. 2013. Toxicity assessment of transgenic papaya ringspot virus 

of 823-22 10 Line Papaya fruits. J. of Agricultural and Food Chemistry 61: 1585-1596. 

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-pesticides. SAP Report No. 99-06A for FIFRA Scientific Advisory Panel Meeting 

held December 8, 1999, held at the Sheraton Crystal City Hotel, Arlington, VA 

EPA-SAP. December 27, 2004. Issues associated with deployment of a type of plantincorporated protectant (PIP), specifically those based on plant viral coat proteins (PVCP-PIPs). 

SAP Report No. 2004-09 for FIFRA Scientific Advisory Panel Meeting held on October 13-15 

2004 at the Holiday Inn - National Airport, Arlington, VA. 

U.S. EPA-SAP. February 28,2006. A set of scientific issues being considered by the 

Environmental Protection Agency regarding: Plant-incorporated protectants based on virus coat 

protein genes: Science issues associated with the proposed rule. SAP Report No. 2006-1 for 

FIFRA Scientific Advisory Panel Meeting held on December 6-8, 2005 at the Holiday Inn -

National Airport, Arlington, VA. 

U.S. EPA 1997. Coat Protein of Papaya Ringspot Virus and the Genetic Material Necessary 

for its Production; Exemption From the Requirement of a Tolerance. 62 FR 44572-44575. 

Gonsalves, D. 1998. Control of Papaya ringspot virus in papaya. Ann. Rev. Phytopath., Vo1.36: 

415-437. 

OECD. 2005. Consensus Document on the Biology of Papaya (Carica papaya). 

10 
Nontarget Organism Testing- Review of Literature To Support Data Requirements (40 

CFR 158.2150; OSCPP Guidelines 885.4000 series 

Viral Coat Protein from Papaya Ringspot Virus; X17-2 Transgenic Line of Papaya (PC 

Code 006701)/EPA File Symboi84427-R-Xl7-2 

Submission No. 960423/ Decision No. 497668/ DP Barcode: DP 424881 

Primary Reviewer: Gail Tomimatsu, Ph.D. ~ 

Secondary Reviewer: Shannon Borges, LeaJ Biologi ~ NOV 0 5 2015 

EXECUTIVE SUMMARY: On behalf of the applicant, the University of FL/lnstitute of Food 

and Agricultural Sciences (Company No. 84427), USDA-IR-4 submitted qualitative summary 

rationale supported by information from peer-reviewed scientific li terature and the 

USDA/APHIS Petition for Non-Regulated Status and Finding ofNo Significant Impact [I] to 

support the nontarget organisms data requirements for the ecological risk assessment of the plant 

incorporated protectant (PIP), a viral coat protein (cp)from papaya ringspot virus (PRSV) 

produced by Event X17-2 transgenic line of papaya (OECD Unique Identifier UFL-Xl7CP-6). 

The weight-of-evidence in the submitted rationale and the cited literature do not justify 

additional nontarget organism testing as recommended in the OCSPP Test Guidelines 885.4000 

series of microbial pesticide data requirements. The USDA-IR-4 submitted rationale supported 

by relevant citations from the open scientifi c literature to conclude that the PRSV coat protein 

that is produced by X 17-2 transgenic papaya does not produce any new, or environmental 

exposures of concern beyond those of papaya trees or fruit naturally infected by the Papaya 

Ringspot virus (PRSV) populations in FL. Notabl y, the PRSY gene used to engineer X 17-2 is 

from a Florida isolate of the virus (H l K, ll J) and reports have shown that similar gene constructs 

give resistance to the disease only from highly similar viral strains (OECD 2005). 

In addition, there are no known adverse effects for the use of a PRSV -resistant transgenic papaya 

in HI, which is also based on a PRSV coat protein, and genetically engineered from a Hawaiian 

PRSY strain [1]. 

CLASSIFICATION: 

Submitted Rationale and Classification of Individual Rationale: 

I. Avian Oral (OPPTS 885.4050) 

Avian Inhalation Toxicity/Pathogenicity, Tier I (OPPTS 885.41 00) 

The applicant claims that data are not needed because there is a long history of consumption of 

the plant virus particles (i.e., papaya ringspot) in food, without causing deleterious effects or 

evidence of toxicity; virus-infected plants are part of the avian diet, and there have been no 

findings which indicate that plant viruses are capable of replication in mammals or other 

vertebrates, thus eliminating the possibility of avian infection [ 11. Furthermore, infectious 

particles are not formed from the transgenic papaya, which has been modified with the portion of 

the genome coding for resistance to Papaya Ringspot virus and subcomponents of the resistance 

gene. The PRSY coat protein is produced by X 17-2 papaya, which relies on a mechanism of 

RNA gene silencing for expressing resistance to PRSV. Since this gene is expressed only within 

plant tissues, exposures are expected to be minimal, and are not likely to impact birds or insects 
Nontarget Organism Testing - Review of Literature To Support Data Requirements (40 

CFR 158.2150; OSCPP Guidelines 885.4000 series 

Viral Coat Protein from Papaya Ringspot Virus; Xl7-2 Transgenic Line of Papaya (PC 

Code 006701)/EPA File Symboi84427-R-Xl 7-2 

Submission No. 960423/ Decision No. 497668/ DP Ba rcode: DP 424881 

that:Serve as a food source for birds; as the genetic material is incorporated into papaya plant 

tissue to overcome PRSV disease, and it is not atomized to create inhalable particles to birds. 

Classification of Submitted Rationale for Avian Oral and Avian Inhalation Data Requirements: 

Acceptable. 

2. Wild Mammal Testing (OPPTS 885.4150) 

The appli cant claims that data are not needed because there is a long history of consumption of 

the plant virus particles (i.e., papaya ringspot) in food, without causing deleterious effects or 

evidence of toxicity; virus-infected plants are part of the mammalian diet, and there have been no 

findings which indicate that plant viruses are capable of replication in mammals or other 

vertebrates, thus eliminating the possibility of wild mammal infection 11). The X17-2 papaya 

does not represent a source of novel potential allergenic or anti-nutrient proteins. 

No deaths or abnormalities were reported in five week-old albino rats ( I 0 males and l 0 females 

per treatment group) when fed papaya fruit powder from 3 transgenic papaya strains (other than 

X 17-2) and one non-transgenic control at a daily dose of 2 g fruit powder/kg body weight/day for 

28 days 12). In addition, there were no significant differences of food consumption, feed 

efficiency and body weights among all treated groups. 

Infectious particles are not formed from the transgenic papaya, which has been modified with the 

portion of the genome coding for resistance to Papaya Ringspot virus and subcomponents of the 

resistance gene. The PRSV coat protein is produced by X 17-2 papaya, which relies on a 

mechanism of RNA gene silencing for expressing resistance to PRSY. 

Classification of Submitted Rationale for Wild Mammal Data Requirement: Acceptable. 

3. Freshwater Fish Testing, Tier 1 (OPPTS 885.4200) 

Estuarine and Marine Animal Testing, Tier 1 (OPPTS 885.4280) 

The appli cant claims that data are not needed because there is no expected exposure to fish, 

estuarine and/or marine animals in water. Since the gene is only expressed within plant tissues, 

the occurrence would be minimal to non-existent. PRSY cp production from transgenic papaya 

X 17-2 does not produce any new environmental exposures compared with PRSV-infected fruit. 

BPPD reviewers disagree that freshwater, estuarine and marine animals would not be exposed to 

the PIP a.i., (PRSV cp-transformed X17-2 tissues) even minimally. Off-site movement of 

transgenic papaya tissues (e.g., primarily seed or fruit) into aquatic ecosystems could result from 

agricultural/horticultural operations (e.g., planting), weather activity (e.g., hurricanes, tropical 

storms uprooting the papaya tree) and animal dispersal of partially-consumed fruit; however 

there is unee1tainty in regards to the significance of these exposures. Nevertheless, BPPD does 

. 2 
Nontarget Organism Testing- Review of Literature To Support Data Requirements (40 

CFR 158.2150; OSCPP Guidelines 885.4000 series 

Viral Coat Protein from Papaya Ringspot Virus; X17-2 Transgenic Line of Papaya (PC 

Code 006701)/EPA File Symboi84427-R-X17-2 

Submission No. 960423/ Decision No. 497668/ DP Barcode: DP 424881 

not anticipate adverse e ffects to aquatic organisms from exposures to the viral coat protein (cp) 

of papaya ringspot virus and other genetic material (CaMV promoter genes from cauliflower 

mosaic virus and npt marker genes) used for the PIP, because no infectious particles are formed 

from the transgenic papaya and the mode of action is specifically for expressing resistance 

through a gene silencing mechanism to PRSV, a plant virus specific to papaya. 

Classification of Submitted Rationale for Freshwater, Estuarine and Marine Animal Data 

Requirements: Supplemental. Some text in the submitted rationale was not supported by 

relevant reference citations. 

4. Freshwater Invertebrate Testing, Tier 1 (OPPTS 885.4240) 

The applicant claims that data are not needed because there are no expected exposures to 

freshwater invertebrates. Since the gene is only expressed within plant tissues, the "[o]ccurrence 

would be minimal to non-existent" . Exposures to plant tissues that express the pesticidal gene are 

anticipated (see text below), however there is uncertainty with respect to the extent and 

persistence of the expressed genes in planta. Because infectious particles are not fonned from 

the transgenic papaya, which has been modified with the portion of the genome coding for 

resistance to PRS V and subcomponents of the resistance gene, adverse effects to aquatic 

invertebrates are not expected. The PRSV coat protein is produced by transgenic 

papaya X 17-2 which relies on a mechanism of RNA gene silencing for expressing resistance to 

PRSV. Papaya Ringspot Virus cp production from transgenic papaya does not produce any new 

environmental exposures compared with PRSV-infected fruit. 

BPPD reviewers disagree that freshwater invertebrates would not be exposed to the PIP a.i. 

PRSV cp-transformed X 17-2 tissues), even minimall y. Off-site movement of transgenic papaya 

tissues (e.g., primari ly seed or fruit) into aquatic ecosystems could result from 

agricultural/horticultural operations (e.g., planting), or weather activity ( e,g, hurri canes, tropi cal 

storms uprooting the papaya tree), and animal dispersal of raw or consumed fruit; however there 

is uncertainty to the significance of these exposures. Nevertheless, BPPD does not 

anticipateadverse effects to freshwater invertebrates from exposures to the viral coat protein (cp) 

of papaya ringspot virus and other genetic material (CaMV promoter genes from cauliflower 

mosaic virus and npt marker genes) used for the PIP, because no infectious particles are formed 

from the transgenic papaya and the mode of action is specifically for expressing resistance 

through a gene silencing mechanism to PRSV, a plant virus specific to papaya. 

Classification of Submitted Rationale for Freshwater, Estuarine and Marine Animal Data 

Requirements: Supplemental. Some text in the submitted rationale was not supported by 

relevant reference citations. 

3 
Nontarget Organism Testing- Review of Literature To Support Data Requirements (40 

CFR 158.2150; OSCPP G uidelines 885.4000 series 

Vira l Coat Protein from Papaya Ringspot Virus; X17-2 Transgenic Line of Papaya (PC 

Code 006701)/EPA File Symbol84427-R-X17-2 

Submission No. 960423/ Decision No. 497668/ DP Barcode: DP 424881 

5. Nontarget Plant Studies, Tier I (OPPTS 885.4300) 

The applicant claims that data are not needed based on several lines of evidence: 

a) "(N]o differences in susceptibility or resistance to other insect pest (two-spotted spider mites 

and papaya fruit fly) or pathogen (anthracnose and powdery mildew) populations compared to 

control non-transgenic plants in these trials conducted on X 17-2 papaya for 7 years in FL [1]. 

The applicant did not address the rationale for why the cited text is relevant. Based on these 

observations, the reviewer opines that there would likely be no signifi cant effect on 

predisposition of nontransgenic papaya to the organisms; and minimal synergy of the PRSV cp 

and the cited organisms. 

b) The cp of PRSV in transgenic papayas may have "[l]ittle influence on soil microorganisms, 

due to low levels or has not been secreted from roots in to soil", and therefore, plants obtaining 

nutrients from the surrounding soil will not be affected [31. The applicant did not address the 

rationale for how the cited text would be relevant towards assessing potential hazards of the viral 

coat protein to nontarget papaya (e.g., organic papaya) or to other nontarget plants in the vicinity 

of plantings of FL X 17-2. The reviewer noted that results of this limited study (conducted on 

transgenic Taiwan papayas) indicated that there was an overall similarity (>80%) of soil 

microorganisms in Taiwan soil profiles of transgenic papaya (with the PRSV cp) and 

nontransgenic papaya. Because these results were extrapolated from results of molecular 

methodologies used to determine microbial "profiles", and that additional experimentation is 

necessary to confirm the reported observations, this part of the rationale is not relevant to 

assessing hazards of the viral coat protein to nontarget plants, including non-transformed papaya. 

c) There is no evidence that the Xl7-2 trait would have any impact on the potential as a weed if 

the PRSV cp escaped into "(w]ild" papaya. The applicant did not provide text from a reference 

citation to support this statement. The reviewer notes that no closely related wild and feral 

relatives coexist in this geographic region [1 ], and risk from gene flow and introgression is, 

therefore, highly unlikely. BPPD concludes that the risk of gene t1ow via pollen transfer from the 

PIP X 17-2 plants resulting in hybridization of a sexually compatible wild relative (SCWR) is 

very unlikely in the United States and its territories but especially in the proposed geographic 

region of commercialization (Florida). 

d) USDA-APHIS was unable to identify any characteristics of X 17-2 that might indicate an 

increased propensity to become weedy or invasive. Feral populations of papaya may exist in 

some locations, however hybridizations with X 17-2 will not I ikely result in production of plants 

with weedy or invasive characteristics [1]. 

e) The PRSV coat protein is produced by Xl7-2 papaya, which relies on a mechanism ofRNA 

gene silencing for expressing resistance to PRSV. Papaya Ringspot Virus cp production from 

X 17-2 papaya does not produce any new environmental exposures compared with PRSVinfected fruit 111 . 

4 
Nonta rget Organism Testing - Review of Litera ture T o Support Data Requirements (40 

CFR 158.2150; OSCPP Guidelines 885.4000 series 

Viral Coa t Protein from Papaya Ringspot Virus; X17-2 Transgenic Line of Papaya (PC 

Code 006701)/EPA File Symbol84427-R-X17-2 

Submission No. 960423/ Decision No. 497668/ DP Barcode: DP 424881 

Classification of Submitted Rationale for Nontarget Plant Data Requirements: Acceptable. 

Although there was no explanation of why some of the submitted text were relevant for the data 

requirement, and some text in the submitted rationale was not supported by relevant reference 

citations, the information provided in the study document is sufficient to conclude that no 

unreasonable, incremental risks are anticipated for nontarget plants, including papaya for organic 

production. 

6. Nontarget Insect Studies, Tier I (OPPTS 885.4340) 

The applicant claims that data are not needed based on several lines of evidence: 

There is no expected exposure to non-target insects. Since the gene is only expressed within 

plant tissues, occurrence would be minimal to non-existent. "Xl7-2 papaya does express 

detectable PRSV [1), but at very low levels compared to papayas infected with PRSV. {These 

levels} do not increase the issue of potential impacts to non-target organisms (i.e., beneficial 

insects such as the ladybird beetl e, green lacewing, parasitic wasp and honeybee) as the PRSV 

coat protein is not known to have any toxic properties [1). 

The applicant did not provide text from a reference citation to support this statement. The 

reviewer does not agree that nontarget insects, e.g., aphids, other sucking insects would not be 

exposed to the PIP a.i., (PRSV cp) even minimally. Aphids spread PRSV in a non-persistent 

manner, the viruses are rapidly acquired from infected plants and can be transmitted to other 

(healthy) papaya within minutes [1]. Furthennore, the applicant noted that the transgenic PRSVresistant cultivar Rainbow (HI transgenic papaya) is susceptible to both the leafhopper and mites, 

but did not provide justification why the cited text is relevant for the nontarget insect data 

requirement. 

The reviewer notes that the PRSV (a plant-pathogenic virus) is pathogeni c to only the papaya 

plant, and insects such as aphids are not adversely affected. Compared to non-transgenic plants, 

there was no change in susceptibility or resistance to two-spotted spider mites and the papaya 

fruit fly in the transgenic papaya plants ( 1). 

In addition, viral coat proteins are routinely ingested by virtually all organisms, including 

humans, when virus-infected fruits and vegetables are consumed. Thus, because of the 

ubiquitous nature ofplant viruses and the likelihood of previous exposure, the likelihood of 

impact to non-target organisms, including benefi cial insects, is virtuall y non-existent [ 1, 5). 

Classification of Submitted Rationale for Nontarget Insect Data Requirements: Supplemental. 

Some text in the submitted rationale was not supported by relevant reference citations. 

5 
Nontarget Organism Testing - Review of Litera ture To Support Data Requirements (40 

CFR 158.2150; OSCPP Guidelines 885.4000 series 

Viral Coat Protein from Papaya Ringspot Virus; Xl 7-2 T ransgenic Line of Papaya (PC 

Code 00670 1)/EPA File Symbol84427-R-X17-2 

Submission No. 960423/ Decision No. 497668/ DP Ba rcode: DP 424881 

7. Honeybee Studies, Tier 1 (OPPTS 885.4380) 

The applicant claims that data are not needed based on several lines of evidence as presented 

above for Item 6, Nontarget Insect Studies, and cited text from the open literature: 

a) Morandin and Winston, 2005 study [6]: One interpretation of this study is that there was a 

similar ' pollinator force' in all "field types" based on observations of"relatively equal number of 

seeds per silique for open-pollinated flowers in organic, conventional, and GM fields. The 

purpose of the cited study was to observe differences between potential and actual pollination 

(i.e., poll ination deficits) and bee abundance in organi c, conventional and herbicide-resistant, 

genetically modified canota fields. The applicant did not explain why the cited text is relevant to 

justify why there would be no adverse effects of PRSV cp to honeybees, if a properly designed 

guideline test were conducted. BPPD concludes that these uses and exposures are substantially 

different than those of a plant virus coat protein; therefore, the citation is irrelevant and this 

portion of the rationale is Unacceptable. 

b) "X 17-2 papaya does express detectable PRSY Ill, but at very low levels compared to papayas 

infected with PRSY''. {These levels} do not increase the issue of potential impacts to non-target 

organisms (i.e., beneficial insects such as the ladybird beetle, green lacewing, parasitic wasp and 

honeybee) as the PRSV coat protein is not known to have any toxic properties [1]. PRSV cp 

production from X 17-2 papaya does not produce any new environmental exposures compared 

with PRSY-infected fruit, which is not known to have adverse (e)ffects on animals or plants to 

which they are exposed. In addition, viral coat proteins are routinely ingested by virtually all 

organisms, including humans, when virus-infected fruits and vegetables are consumed. Thus, 

because of the ubiquitous nature of plant viruses and the likelihood of previous exposure, the 

likelihood of impact to non-target organisms, including the honeybee, is vi1tually non-existent 

[1 , 5, 61 

Classification of Submitted Rationale for Nontarget Insect Data Requirements: Supplemental. 

Some text in the submitted rationale was not supported by relevant reference citations or 

considered irrelevant. The reviewer notes that the PRSV (a plant-pathogenic virus) is pathogenic 

to only the papaya plant, and honeybees are not anticipated to be adversely affected. 

References and Literature Cited: 

I. USDA-APHIS. 2009. Draft Environmental Assessment, Finding of No Significant Impact. In 

response to Univ. FL Petition 04-337-0IP seeking a Determination of Nonregulated Status for 

X 17-2 Papaya Resistant to Papaya Ringspot Virus. 

2. Lin, H-T., G-C.Yen, T-T.Huang, L-F.Chan, Y-H, Cheng, J-H. Wu, S-D.Yeh, S-Y.Wang, and 

J-W.Liao. 2013. Toxicity assessment of transgenic papaya ringspot virus of 823-2210 Line 

Papaya fruits. J. of Agricultural and Food Chemistry 61: 1585-1596. 

6 
Nonta rget Organism Testing- Review of Literature To Support Data Requirements (40 

CFR 158.2150; OSCPP Guidelines 885.4000 series 

Viral Coat Protein from Papaya Ringspot Virus; X17-2 Transgenic Line of Papaya (PC 

Code 006701)/EPA File Symboi84427-R-X17-2 

Submission No. 960423/ Decision No. 497668/ DP Barcode: DP 424881 

3. Hsieh, Y.T. and T.M.Pan. 2006. Influence of planting papaya ringspot virus resistant 

transgenic papaya on soil microbial biodiversity. J .Agric. Food Chem.54: 130-137. 

4. OECD. 2005. Consensus Document on the Biology of Papaya (Carica papaya). 

5. EPA 1997. Coat Protein of Papaya Ringspot Virus and the Genetic Material Necessary 

for its Production; Exemption From the Requirement of a Tolerance. 62 FR 44572. 

6. Morandin, L.A. and M.L.Winston.2005. Wild bee abundance and seed production in 

conventional, organic and genetically modified canola. Ecological Applications 15(3):87 1-88 1. 

7