## B IOPESTICIDES REGISTRATION ACTION DOCUMENT PROPOSED DECISION



## Coat Protein Gene of Plum Pox Virus

PC Code: 006354

U.S. Environmental Protection Agency Office of Pesticide Programs Biopesticides and Pollution Prevention Divis ionCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

2 TABLE OF CONTENTS BIOPESTICIDE S REGISTRATION ACTION DOCUMENT TEA M 4 GLOSSARY OF ACRONYMS AND ABBREVIATIO NS 5 I. OVERVIEW A . Executive Summary 6 B. Use Profile 8 C. Regulatory Background 8 I I . RISK A SSESSMENT SUMMARIES A . Product Characterization 9 B. Human Health Assessment 10 C. Environmental Effects Assessment 1 1 III . ENVIRONMENTAL JUSTICE 1 2 I V. BENEFITS AND PUBLIC INTEREST FINDING 1 2 V . RISK MANAGEMENT AND PROPOSED REGISTRATION DECISION A. Determination of Eligibility 1 3 B. Proposed Regulatory Decision 1 4 VI. ACTIONS REQUIRED BY THE APPLICANT A. Satisfaction of the Conditions of Registration 1 4 B. Reporting Requirements 1 4 APPENDIX A: Product Characterization I. Manufacturing Process 1 6 A . Inert Ingredients 1 7 B. Active Ingredient 18 II. P lum P ox V irus Resistance and Mode of Action 18 III. Enforcement Analytical Method 19Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

3 APPE NDIX B: Human Health Assessment I. Toxicological Profile 20 A. Data Waivers
– Justification 20 B. Previous Scientific Findings 21 C. Toxicity and Allergenicity Assessment 23 II. Aggregate Exposures 23 III. Cumulative Effe cts 24 IV. Endocrine Disrupt ors 24 APPENDIX C: Environmental Effects Assessment I. Non - Target Species Effects 25 A. Data Waivers
– Justification 25 II. E ndangered Species Consideration 27 BIBLIOGRAPH Y I. Data Subm issions Received and Reviewed by the Agency 2 8 II. EPA Risk Assessment Memoranda 29 III. Other References 29Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

4 BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM

Office of Pesticide Programs Biopesticides and Pollution Prevention Divis ion

Microbial Pesticides Branch Science Reviews Joel V. Gagliardi, Ph.D. Product Characterization , Human Health Assessment, and Environmental Effects Assessment Regulations Sheryl K. Reill y, Ph.D. Chief, Microbial Pesticides Branch Denise Greenway Regulatory Action LeaderCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

5 GLOSSARY OF ACRONYMS AND ABBREVIATIONS APHI S Animal and Plant Health Inspection Service (USDA) BPPD Biopesticides and Pollution Prevention Division CBI Confidential Business Information 40 CFR Title 40 of the Code of Federal Regulations C5 or C5 plum C5 Honey Sweet Plum CPG - PPV Coat Protein Gene of Plum Pox Virus cDNA Copied (or Copy) DNA dsRNA Double
- Stranded RNA cos Cosmid o C Temperature in Centigrade or Celsius Degrees DNA Deox yribonucleic Ac id EPA Environmental Protection Agency (the “Agency”) ELISA Enzy me - Linked Immunosorbent Assay FFDCA Federal Food, Drug, and Cosmetic Act FIFRA Federal Insecticide, Fungicide, and Rodenticide Act FQPA Food Quality Protection Act FR Federal Regis ter g Gram IR4 Interregional Research Project Number 4 kg kilogram L Liter MRID No. Master Record Identificati on Number mg M illigram mL M illiliter μ g Microgram MP M anufacturing
- U se P roduct mRNA Messenger RNA NE No Effect NI OSH Natio nal Institute for Occupational Safety and Health OPP Office of Pesticide Programs OPPTS Office of Prevention, Pesticides, and Toxic Substances PCR Poly merase Chain Reaction PIP Plant
- In corporated Protectant PPV Plum Pox Virus PTGS Post - Transcriptional Gene Silencing PVCP Plant Virus Coat Protein RNA Ribonucleic Acid rRNA Ribosomal RNA TGAI Technical Grade of the Active Ingredient USDA United States Department of AgricultureCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

6 I. OVERVIEW The Environmental Protection Agency (EPA, Agen cy) is providing a 30
- day public comment period before making its final decision to register a pesticide product, C5 Honey Sweet Plum, containing the new plant
- incorporated protectant (PIP), Coat Protein Gene of Plum Pox Virus (CPG
- PPV) . The registration o f this PIP will qualify as a new active ingredient, a new food use, and a first outdoor use under a new policy established by the Agency

on October 1, 2009, intended to inform and provide the public an opportunity to comment on such major registration deci sions for 30 day s before they occur. All comments will be included in the docket (EPA
- HQ OPP - 2008 - 0742) as they

are received. While a final decision on registration is contingent upon review and consideration of public comments, the Agency

believes that , based upon its assessment of the data and information submitted in support of the registration , it is in the best interest of the public and the environment to issue the registration. If the Agency receives comments during the 30 day public comment perio d that change EPA’s initial decision, EPA will address such new information and take appropriate action. A. Executive Summary Plum Pox Virus (PPV) is a plant virus that reduces the quality of stone fruits, and eventuall y renders infected trees to be in capable of producing fruits. First described in Europe in 1915, where it is considered to be the most devastating viral disease of stone fruit, PPV is also present in the United S tates . Current distribution of PPV along the Canadian border and recent outbreaks in New York and Michigan underscore that PPV is becoming endemic despite containment efforts (bulldozing and disposal of infected vegetation, moratoria on the movement/transport of infected plant materials, and control of insect vector s ). PPV is an a gricultural pest that causes significant economic losses to the stone fruit industry, since the primary

effect s of the infection are reduc tion of fruit quality and crop yi eld. Stone fruits (including native or wild Prunus species) that are affected b y PPV include plums, peaches, almonds, nectarines, and sweet and sour cherries . [ http://www.apsnet.org/online/feature/PlumPox/plumpoxfs.pdf ]. V arious other non
-Prunus dicotyledonou s plants have been infected under experimental conditions [ http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/00.057.0.01.054.htm ]. When PPV infects a plant, its genetic material ( a singl e strand of RNA) is inserted into the plant cells . This strand of RNA contains the genes needed to make new virion s. One of these genes codes for the PPV coat protein

– CPG - PPV. The infected cell makes viral coat protein in a similar manner as it produce s its own plant proteins. RNA coding for the CPG - PPV are translated into the amino acid sequences that make up the protein. During virus replication , segments of double
- stranded RNA are produced, but , ultimately , exact copies of the original single stranded virus RNA chromosome are formed and packaged together with the coat proteins into new viruses. S mall segments of double stranded RNA (dsRNA) are formed by reacting host defenses initiat ing a defense mechanism within the host plant , known as post
- tran scriptionalCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

7 gene silencing (PTGS) . During this process, the plant recognizes the viral gene transcription process that produces the dsRNA as abnormal, and blocks the transcription as well as the production of viral proteins and RNA. T his sequence of events results in the development of natural resistance to further PPV infection s, but not before fruit degradation, leaf chlorosi s, and other serious damage caused by the virus has occurred . Uninfected plum trees can be genetically engineered to express the CPG - PPV . But, because the CPG - PPV is responsible for only one component needed for the production of new virions, the se engineered plum tree s cannot produce the virus. The U.S. Department of Agriculture, Agricultural Research Service
- Appalachian Fruit Re search Station (the applicant) has developed a genetically

engineered plum tree, called the C5 Honey Sweet Plum (C5 or C5 plum), which expresses the CPG - PPV and is resistant to PPV infection. To create the C5 plum, the CPG - PPV is first isolated then copied into DNA . The copied DNA is inserted into the plant genome. During the plant's naturally occurring cellular processes, the transgenic CPG
- PPV gene is transcribed. The mRNA copied from the inserted viral coat proteins genes forms abnormal regions of ds R

NA, and the PTGS mechanism recognizes the abnormality and destroy s segments with the same sequence . This process establishes the ability of the plant to respond quickly to a PPV infection, blocking the production of new virions and spread of the disease. When purposely transferred to uninfected plants to make them resistant to PPV infection, the CPG - PPV is classified as a plant
- incorporated protectant (PIP) , and require s registration by the EPA. All of the data requirements for registration of this PIP h ave been satisfied, except for the submission of data required from an independent laboratory validation of the applicant’s anal yt ical method that detect s residues of the PI P in fresh and processed plum commodities for enforcement purposes. Insufficient t ime has elapsed since the requirement was imposed for the applicant to comply . In view of the clear benefits and minimal risks to human health and the environment, EPA has determined that the use of the CPG - PPV in C5 Honey Sweet Plum for the time it would take for the applicant to comply with the requirement would not result in unreasonable adverse effects to the environment , and proposes that a time
- limited registration of one year should be issued under FIFRA Section 3(c)(7)(C).Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

8 B. Use Profile Active Ingredient: Coat Protein Gene of Plum Pox Virus (CP G - PPV) Office of Pesticide Programs (OPP) Chemical Code: 006354 Product Name: C5 Honey Sweet Plum (C5) EPA File Sy

mbol: 11312 - I Patent Number US PP15,154 P2 Applicant/ Manufacturer: U.S. Department of Agriculture, Agricultural Research Service
Appalachian Fruit Research Station 2217 Wiltshire Road, Kearney sville, WV 25430 Ty pe of Pesticide: Plant
- In corporated Protectant (PIP) Use: Stone Fruits and Almond Target Pest: Plum Po x Virus (PPV), a.k.a. Sharka disease C . Regulatory Background On October 29, 2008, EPA published in the Federal Register (73 FR 64325) a Notice of Receipt announcing that Interregional Research Project Number 4 (IR
- 4), Rutgers University , 500 College Rd. East, Suite 201 W, Princeton, NJ, 08540 submitted on behalf of the United States Department of Agriculture, Agricultural Research Service
- Appalachian Fruit Research Station (the applicant) an application to register a pesticide product containing a new active ingredient not included in any currently registered pesticide products. Four favorable comments were received during a 30 day

comment period following the publication of this notice. A petition (7E7231) seeking an exemption from the requirem ent of a tolerance for residues of the C oat Protein of Plum Pox Virus, in or on stone fruit and almond, was filed by I R - 4 on behalf of the United States Department of Agriculture, Agricultural Research Service
- Appalachian Fruit Research Station . The EPA p ublished a Notice of Filing of the petition in the Federal Register on November 14, 2008 (73 FR 67512) and the public was given 30
- day comment period. No comments were received. A final rule establishing the exemption from tolerance is pen ding the Agency ’s approval , the tolerance exemption will be codified under 40 CFR § 174 .Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

9 II. RISK ASSESSMENT SUMMARIES Described below are summaries of EPA’s assessment of the product characterization, human health, and environmental risks from the use of the coat prot ein gene of the Plum Pox Virus ( CPG - PPV ) as a plant
- incorporated protectant. Inits assessment, the EPA relied upon data and other information submitted by the applicant. A more detailed description of the assessments can be found in the Appendices. A . Product Characterization The C5 Honey S weet Plum (C5) is one of a number of clones resulting from research that geneticall y engineered the CPG - PPV into the plum tree, Prunus domestica L. The C5 clone was sele cted for commercial development because of i ts stability and durability

to remain resistant to PPV under a variety of environmental conditions, exposure to different virus strains, and mixtures of virus strains, the absence of coat protein production, and the excellent quality of the fruit. When fi eld trials were conducted, the C5 trees remained sy mptom - free following infection ( either via aphid vectors or by grafting of infected plant tissues onto the trees ) . The genetic inserts into plum tree DNA were accomplished using an Agrobacterium
- mediated transformation, and all aspects of the development of the C5 Honey Sweet Plum trees have been published in peer - reviewed manuscripts (s ee “Application for Determination of Non
- Regulatory Status for C5 (“Honey Sweet”) Plum ( Prunus domestica L.) Resistant to P lum Pox Virus,” (http://www.aphis.usda.gov/brs/aphisdocs/04_26401p.pdf ). As discussed previousl y, the mode of operation postulated for PPV resistance in C5 trees relies on the plant’s natural PTGS defense mechanism. When the C5 DNA is being transcribed into m essenger RNA (mRNA) , the strand carrying the inserted DNA cop y of the viral coat protein gene is also trans cribed into mRNA. Insome areas of the C5 DNA insert in the plant, mirror image copies of the PPV
- coat protein mRNA are produced that naturall y bind each other, forming areas of dsRNA that the plant senses as abnormal and to wh ich plant host defenses react. The plant PTGS response to ds RNA corresponding to the PPV
- coat prote in produces shorter segments (approximately twenty
- three nucleotide base pairs in length) of double
- strand ed RNA , and these segments are used as signals by the cell to initiate an immune
- ty pe response to any matching sequences of dsRNA in the plant . The plant’s PTGS defense mechanism acts quickly to degrade matching dsRNA sequences and remains capable of destroying the viral genome if the plant becomes infected with PPV . Plant host defenses, once activated, also down
- regulate expression of the DNA inserted into C5 so that the plant likely no longer expresses this gene and further transcription in RNA, and hence translation into coat proteins is not expected to occur. For a more comprehensive discussion of the Agency ’s assessment of the product characterization data submitted in support of the registration of the CPG - PPV , refer to APPENDIX A.Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

10 B. Human Health Assessment Human exposure to a variety of natural plant viruses and plant viral proteins is common in the diet , and exposure includes plant virus coat proteins (PVCPs) . Plant viruses are not pathogenic to humans; in fact, a recent study ( Zhang, et al., 2006 ) demonstrated that the human gastrointestinal tract harbor s a wide variety of plant viruses within the intesti nes . EPA reviewed the availab le scientific data and other relevant information submitted by the applicant in support of the registration of the PIP, CPG - PPV , and considered its validity , completeness and reliability , and the relationship of this information to human risk. EPA also co nsidered the available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children. T he CPG - PPV is a PIP that consist s of nucleic acids, and as such, is exempt from the requireme nt of a tolerance (40 CFR 174.507). Although PP V coat protein generated from the PIP itself has not been detected in C5 plums , the Agency still considered whether there would be any dietary risks, especially

toxicity , allergenicity, and anti
- nutrient prop erties in the unlikely event that the coat protein is produced in the fruit. Inits analy sis, EPA considered the safety of dietary exposure to plant viruses and PVCPs, and the following three sections summarize the Agency ’s conclusions.

i. Plants infect ed with plant viruses have always been part of food supply without adverse effects . Virus
- infected food plants have alway s been a part of the human and domestic animal food supply , and components of plant viruses, including coat proteins, are often found in the produce of many typ es of crop s . For example, at the beginning of this century virtually every commercial cultivar of potatoes grown in the United States and Europe was infected with at least one potato virus. Even as ymptomatic plants are often fo und to be infected . A common agricultural practice ( since the 1920s ) involves the intentional inoculation of health y plants with a mild form of a virus in order to prevent infection b y a more virulent form. To date , there have been no reports of adverse health effects in human s or animal s associated with consumption of plant viruses in food. Experiments have shown that viral coat protein levels expressed in plants that are geneticall y engineered to resist a virus infection can be 100 - 1000 times lower in concentration than in plants naturally infected by the virus. In the C5 plum, the re is little to no detectable virus coat protein produced, since the inserted gene initiates the plant’s natural defense mechanism before virus proteins can be manufactured.

ii. Plant viruses are n ot infectious to humans and animals . Any virus/host relationship is characterized by a high degree of specificity . Plant viruses do not infect humans or other vertebrates, and usuall y only infect plants within a certain taxono mic group . In order to replicate, a plant vi rus usuall y relies on an insect or some other mechanicalCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

11 vector to insert its genome through plant cell walls . Plant virus replication depends on either specific virus
- encoded enzy mes , or plant enz ym es, to tran scribe the viral genes, produce the viral components and package them i nto new virus. Human s and other vertebrates lack the corresponding cellular “machinery” to transcribe, translate, and package plant viruses . Thus, it is reasonable to assume that a si ngle component of PPV , the CPG - PPV , will not cause a viral infection or replication in humans and animals.

iii. Plant viruses are not toxic to humans and animals . As stated previousl y, f

ood from crops infected with plant viruses is (and has long been) consumed by human s and animal s without any known toxicity or other adverse effects. Additional evidence of the lack of toxicity by a different route of exposure comes from the common practice of injecting laboratory animals with purified plant virus preparations to generate antibodies used for anal yt ical tests (for example, to identify and confirm plant virus infections), without any

adverse effects on the animals. The Agency

is not aware of any

plant virus coat protein s that have been identified as a human food allergen. To determine whether the PPV coat protein would potentially cause toxicity

or hy persensitivity , its amino acid structure was compared with known food allergens and toxins. N o amino acid segments triggering a concern were identified. The lack of production of PPV coat proteins by the C5 plum, together with reports indicating that there have been no h yp ersensitivity incidents or other adverse effects among researchers handling the trees, fruits and other plant tissues since experiment ati on with C5 plum began in 1992, support the Agency’s conclusion that , although unlikel y to be produced in foods derived from C5 plum , PPV coat protein expressed in the C5 plum is safe for human dietary consumption. For a more comprehensive discussion of the Agency ’s assessment of the data and information concerning human health submitted in support of the registration of the CPG - PPV , refer to APPENDIX B. C . Environmental Effects Assessment EPA considered all possible effects the CPG - PPV might have on mammalian, avian, f ish, terrestrial and aquatic invertebrate, and plant non - target species. Since the PIP consists solely of a viral gene insert, and does not result in expressed viral coat proteins or produce new viruses , no adverse effects to species that interact with agriculture is expected. For non - target plants, the Agency considered whether the gene could be transferred by hy bridization of the C5 plum with other Prunus species. Successful gene flow in the environment, given the inefficiency o f artificial intentional crosses from the hexaploid Prunus domestica , is considered highl y unlikely. In the event hy bridization occur

red and the co at protein gene was transferred to a non
- target plant, it is possible that any resulting resistance might be beneficial .Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

12 For a more comprehensive discussion of the Agency ’s assessment of the data and information concerning environmental risks submitted in support of the registration of the CPG - PPV , refer to APPENDIX C . III. ENVIRONMENTAL JUSTICE EPA seeks t o achieve environmental justice, the fair treatment and meaningful involvement of all people, regardless of race, color, national origin, or income, in the development, implementation, and enforcement of environmental laws, regulations, and policies. To he lp address potential environmental justice issues, the Agency seeks information on any groups or segments of the population who, as a result of their location, cultural practices, or other factors, may have aty pical, unusually

high exposure to CPG - PPV comp ared to the general population. Please comment if you are aware of any sub
- populations that may have atypical, unusually high exposure compared to the general population. For additional information regarding environmental justice issues, please visit EPA ’s web site at http://www.epa.gov/compliance/environmentaljustice/index.html . IV. BENEFITS AND PUBLIC INTEREST FINDING All of the data requirements for the registration of th e use of the CPG
- PPV as a plant
incorporated protected in C5 Honey Sweet Plum have been satisfied, except for an independent laboratory

validation of the applicant’s enforcement analy tical method, as discussed previousl y. Since the applicant has not had su fficient time to comply with this requirement, EPA considered whether a conditional registration, issued in accordance with FIFRA Section 3(c)(7)(C), would be in the public interest. EPA determines whether a conditional registration of a pesticide is in the public interest in accordance with the criteria set forth in the Federal Register dated March 5, 1986 ( 58 FR 7268 ). There is a presumption that registration of a pesticide is in the public interest if (1) the use is for a minor crop, (2) the use is a replacement for another pesticide that is of continuing concern to the Agency , (3) the use is one for which an emergency exemption under FIFRA section 18 has been granted (i.e., the basis for the exemption was lack of a registered alternative product), or (4) the use is against a pest of public health significance. In 2009, the U.S. plum acreage (reported in CA, ID, MI , OR, WA acres, representing approximately

95% of all acreage in the U.S., including plums and prunes) was 93,790 acres, well
- below the 300,000 acre maximum for defining a minor/specialty crop. (http://usda.mannlib.cornell.edu/usda/current/NoncFruiNu/NoncFruiNu
- 01 - 22 - 2010_revision.pdf

). Ninety nine percent of the U.S. supply of dried plums is produced in California, and that state exports 46% of its production, which (although a minor crop) represents 70% of the world suppl y. Since the aphid vectors of the disease are common throughout the U.S., the occurrence of PPV in this major plum
- producing area could devastate the industry andCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

13 affec t world supplies of product. This devastation has been the case in countries where PPV has already spread. In fact, the disease is classified as an invasive species in the U.S. because of the significant economic losses that result to the orchard industry . If eradication of PPV was achieved in the U.S., this minor crop would remain vulnerable to reintroduction as long as the virus exists elsewhere. Additionally , man y Prunus species have ornamental value, and many of these are susceptible to PPV. Spr ead of the disease into high value ornamental nursery stock could cause significant losses to the nursery

trade and its clients. Furthermore, wild species of Prunus provide food and shelter to wildlife, and natural habitat could be disrupted should those wild stone fruit species become infected. Current control practices include the use of chemical pesticides to control aphids, which transmit (vector) the virus to the plants. Aphid infestations can be difficult to manage, so repeated applications of pe sticides are usuall y necessary . Often, infected orchards must be destroyed, resulting in further economic losses. In Europe, over 100 million trees have been lost to this devastating virus. C5 Honey Sweet Plum offers several benefits, including better crop production (yield) in a minor/specialty

crop, high quality

fruit, and a reduction in the amount of chemical pesticides needed to control aphids that are vectors for PPV. Control of PPV via C5 Honey Sweet Plum may contribute to protecting U.S. agriculture, as well as our managed landscapes and natural areas. Based on this information, EPA presumes this registration will be in the public interest. V. RISK MANAGEMENT AND PROPOSED REGISTRATION DECISION A. Determination of Eligibility EPA is proposing a conditional registration for th e CPG - PPV , expressed in C5 HoneySweet Plum . Pursuant to FIFRA Section 3(c)(7)(C), EPA may conditionally

register a new pesticide active ingredient for a period of time reasonably sufficient for the generation and submissio n of required data that are lacking because insufficient time has elapsed since the imposition of the requirement for those data to be developed. EPA may grant such conditional registration only if EPA determines that (1) the use of the pesticide product during the period of the conditional registration will not cause an y unreasonable adverse effect on the environment, and (2) the registration and use of the pesticide during the conditional registration is in the public interest. EPA believes these criter ia have been met. EPA concludes that i nsufficient time has elapsed since the imposition of the requirement for the applicant to submit data from an independent lab validation of the enforcement anal yt ical method for the detection of the CPG - PPV in fresh o r processed plum commodities . The first criterion is met, because the applicant provided sufficient data and other relevant information for EPA to determine that the use of the CPG - PPV in C5 Honey Sweet Plum during the period of the conditional registratio n (one year) will not result in unreasonable adverse effects to theCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

14 environment. Th e second criterion was met, because the use of the PIP in the C5 plum would be in the public interest, as described in section IV, above. B. Proposed R egulatory Decision On October 1, 2009, EPA announced a new policy to provide a more meaningful opportunity for the public to participate on major registration decisions before they occur. According to this new policy , EPA intends to provide a public comment period prior t o making a registration decision for, at minimum, the following types of applications: new active ingredients; first food use; first outdoor use; first residential use; and other actions for which the Agency believes there will be significant public inter est. Accordingl y, this pesticide is subject to a 30
- day comment period as a new active ingredient with both food and outdoor uses. Any regulatory

decision will be informed b y the comments EPA receives. The applicant provided sufficient data and other re levant information for EPA to determine that conditional registration of the CPG - PPV in C5 Honey Sweet Plum under FIFRA 3(c)(7)(C) for one year will not result in unreasonable adverse effects to the environment. The human health and non
- target organism dat a requirements are satisfied for the period of the conditional registration. EPA will impose the requirement for the applicant to submit data from an independent lab validation of its enforcement analytical method as a condition of registration. Therefore, EPA is providing notice of its intent to grant a one
- year conditional registration under Section 3(c)(7)(C) of FIFRA for the plant
- incorporated
- protectant CPG - PPV in C5 Honey Sweet Plum. VI. ACTIONS REQUIRED BY THE APPLICANT A. Satisfaction of the Con ditions of Registration The applicant must submit acceptable data generated from an independent laboratory validation of the enforcement anal ytical method within one year of the date the conditional registration is issued.B . Reporting Requirements No twithstanding the information stated in the previous paragraph, it should be clearly understood that certain, specific data are required to be reported to the Agency as a r equirement for maintaining the f

ederal registration for a pesticide product. A brie f summary of these types of data are listed below.

i . Adverse Effects Reports of all incidents of adverse effects to the environment must be submitted to the Agency under the provisions stated in FIFRA, Section 6(a)(2).Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

15

ii . Hypersensitivity Incidents All incidents of hy

persensitivity

(including both suspected and confirmed incidents) must be reported to the Agency

under the provisions of 40 CFR § 158.2050(d).Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

16 APPENDIX A: PRODUCT CHARACTERIZATION ( 40 CFR § 158.2120 ) I. Manufacturing Process Slices of the hy

pocotylsegment from fresh (or recently stored at 4 o C) plum (

Prunus domestica ) cultivar ‘BlueBy

rd’ seeds are removed under sterile conditions for transformation. Approximately

8% of slices incubated on selective
- differential growth media (50 μ g/mL k anam ycin and GUS at 37 o C) were able to produce shoots and roots following Agrobacterium
mediated transformation with binary plasmid pGA482GG/PPV
- CP - 33. The cloned plum pox virus coat protein was under the PPV
- CP - 33 plasmid
- carried CaMV 35s promote r subcloned into the pGA482GG plasmid HindIII site after restriction enzy me digestion, then transformed to Agrobacterium tumefaciens strain C58.Z707 and grown in selective media using kanamycin (50 μ g/mL) and gentamycin (50 μ g/mL) and tested for GUS activi ty with an X
- Glu solution. Presumptive transformants containing the desired cassette (MRID 471573
- 01 ) were confirmed by multiplex PCR for the PPV
- CP, nptII and GUS genes as inserted. In all there are 5 mapped inserts into the selected plum event (C5) inc luding one complete and four partial inserts. There is one complete, and one doubled (tail
- to - tail) PPV
- CP insert (MRID 471573 - 01). Each PPV
- CP insert has a leading 35s CaMV promoter and short untranslated region from the TMV virus in the polycloning sit e present (MRID 471573 - 01). Potentially this would produce 3 copies of mRNA for the PPV
- CP from the PPV
- D strain with an added ATG start codon and TMV leader sequence. Stability

was assessed repeatedl y from 1990
- 2005 in propagated transformants and proge ny to confirm retention of the inserts, all of which appear to be linked but are an unknown distance from each other on the chromosome. RNA transcripts were characterized using blots, aimed at the expected 1.4 kb PPV
- CP target. Transformants C2, C3 and C4 produced detectible transcripts within 5 hours with blot exposure (32
- P hy bridization). C5 transcript was barel y detected after 40 hours when compared to untransformed controls. No transcript was detected for C6 and no coat protein production was found in either the C5 or C6 events. Anal ys is of inserts by restriction enz yme digestion and DNA gel blot showed the expected internal PPV
- CP BamHI 1.2 kb fragment plus an approximately

double
- sized fragment from C5. Several EcoRI digest bands besides the e xpected 7 kb band further indicated multiple, and different, insertions compared to the pGA482GG/PPV
CP - 33 plasmid control. In C5 EcoRIbands at 1.9, 3, 5, 7 and 10 kb hy bridized with PPV
- CP; the 5 and 10 kb bands also hy bridize with a probe for nptII and the 7 kb band with uidA. Another 20 kb fragment only hy bridized with nptII. Sequencing of the inserts was accomplished but only about 80% determined “due to sequence repeats, DNA meth yl ation, and the presence of an origin of replication in the insert . ” The proposed structures ( MRID 471573 - 01) as discussed above are in five parts, one of them complete the others containing duplications or rearrangements and one is listed as an inverted repeat of the PPV coat protein. The mechanism for inversion is not stated though this insert “may be critical for providing PPV resistance . ”Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

17 A . Inert Ingredients An ampicillin resistance gene ( β
- lactamase) is present as part of the construct from plasmid pBR322 fragments engineered into plasmid pGA482GG
- PPV - CP. However the gene has a bacterial insert containing a cos site (from cosmid MUA10 as derived from pBR322), and is inactivated. Non
- func tionality of this gene was demonstrated by RNA extraction, and reverse
transcriptase PCR using a reverse primer and spanning the bacterial insert. Analysis of the original plasmid construct but in E. coli DH5 - α using gentamycin to select for the plasmid, and control E. coli DH5 - α grown in broth but without the plasmid, were plated to LB agar containing 100 mg/L ampicillin. This experiment was replicated twice. Each trial resulted in five colonies of E. coli DH5 - α containing plasmid pGA482GG
- PPV - CP that r everted to functional β
- l actamase b y deletion of the cos insert. No spontaneous ampicillin
- resistant colonies resulted from E. coli DH5 - α without the plasmid. Ten C5 plum leaf samples taken from 1997
- 2005 were assayed for β
- lactamase mRNA using reverse primers specific to a 532 bp region spanning the cos site. No mRNA was detected from the samples (archived at
- 80 o C) taken at various months throughout those ye ars. Positive controls were the uidA sequence for the GUS transgene, and CAB for the plant ch lorophyll A/B

– both produced positive PCR results in all 10 samples. Supplemental information provided during USDA’s Animal and Plant Health Insp e ction Service (APHI S) review confirmed by PCR that the cos site remained in the C5 inserted gene. It is evi dent that even in the highl y selective environment of fast
- growing E. coli cells in presence of ampicillin and harboring a high
- cop y- number plasmid, that the mutation rate to active amp
- R is exceedingl y low. None of these conditions is expected to occur f or C5 Honey Sweet plum trees or fruit. Other selective marker antibiotic genes present on the original plasmid were tetracycline and gentamycin resistances, present on the part of the plasmid under a bacterial promoter. EPA requested that the applicant provide proof that these genes were absent in the C5 plum trees. On July 10, 2008 the applicant supplied new laboratory tests on extracts of tree fruit and leaves, both the wild
- ty

pe ( BlueBy

rd) and the PIP (C5). The positive control, plasmid pGA482GG
- PPV - C P response is clear, while all lanes of test sample PCR product is negative for the tetracycline and gentamycin resistance genes. PPV
- CP DNA is detected from the plasmid and C5 leaves but not from C5 fruit (MRID 474749
- 02). All genes were absent from the wild
- ty pe BlueBy

rd fruit and leaf samples. The applicant reports that simultaneous tests for nptII, PPV
- CP, uidA and 26s rDNA produced requisite PCR products though these results were not shown on these gel photos (page 8, MRID 474749
- 03). The gene for nptII (neomycin phosphotran s ferase II, or kanamycin resistance) is present and does produce both mRNA and protein from the inserted Agrobacterium tumefaciens NOS promoter. There are four copies of the nptII gene inserted into the C5 chromosome, of which a t least three are thought functional. There is a tolerance exemption for neomycin phosphotransferase II at 40 CFR § 174.521. Another inert is the protein from the uidA gene, GUS (E. coli β
- D - glucuronidase), under direction of a CaMV 35s promoter. There are two complete copies of the uidA gene on separate inserts, and three fragments on two of the other inserts. GUS has an existing tolerance exemption at 40 CFR § 174.525.Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

18 B. Activ e Ingredient The PIP active ingredient as inserted is a reverse transcrip tion derivation of the virus coat protein RNA, inserted with a 3’ untranslated region with fusion of a start codon and short leader sequence and an Agrobacterium tumefaciens NOS terminator under direction of a CaMV 35s promoter. There is one complete cop y of the PPV
- CP gene, a small fragment of the PPV
- CP 35s promoter on another insert and a third insert that is a “3’
- 3’ tail
- to - tail cop y of the PPV
- CP with the 35S promoter for each copy and a portion of GUS sequence flanking each PPV
- CP copy . The insert is flanked b y plum DNA. II. P lum P ox V irus Resistance and Mode of Action The Agency

postulates that the C5 honey sweet plum transcribes both a readable (translatable) copy of the mRNA and a complementary cop y of that mRNA. These two “ complementary” transcripts bind to form a double
- stranded RNA molecule that triggers the plant's inherent virus defense mechanism based on post
- transcriptional gene silencing (PTGS). C5 plum was shown to be resistant to the four major serotypes of PPV (Ravelonandro et al. 2001) by a method other than PPV coat protein production in the plant. Subsequent work narrowed the mechanism of resistance to PTGS noting there were low mRNA levels and high methylation of CPG - PPV DNA sequences relating to resistance, upon challenge with PPV (Scorza et al. 2001). C5 trees were selected from testing among five transgenic plums from the same event - C2, C3, C4, C5, C6 - (Scorza et al. 1994) all confirmed b y multiplex PCR for the PPV - CP, nptII and GUS genes as inserted (see above). Field experiments in Poland with replicates of each transgenic plum and a wild
- ty pe, were chip
- bud inoculated in 2 out of 10 replicates, then exposed to natural aphid populations over two years (2003, 2004); as a result, non - inoculated C5 trees were the onl y one s not infected in the field (Hil y et al. 2004). Chip
- bud inoculated C5 replicates showed mild sy mptoms along the chip
- bud branches with sy mptoms decreasing over several more years. Though s ymptoms had abated, ELISA and IC
- RT - PCR testing showed presence o f the Plum pox virus in a few samples of C5, mainly those still showing s ymptoms of infection. The other susceptible transgenic plums and the wild
- ty pe (highl y susceptible B70146) all developed progressivel y worsening s ymptoms. B70146 had readily visib le chlorotic sy

mptoms within 1
- 2 years. Another field trial, in Spain, tested variations of non
- transgenic, wild
- ty pe plum and fewer of the transgenic plums were tested in Poland. Results confirmed that C5 had durable re sistance to inoculated or field
- ac quired PPV. Even when infected through rootstock, C5 trees were able to keep the spread of the virus in check, and s ymptoms of the virus were onl y observed near the site of the graft. Further testing of hy brids and seedlings showed that “the multiple transgene inserts of C5 are closely linked and are transmitted as a single dominant gene (locus)” (Scorza et al. 1998) , al though PTGS
- based resistance in germinated seedlings may be delayed up to a month . In MRID 474749 - 02 , the inverted repeat of the CPG - PPV was further clarified: a piece of the 35s PPV
- CP promoter is present on either end followed b y a portion of the PPV
- CP DNA without the NOS terminator. It is unknown if the sense strand of this insertCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

19 can produce mRNA or protein, though as discussed above the antisense strand may produce mRNA from a plant open reading frame.

Table 1: Certified Limits and Nominal Concentrations In gredients (CAS number) PC Code Purpose Concentration (% b y weight) Nominal Lower Upper Active Ingredient Plum Pox Virus Resistance Gene (Plum Pox Viral Coat Protein Gene) DNA 006354 Active ingredient 0.0005 0.00045 0.00055 In ert Ingredients Neomycin phosphotrasnferase nptII * CAS number 62213
- 36 - 9 806304 Selective marker / In ert ingredient CBI CBI CBI Beta - Glucuronidas e GUS * CAS number 9001
- 45 - 0 829082 Differential marker / Inert ingredient CBI CBI CBI

III. Enforcement Analytical Method As provided in MRID 474749
- 02, the C5 transformation event of BlueBy rd may be differentiated from untransformed trees using the following PCR primers for the inserted coat protein gene: PPV - CP (1 kbp) Forward: 5’
- AAGCTGACGAAAGAGAGGACGAG - 3’ Reverse: 5’ - CTACACTCCCCTCACACCGAGGAA - 3’ PPV - CP (~70 bp) Forward: 5’
- GCAGGCAAGCCGATTGTAGT - 3’ Reverse: 5’ - TGTATGACTGGAGGTGGTTGAAGT - 3’ The applicant submitted a proposed anal yt ical method based on the use of these primers and a poly merase chain reaction (PCR) technique that should distinguish the transformed C5 Honey Sweet plum from non
- transformed varieties. The Agency is requiring the applicant to provide data generated by and independent laboratory to validate this method.Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

20 APPENDIX B: HUMAN HEALTH ASSESSMENT (40 CFR § 158.2140) Section 408(c)(2)(A)(i) of the FFDCA allows EPA to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) onl y if EPA determines that the exemption is “safe.” Section 408(c)(2)(A)(ii) of the FFDCA defines “safe” to mean that “there is a reasonable certainty that no harm will result f rom aggregate exposure to the pesticide chemical residue, including all anticipated dietary exposures and all other exposures for which there is reliable information.” This includes exposure through drinking water and in residential settings, but does not include occupational exposure. Pursuant to section 408(c)(2)(B), in establishing or maintaining in effect an exemption from the requirement of a tolerance, EPA must take into account the factors set forth in section 408(b)(2)(C), which require EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance and to “ensure that there is a reasonable certainty that no harm will result to infants and children from aggregate exposure to the pesticide chemical residue... .” Additionally , section 408(b)(2)(D) of the FFDCA requires that the Agency consider “available information concerning the cumulative effects of a particular pesticide’s residues” and “other substances that have a common mechani sm of toxicity .” EPA performs a number of analy ses to determine the risks from aggregate exposure to pesticide residues. First, EPA determines the toxicity of pesticides. Second, EPA examines exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings. I. Toxicological Profile Consistent with section 408(b)(2)(D) of the FFDCA, EPA has reviewed the available scientific data and other relevant information submitted in support of this action and considered its validity , completeness and reliability , and the relationship of this information to human risk. EPA has also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children . A. Data Waivers - Justification The applicant submitted requests to waive the Tier 1 mammalian toxicity data requirements , except for the requirement to report hy persensitivity

incidents, for the registration of the CPG PPV , listed below. The Agency granted the requests, based on the following rationales .  There is a long history of consumption of plant virus particles in food without any known toxicity or other deleterious human health effects. Although plant viruses have been found in the intestin es (Zhang, et al., 2006) , plant viruses have not been shown to replicate in humans and other vertebrates.

.  Non - occupational exposure is minimal to non
- existent since the gene is only expressed withi n plant tissues.Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

21  The C5 plum does not represent a source of new, potentially allergenic or anti
- nutrient proteins. Due to the plant’s earl y defense mechanism (PTGS), mRNA transcribed from the CPG - PPV is destroyed. Therefore, it is highl y unlikely that an y viral coat proteins will be produced in the C5 plum. This conclusion is supported by field expression data submitted by

the applicant that found no viral protein TABLE 2: Tier 1 Mammalian Toxicology Data Requirements

and Associated OPPTS Test Guidelines Acute Oral Toxicity

/Pathogenicity OPPTS 885.3050 Acute Dermal Toxicity OPPTS 885.3100 Acute Pulmonary

Toxicity /Pathogenicity OPPTS 885.3150 Acute Injection Toxicity

/Pathogenicity OPPTS 885.3200 Hy persensitivity

Incidents 

### * OPPTS 885.3400 Cell Culture OPPTS 885.3500 Acute Oral Toxicity OPPTS 870.1100 Acute Dermal Toxicity OPPTS 870.1200 Acute Inhalation Toxicity OPPTS 870.1300 Acute E ye Irritation OPPTS 870.2400 Primary Dermal Irritation OPPTS 870.2500 

### * Hypersensitivity Incidents : Reporting is required when incidents occur. No hypersensitivity incidents, including immediate
- type or delayed
- type reactions in humans or animals, during the 18 years that development of the C5 HoneySweet Plum occurred. Should any future hypersen si tivity incidents occur, they must be reported to the Agency. B. Previous Scientific Findings As part of its consideration of the proposed use of the Coat Protein Gene of Plum Pox Virus as a PIP in the C5 Honey Sweet Plum, EPA reviewed previous information r egarding the safety of exposure for plant expression of plant virus components, especiall y the coat proteins. The Agency has previousl y registered and established food tolerance exemptions for plant virus coat proteins and genes as parts of PIPs. This ba se of knowledge and experience led to the following three conclusions on which the Agency would rely to support a tolerance exemption for the Coat Protein of Plum Pox Virus, in the remote possibility that an y currently undetectable residues of this protein are produced in the C5 plum. i . Plants infected with plant viruses have always been part of food supply without adverse effects . Virus
- infected food plants have alway s been a part of the human and domestic animal food supply , and components of plant viruses, including plant virus coat proteins (PVCPs) , are often found in the produce of many types of crops. For example, at the beginning of this centuryCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

22 virtually every commercial cultivar of potatoes grown in the United States and Europe was infected w ith at least one potato virus. Even as ymptomatic plants are often found to be infected. A common agricultural practice (since the 1920s) involves the intentional inoculation of healthy plants with a mild form of a virus in order to prevent infection by a more virulent form. To date, there have been no reports of adverse health effects in humans or animals associated with consumption of plant viruses in food. The National Research Council (NRC) observed in its 2000 report that “[h]uman or animal consump tion of plants with viral coat proteins is widely considered to be safe, on the basis of common exposure to these ty pes of proteins in non
- transgenic types of food.” The FIFRA Science Advisory

Panel (SAP) also addressed the issue of dietary risk posed by plant viruses and components in December, 1992. In its report from the meeting, the SAP stated that “[s]ince viruses are ubiquitous in the agricultural environment at levels higher than will be present in transgenic plants, and there has been a long histo ry of ‘contamination’ of the food supply by virus coat protein, there is scientific rationale for exempting transgenic plants expressing virus coat protein from the requirement of a tolerance.” The FIFRA SAP again discussed PVCPs on October 11
- 13, 2004, and “agreed that (because of the human history of consuming virus infected food), unaltered PVCPs do not present new dietary exposures.” Experiments have shown that PVCP levels expressed in plants engineered to resist a virus can be 100 - 1000 times lower i n concentration than in plants naturally infected by

the virus. In the C5 plum, no detectable levels of PPV coat protein were found , therefore, if any is produced, it is below the current levels of detection. However, as discussed previously , the C5 plum likely is incapable of producing viral coat protein from the Coat Protein Gene of PPV, since the inserted gene initiates the plant’s natural defense mechanism before the protein can be manufactured.

ii . Plant viruses are not infectious to humans and animals. Any virus/host relationship is characterized by a high degree of specificity . Plant viruses do not infect humans or other vertebrates, and usuall y only infect plants within a certain taxonomic group. In order to replicate, viruses must insert into the plant genome by means of an insect or other mechanical vector . Plant virus replication depends on components of the viral genome and the plant’s ability

to transcribe its genes, produce the components and package them into new virus ; human and an imals lack the corresponding cellular “machinery” for these processes . Thus, it is reasonable to assume that a single component of PPV, the CPG - PPV , will not cause a viral infection in humans and other animals.

iii . Plant viruses are not toxic to humans and animals. As stated previousl y, food from crops infected with plant viruses has always been consumed without human or animal toxicity related to these viruses. Additional evidence of the lack of toxicity by a different route of exposure comes from th e common practice of injecting laboratory animals with purified plant virus preparations to generate antibodies used for anal yt ical tests (for example, to identify

and confirm plant virus infections), without any adverse effects on the animals.Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

23 C. Toxici ty and Allergenicity Assessment The Agency

is not aware of any PVCPs that have been identified as human food allergen s . To determine whether the PPV coat protein or the gene insert would potentially cause toxicity

or hy persensitivity , they

were sequenced and anal yz ed for homology with known food allergens and toxins. No alignments that would trigger a concern were identified. The lack of production of PPV coat protein by the C5 plum, together with reports indicating there have been no h yp ersensitivity incidents or other adverse effects in researchers handling the trees, fruits and other plant tissues since experimentation with C5 plum began in 1992, support the Agency ’s conclusion that the CPG - PPV expressed in the C5 plum is safe for human dietary cons umption. No further testing, including an in vitro digestibility assay , is required. The safety finding for human consumption of the Coat Protein and the CPG - PPV holds equally for all foods in the stone fruit crop group, and also in almond. II. Aggre gate Exposures Pursuant to FFDCA section 408(b)(2)(D)(vi), EPA considers available information concerning aggregate exposures from the pesticide residue in food and all other non
- occupational exposures, including drinking water from ground water or surfac e water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses). The Agency

has considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue and to other related substances. These considerations include d dietary exposure under the proposed tolerance exemption for the Coat Protein of Plum Pox Virus, all other toleran ces or exemptions in effect for r esidues of v irus coat proteins and viral coat protein gene PIPs, and non - occupational exposure . Exposure via the skin or inhalation is not likely , since the PIP , which is contained within the genome of the C5 plum plant cells, does not produce PPV coat protein . Further, the C5 plum’s resistance to PPV infection reduces or eliminates the production of PPV coat protein following natural infection with the virus. Although the Agency ’s allergenicity assessment focused on the potential of the Coat Protein of Plum Pox Vir us to be a food allergen, the data also indicated a low potential for the PIP to be an allergen. Even if exposure occurred through an unlikely

route, such as inhalation, the potential for the Coat Protein of Plum Pox Virus to be an allergen is low, as evi denced b y the lack of h ypersensitivity in researchers handling C5 plum trees for eighteen ye ars, discussed previousl y. Exposure of infants and children to the Coat Protein of Plum Pox Virus from residential or lawn use is not expected, because the use s ite for the C5 plum is agricultural. Inthe unlikely event that the C5 plum expresse s an y PPV coat protein, o ral exposure could occur from eating fresh and processed C5 plum products . The level of the coat protein would be extremely low, as indicated b y the mode of action of the PIP and the lack of detection of the coat protein inCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

24 C5 plums , discussed previously . The same evidence supports the Agency ’s conclusion that oral exposure from drinking water would be highl y unlikely . III. Cumulative Effects Section 408(b)(2)(D)(v) of FFDCA requires the Agency , when considering whether to establish, modify , or revoke a tolerance, to consider “available information” concerning the cumulative effects of pesticide residues and “other substances that have a commo n mechanism of toxicity .” These considerations include the cumulative effects of such residues on infants and children. Because there is no indication of mammalian toxicity from CPG - PPV , the Agency concludes that CPG - PPV does not share a common mechanism of toxicity with other substances. Therefore, section 408(b)(2)(D)(v) does not apply . Determination of Safety

for U.S. Population, Infants and Children FFDCA section 408(b)(2)(C), as amended b y the Food Quality Protection Act (FQPA) of 1996, provides th at EPA shall assess the available information about consumption patterns among infants and children, special susceptibility of infants and children to pesticide chemical residues and the cumulative effects on infants and children of the residues and other substances with a common mechanism of toxicity . In addition, FFDCA section 408(b)(2)(C) also provides that EPA shall apply

an additional tenfold margin of safety for infants and children in the case of threshold effects to account for prenatal and postnata l toxicity and the completeness of the database unless EPA determines that a different margin of safety will be safe for infants and children. Based on its review and consideration of all of the data and other information submitted by the applicant, in ad dition to its previous knowledge of plant viruses, including for plant virus coat proteins , EPA concludes that there is a reasonable certainty that no harm will result to the United States population, including infants and children, from aggregate exposure to residues of Coat Protein of Plum Pox Virus. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. The Agency has arrived at this conclusion because the data available on the CPG - PPV demonstrate a lack of toxicity and pathogenicity. The PIP active ingredient, CPG - PPV , is not known to produce an y recognized toxins, novel proteins, anti
- nutrients, virulence factors or enzy mes normally

associated with pathogen invasiveness or toxicity

in mammals . Thus, there are no threshold effects of concern and, as a result, the Agency

has concluded that an additional tenfold margin of safety for infants and children is unnecessary

in this instance. IV. Endocrine Disrupt ors The active ingredient, CPG - PPV is comp osed of nucleic acids (RNA), and is not known to exert any influence on the endocrine s ys tem. To date, there is no evidence to suggest that the CPG PPV affects the immune sy stem, functions in a manner similar to any known hormone, or that it acts as an en docrine disruptor. It is unlikel y that this PIP will have estrogenic or endocrine effects. Therefore, the Agency is not requiring information on endocrine effects at this time.Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

25 APPENDIX C: ENVIRONMENTAL ASSESSMENT (40 CFR § 158.2150) I. Non - Target Spec ies Effects All possible and likely

effects on mammal ian , avian , fish, terrestrial and aquatic invertebrate non - target species were considered. The Agency determined that an y hazard to terrestrial or aquatic wildlife when the product is grown is unlikely to exceed the Agency ’s level of concern (LOC) for non
- target organisms. A review of submitted data and published literature showed no evidence of toxicity

or pathogenicity from the CPG - PPV or the inert ingredients to wildlife, including those related to federally listed endangered or threatened species, nor any effects on critical habitats, when the PIP is produced in growing plants. No adverse indirect effects are expected from the registered uses of C5 Honeysweet plum trees. The data requirements fo r non - target effects are listed in the table below. The OPPTS Guideline refers to the Agency ’s documents that contain protocols for conducting the studies. T ABLE 3: Non - Target Organisms and Environmental Fate Tier I Data Requirements and Associated O PPTS Test Guidelines Avian Oral Toxicity OPPTS 885.4050 Avian Inhalation Toxicity /Pathogenicity OPPTS 885.4100 Wild Mammal Toxicity

/Pathogenicity OPPTS 885.4150 Freshwater Fish Toxicity /Pathogenicity OPPTS 885.4200 Freshwater Invertebrate Toxicity /Pathogenicity OPPTS 885.4240 Estuarine/Marine Fish Testing Estuarine and Marine Invertebrate Testing OPPTS 885.4280 Non - Target Plant Testing OPPTS 885.4300 Non - Target Insect Testing OPPTS 885.4340 Honey Bee Testing OPPTS 885.4380 A . Data Waiver s - Just ification Prior to submitting its application for registration, the applicant met with Agency staff, and during the pre
- submission meeting it was determined that most of the non
- target species data requirements, except for non
- target plant testing, were n ot required for the registration of C5 plum . The decision to waive the data requirements for all non
- target organisms is based upon the following justificationsCoat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

26

i. Non - Target Plants The waiver of non - target plant data requirement was requested b y the applicant, based upon information from references dating from the 19th
- 21st centuries on breeding in Prunus species. T he following summarizes the information that supported the Agency ’s acceptance of the applicant’s requested waiver for conducting a non
- t arget plant test study .  P. domestica is naturall y incompatible with most other Prunus species . The genomes of native species are mainl y diploid , while the genome for C5 is hexaploid.  Forced or artificial h ybridation between other Prunus species with C5 r esults in very low percentages of fruit set .  There is a greater tendency of hy brids to be produced when P. domestica is the female parent , rather than as a male (pollen
- producing) parent in  The extremely

low hy bridization rate with self

- incompatible P. do mestica (‘Honey sweet’ is self

- incompatible);  The low vigor of h ybrid seedlings; and  The low fertility

of surviving hy

brids Specificall y, viable cross es made with P. domestica are unlikel y and are often not vigorous or fertile (artificial crosses with su ccess rates up to 1.3% are reported). T he only known cross in cultivation, “ Alhambra ,” is three generations removed from an original crossing with P. domestica . The inert ingredients, beta
- glucuronidase (GUS) and neomycin phosphotransferase (nptII), have food tolerance exemptions and are not expected to provide any environmental benefit or hazard. While data on the ability

of C5 plum to cross with indigenous plum and other Prunus species is not readily

available or is incomplete, the genome incompatibili ty (mainly diploid for natives, hexaploid for C5 , as stated above ), the low fertility of any successful crosses, and the very low frequency of artificial (forced) crosses make this a very small probability . If a successful cross were to occur, in the abse nce of a PPV infection no benefit or adverse effects from the resistance gene is expected. However, i n light of the ongoing invasion of PPV into the U.S. it would likely be beneficial to increase PPV resistance in potential virus hosts , to lower the incid ence of virus and of the virus “ sinks ” (non
- agricultural species that are infected with PPV can act as reservoirs for the virus) that enable spread of the virus b y aphids .

ii . Other Non - Target Organisms During a meeting with the applicant prior to subm ission of the application for registration of the C5 Honey Sweet Plum, the Agency determined that sufficient data from peer
- reviewed scientific literature, in addition to its own knowledge base (discussed in Appendix B ) concerning plant viruses and plant vi ral coat proteins, would provide justification to waive the requirements of all non - target species testing, except for no n - target plants. The following summarizes the information that supported the Agency ’s acceptance of the applicant’s requested waiver f or conducting a non
- target plant test study .Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

27  There is a long history of consumption of plant viruses (including PVCPs) in foods consumed b y animals, including birds, mammals, and other vertebrates, without deleterious effects or evidence of toxicity .  No e xposure to aquatic species ( including vertebrate and invertebrate species) is expected since gene is onl y expressed (as DNA) within the plant genome.  Exposure to non - target insects, including honey bees, is not expected since the gene is only expressed (as DNA) within the plant genome.  The gene is incapable of forming infectious virus particles.  C5 plum is not likely

to produce an y viral coat protein or other novel protein that would be toxic or infectious to non
- target species.  Even if PPV coat protein is produced in C5 plum, it would be at a very low levels. PPV coat protein and the CPG
- PPV sequences have been compared with sequences of known toxins and food allergens, and no sequence matches were found that would raise any concerns. II. E ndangered Speci es Consideration Prunus geniculata is the only species of plum listed by the U.S. Fish and Wildlife Service as endangered . This species is found only in patches of remaining scrub areas in parts of Florida , and its level of natural resistance to PPV is unknown. P. geniculata is known to thrive after fire, and its habitat is reportedly being deprived of this condition, which has decreased its range and populations. Prunus domestica is not found or cultivated in Florida , so P. geniculata will not be expos ed to C5 plum trees. Since EPA has determined that no effects are anticipated for any non - target species exposed to CPG - PPV as a result of labeled uses, effects to threatened and endangered species and their designated critical habitats are also not exp ected. Therefore, a “No Effect” determination is made for direct and indirect effects to listed species and their designated critical habitats resulting from the registered uses of CPG - PPV , as labeled.Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

28 BIBLIOGRAPHY I. Data Submissions Received and Rev iewed by the Agency MRID No. Citation 47157300 Interregional Research Project No. 4 (2007) Submission of Product Chemistry and Toxicity

Data in Support of the Application for Registration of C5 Honey sweet Plum. Transmittal of 3 Studies. 47157301 Braverm an, M. (2007) Group A
- Product Analy sis Test Guidelines: C5 Honey sweet Plum.

Project Number: 0377B. Unpublished study prepared by

I R - 4 Project. 89 p. 47157302 Scorza, R.; Braverman, M. (2007) Plum Pox Viral Coat Protein
- Non - Target Plant Waiver

Requests . Project Number: 0377B. Unpublished study prepared by

Interregional Research Project No. 4. 259 p. 47157303 Scorza, R.; Braverman, M. (2007) C5 Honey sweet Plum Resistant to Plum Pox Virus (Plum

Pox Viral Coat Protein Gene). Project Number: 0377B. Unpubli shed study prepared by Interregional Research Project No. 4. 243 p. 47474900 USDA - ARS (2008) Submission of Toxicity and Product Chemistry

Data in Support of the Application for Registration of C5 Honey Sweet Plum and the Petition for Tolerance of Plum Pox Virus Resistance Gene. Transmittal of 2 Studies. 47474902 Scorza, R.; Braverman, M. (2008) C5 Honey Sweet Plum Resistance to Plum Pox Virus

(Plum Pox Viral Coat Protein Gene): Amendment No. 1 to MRID 47157303. Project Number: 0377B. Unpublished study prepared by

USDA - ARS Appalachian Fruit Research Station and IR - 4 Project. 38 p. 47474903 Scorza, R.; Braverman, M. (2008) Responses to Questions Concerning Plum Pox

VirusResistant Honey Sweet Plum: C5 Honey Sweet Plum. Project Number: 0377B. Unpublished study

pr epared b y USDA - ARS Appalachian Fruit Research Station and IR - 4 Project. 100 p. 47490600 Interregional Research Project No. 4 (2008) Submission of Product Chemistry Data in

Support of the Application for Registration of C5 Honey Sweet Plum. Transmittal of 1 Study . 47490601 Braverman, M. (2008) Group A
- Product Analy sis Test Guidelines: Amendment Number 1

to MRID 471573 - 01: C5 Honey Sweet Plum: Resistant to Plum Pox Virus (Plum Pox Viral Coat Protein Gene). Project Number: 0377B. Unpublished study prepared by Interregional Research Project No. 4. 844 p.Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document Proposed Decision March 29, 2010

29 II. EPA Risk Assessment Memoranda Gagliardi, J. V. Review (of) submitted registration and food tolerance related materials for registration of C5 HoneySweet Plum resistant to the Plum Pox Poty virus. Memorandum dated March 19, 2009. Gagliardi, J. V. Environmental Risk Assessment for C5 Honey Sweet Plum containing the coat protein gene of Plum Pox Virus. Memorandum dated April 22, 2009. Gagliardi, J. V., Review [of] Submitted draft "Protocols for the Detection of C5 'Honey Sweet' Plum DNA" to support registration. Memorandum dated December 3, 2009. III. Other References Hily , J.M., R. Scorza, T. Malinowski, B. Zawadzka and M. Ravelonandro. 2004. Stability of Gene Silencing
- Based Resistance to Plum pox virus in Transgenic Plum ( Prunus domestica L.) under field conditions. Transgenic Res. 13: 427
- 436. Ravelonandro, M., P. Briard, R. Renaud and R. Scorza. 2001. Transgene
- Based Resistance to Plum Pox Virus (Sharka Disease) Transferred Through Interspecific Hy bridization in Prunus . Proc. Int. Sy

mp. On Molecular Markers (Dore, Dosba and Baril, Eds.). Acta Hort. 546, I SHS 2001. Ravelonandro, M., P. Briard and R. Scorza. 2001. Significant Resi stance of Transgenic Plums Against the Four Seroty

pes of Plum Pox Poty

virus. Proc. 18th Int. S ymp. On Fruit Tree Virus Diseases (M.F. Clark ed.), Acta Hort. 550, I SHS 2001. Scorza, R. M. Ravelonandro, A.M. Callhan, J. Cordts, M. Fuchs, J. Dunez and D. Gon salves. 1994. Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene. Plant Cell Reports 14: 18
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