BIOPESTICIDES 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 Division
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TABLE OF CONTENTS

BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM 4

GLOSSARY OF ACRONYMS AND ABBREVIATIONS 5

I. OVERVIEW

A. Executive Summary 6

B. Use Profile 8

C. Regulatory Background 8

II. RISK ASSESSMENT SUMMARIES

A. Product Characterization 9

B. Human Health Assessment 10

C. Environmental Effects Assessment 11

III. ENVIRONMENTAL JUSTICE 12

IV. BENEFITS AND PUBLIC INTEREST FINDING 12

V. RISK MANAGEMENT AND PROPOSED REGISTRATION DECISION

A. Determination of Eligibility 13

B. Proposed Regulatory Decision 14

VI. ACTIONS REQUIRED BY THE APPLICANT

A. Satisfaction of the Conditions of Registration 14

B. Reporting Requirements 14

APPENDIX A: Product Characterization

I. Manufacturing Process 16

A. Inert Ingredients 17

B. Active Ingredient 18

II. Plum Pox Virus Resistance and Mode of Action 18

III. Enforcement Analytical Method 19
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APPENDIX 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 Effects 24

IV. Endocrine Disruptors 24

APPENDIX C: Environmental Effects Assessment

I. Non-Target Species Effects 25

A. Data Waivers – Justification 25

II. Endangered Species Consideration 27

BIBLIOGRAPHY

I. Data Submissions Received and Reviewed by the Agency 28

II. EPA Risk Assessment Memoranda 29

III. Other References 29
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BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM

Office of Pesticide Programs

Biopesticides and Pollution Prevention Division

Microbial Pesticides Branch

Science Reviews

Joel V. Gagliardi, Ph.D. Product Characterization, Human Health

Assessment, and Environmental Effects Assessment

Regulations

Sheryl K. Reilly, Ph.D. Chief, Microbial Pesticides Branch

Denise Greenway Regulatory Action Leader
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GLOSSARY OF ACRONYMS AND ABBREVIATIONS

APHIS 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 HoneySweet Plum

CPG-PPV Coat Protein Gene of Plum Pox Virus

cDNA Copied (or Copy) DNA

dsRNA Double-Stranded RNA

cos Cosmid

oC Temperature in Centigrade or Celsius Degrees

DNA Deoxyribonucleic Acid

EPA Environmental Protection Agency (the “Agency”)

ELISA Enzyme-Linked Immunosorbent Assay

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

FQPA Food Quality Protection Act

FR Federal Register

g Gram

IR4 Interregional Research Project Number 4

kg kilogram

L Liter

MRID No. Master Record Identification Number

mg Milligram

mL Milliliter

μg Microgram

MP Manufacturing-Use Product

mRNA Messenger RNA

NE No Effect

NIOSH National Institute for Occupational Safety and Health

OPP Office of Pesticide Programs

OPPTS Office of Prevention, Pesticides, and Toxic Substances

PCR Polymerase Chain Reaction 

PIP Plant-Incorporated 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 Agriculture
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I. OVERVIEW

The Environmental Protection Agency (EPA, Agency) is providing a 30-day public comment 

period before making its final decision to register a pesticide product, C5 HoneySweet Plum, 

containing the new plant-incorporated protectant (PIP), Coat Protein Gene of Plum Pox Virus

(CPG-PPV). The registration of 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 

decisions for 30 days before they occur. All comments will be included in the docket (EPA-HQOPP-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 

period 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 eventually 

renders infected trees to be incapable 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 States. 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 vectors). 

PPV is an agricultural pest that causes significant economic losses to the stone fruit industry,

since the primary effects of the infection are reduction of fruit quality and crop yield. Stone 

fruits (including native or wild Prunus species) that are affected by PPV include plums, peaches, 

almonds, nectarines, and sweet and sour cherries. 

[http://www.apsnet.org/online/feature/PlumPox/plumpoxfs.pdf]. Various other non-Prunus

dicotyledonous 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 single strand of RNA) is inserted into the plant 

cells. This strand of RNA contains the genes needed to make new virions. 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 produces 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. Small segments of double stranded RNA (dsRNA) are formed by reacting host 

defenses initiating a defense mechanism within the host plant, known as post-transcriptional 
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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. This sequence of events results in the development of 

natural resistance to further PPV infections, but not before fruit degradation, leaf chlorosis, 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, these

engineered plum trees cannot produce the virus. The U.S. Department of Agriculture, 

Agricultural Research Service-Appalachian Fruit Research Station (the applicant) has developed 

a genetically engineered plum tree, called the C5 HoneySweet 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 dsRNA, and the PTGS mechanism recognizes the abnormality and destroys 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 requires registration by the 

EPA. All of the data requirements for registration of this PIP have been satisfied, except for the 

submission of data required from an independent laboratory validation of the applicant’s

analytical method that detects residues of the PIP in fresh and processed plum commodities for

enforcement purposes. Insufficient time 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 HoneySweet 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).
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B. Use Profile

Active Ingredient: Coat Protein Gene of Plum Pox Virus (CPG-PPV)

Office of Pesticide 

Programs (OPP)

Chemical Code: 006354

Product Name: C5 HoneySweet Plum (C5)

EPA File Symbol: 11312-I

Patent Number US PP15,154 P2

Applicant/

Manufacturer: U.S. Department of Agriculture, Agricultural Research ServiceAppalachian Fruit Research Station

2217 Wiltshire Road, Kearneysville, WV 25430

Type of Pesticide: Plant-Incorporated Protectant (PIP)

Use: Stone Fruits and Almond

Target Pest: Plum Pox 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 requirement of a tolerance for residues of the 

Coat Protein of Plum Pox Virus, in or on stone fruit and almond, was filed by IR-4 on behalf of 

the United States Department of Agriculture, Agricultural Research Service-Appalachian Fruit 

Research Station. The EPA published 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 pending the 

Agency’s approval, the tolerance exemption will be codified under 40 CFR §174.
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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 protein gene of the Plum Pox Virus 

(CPG-PPV) as a plant-incorporated protectant. In its 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 HoneySweet Plum (C5) is one of a number of clones resulting from research that 

genetically engineered the CPG-PPV into the plum tree, Prunus domestica L. The C5 clone was 

selected for commercial development because of its 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 field trials were conducted, the C5 trees remained symptom-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 HoneySweet Plum trees have been 

published in peer-reviewed manuscripts (see “Application for Determination of Non-Regulatory

Status for C5 (“HoneySweet”) Plum (Prunus domestica L.) Resistant to Plum Pox Virus,”

(http://www.aphis.usda.gov/brs/aphisdocs/04_26401p.pdf ).

As discussed previously, 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

messenger RNA (mRNA), the strand carrying the inserted DNA copy of the viral coat protein 

gene is also transcribed into mRNA. In some areas of the C5 DNA insert in the plant, mirrorimage copies of the PPV-coat protein mRNA are produced that naturally bind each other, 

forming areas of dsRNA that the plant senses as abnormal and to which plant host defenses react. 

The plant PTGS response to dsRNA corresponding to the PPV-coat protein produces shorter 

segments (approximately twenty-three nucleotide base pairs in length) of double-stranded RNA ,

and these segments are used as signals by the cell to initiate an immune-type 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.
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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 harbors a wide variety of plant viruses within the intestines. 

EPA reviewed the available 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 

considered the available information concerning the variability of the sensitivities of major 

identifiable subgroups of consumers, including infants and children.

The CPG-PPV is a PIP that consists of nucleic acids, and as such, is exempt from the 

requirement of a tolerance (40 CFR 174.507). Although PPV 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 properties in the unlikely

event that the coat protein is produced in the fruit. In its analysis, EPA considered the safety of 

dietary exposure to plant viruses and PVCPs, and the following three sections summarize the 

Agency’s conclusions.

i. Plants infected with plant viruses have always been part of food 

supply without adverse effects.

Virus-infected food plants have always 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 types of crops. 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 asymptomatic 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. 

Experiments have shown that viral coat protein levels expressed in plants that are genetically 

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, there 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 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 usually only infect plants within a certain taxonomic 

group. In order to replicate, a plant virus usually relies on an insect or some other mechanical 
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vector to insert its genome through plant cell walls. Plant virus replication depends on either 

specific virus-encoded enzymes, or plant enzymes, to transcribe the viral genes, produce the viral 

components and package them into new virus. Humans and other vertebrates lack the 

corresponding cellular “machinery” to transcribe, translate, and package plant viruses. Thus, it is 

reasonable to assume that a single 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 previously, food from crops infected with plant viruses is (and has long been)

consumed by humans and animals 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 analytical 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 proteins that have been identified as a human 

food allergen. To determine whether the PPV coat protein would potentially cause toxicity or 

hypersensitivity, its amino acid structure was compared with known food allergens and toxins. 

No 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 hypersensitivity incidents or other adverse effects among researchers 

handling the trees, fruits and other plant tissues since experimentation with C5 plum began in 

1992, support the Agency’s conclusion that, although unlikely 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, fish, 

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

hybridization of the C5 plum with other Prunus species. Successful gene flow in the 

environment, given the inefficiency of artificial intentional crosses from the hexaploid Prunus 

domestica, is considered highly unlikely. In the event hybridization occurred and the coat 

protein gene was transferred to a non-target plant, it is possible that any resulting resistance 

might be beneficial. 
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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 to 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 help 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 

atypical, unusually high exposure to CPG-PPV compared 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 the use of the CPG-PPV as a plantincorporated protected in C5 HoneySweet Plum have been satisfied, except for an independent 

laboratory validation of the applicant’s enforcement analytical method, as discussed previously. 

Since the applicant has not had sufficient 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 supply. 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 and 
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affect 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, many Prunus species have ornamental value, and many of these are susceptible to 

PPV. Spread 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 pesticides are usually 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 HoneySweet 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 HoneySweet 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 the 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 submission 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 any 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 criteria have been met.

EPA concludes that insufficient time has elapsed since the imposition of the requirement for the 

applicant to submit data from an independent lab validation of the enforcement analytical 

method for the detection of the CPG-PPV in fresh or 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 HoneySweet Plum during the period of the 

conditional registration (one year) will not result in unreasonable adverse effects to the 
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environment. The 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 Regulatory 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 to 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 interest. Accordingly, 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 by the comments EPA receives.

The applicant provided sufficient data and other relevant information for EPA to determine that 

conditional registration of the CPG-PPV in C5 HoneySweet 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 data 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 HoneySweet 

Plum.

VI. ACTIONS REQUIRED BY THE APPLICANT

A. Satisfaction of the Conditions of Registration

The applicant must submit acceptable data generated from an independent laboratory validation

of the enforcement analytical method within one year of the date the conditional registration is 

issued. 

B. Reporting Requirements

Notwithstanding 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 requirement for 

maintaining the federal registration for a pesticide product. A brief 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).
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ii. Hypersensitivity Incidents

All incidents of hypersensitivity (including both suspected and confirmed incidents) must be 

reported to the Agency under the provisions of 40 CFR §158.2050(d).

 
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APPENDIX A: PRODUCT CHARACTERIZATION (40 CFR § 158.2120)

I. Manufacturing Process

Slices of the hypocotyl segment from fresh (or recently stored at 4oC) plum (Prunus domestica) 

cultivar ‘BlueByrd’ seeds are removed under sterile conditions for transformation. 

Approximately 8% of slices incubated on selective-differential growth media (50 μg/mL 

kanamycin and GUS at 37oC) were able to produce shoots and roots following Agrobacteriummediated 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 promoter subcloned into 

the pGA482GG plasmid HindIII site after restriction enzyme digestion, then transformed to 

Agrobacterium tumefaciens strain C58.Z707 and grown in selective media using kanamycin (50 

μg/mL) and gentamycin (50 μg/mL) and tested for GUS activity 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) including 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 site 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 repeatedly from 1990-2005 in propagated transformants and 

progeny 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 hybridization). C5 transcript was barely 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. Analysis of inserts by restriction enzyme 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 expected 

7 kb band further indicated multiple, and different, insertions compared to the pGA482GG/PPVCP-33 plasmid control. In C5 EcoRI bands at 1.9, 3, 5, 7 and 10 kb hybridized with PPV-CP; 

the 5 and 10 kb bands also hybridize with a probe for nptII and the 7 kb band with uidA. 

Another 20 kb fragment only hybridized with nptII. 

Sequencing of the inserts was accomplished but only about 80% determined “due to sequence 

repeats, DNA methylation, 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.”
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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-functionality of this gene was demonstrated by RNA extraction, and reversetranscriptase 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 reverted to functional 

β-lactamase by 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 oC) taken at various months 

throughout those years. Positive controls were the uidA sequence for the GUS transgene, and 

CAB for the plant chlorophyll A/B – both produced positive PCR results in all 10 samples. 

Supplemental information provided during USDA’s Animal and Plant Health Inspection Service 

(APHIS) review confirmed by PCR that the cos site remained in the C5 inserted gene. It is 

evident that even in the highly selective environment of fast-growing E. coli cells in presence of 

ampicillin and harboring a high-copy-number plasmid, that the mutation rate to active amp-R is 

exceedingly low. None of these conditions is expected to occur for C5 HoneySweet 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-type (BlueByrd) and the PIP (C5). The positive control, plasmid pGA482GG-PPV-CP 

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-type BlueByrd 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 phosphotransferase 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 at 

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.
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B. Active Ingredient

The PIP active ingredient as inserted is a reverse transcription 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 copy 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 copy 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 by plum DNA. 

II. Plum Pox Virus 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 copy 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 by 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-type, 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 only ones not infected in the field (Hily et al. 2004). Chip-bud 

inoculated C5 replicates showed mild symptoms along the chip-bud branches with symptoms 

decreasing over several more years. Though symptoms had abated, ELISA and IC-RT-PCR 

testing showed presence of the Plum pox virus in a few samples of C5, mainly those still 

showing symptoms of infection. 

The other susceptible transgenic plums and the wild-type (highly susceptible B70146) all 

developed progressively worsening symptoms. B70146 had readily visible chlorotic symptoms 

within 1-2 years. Another field trial, in Spain, tested variations of non-transgenic, wild-type 

plum and fewer of the transgenic plums were tested in Poland. Results confirmed that C5 had 

durable resistance to inoculated or field-acquired PPV. Even when infected through rootstock, 

C5 trees were able to keep the spread of the virus in check, and symptoms of the virus were only 

observed near the site of the graft. Further testing of hybrids 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), although 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 by a portion of 

the PPV-CP DNA without the NOS terminator. It is unknown if the sense strand of this insert 
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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

Ingredients (CAS number) PC Code Purpose Concentration (% by 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

Inert Ingredients

Neomycin phosphotrasnferase nptII 

* CAS number 62213-36-9

806304 Selective marker / 

Inert ingredient CBI CBI CBI

Beta-Glucuronidase 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 BlueByrd 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 analytical method based on the use of these primers and a 

polymerase chain reaction (PCR) technique that should distinguish the transformed C5 

HoneySweet plum from non-transformed varieties. The Agency is requiring the applicant to 

provide data generated by and independent laboratory to validate this method.
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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) only 

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 from 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 mechanism of toxicity.” EPA performs a number of analyses 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 hypersensitivity incidents, for the registration of the CPGPPV, 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 intestines (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 

within plant tissues.
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 The C5 plum does not represent a source of new, potentially allergenic or anti-nutrient 

proteins. Due to the plant’s early defense mechanism (PTGS), mRNA transcribed from 

the CPG-PPV is destroyed. Therefore, it is highly unlikely that any 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

Hypersensitivity 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 Eye 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 hypersensitivity 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 HoneySweet Plum, EPA reviewed previous information regarding the safety of 

exposure for plant expression of plant virus components, especially the coat proteins. The 

Agency has previously registered and established food tolerance exemptions for plant virus coat 

proteins and genes as parts of PIPs. This base 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 any 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 always 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 century 
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virtually every commercial cultivar of potatoes grown in the United States and Europe was 

infected with at least one potato virus. Even asymptomatic 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 

consumption of plants with viral coat proteins is widely considered to be safe, on the basis of 

common exposure to these types 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 history 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 in 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 usually 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 animals 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 previously, 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 the common practice of injecting laboratory 

animals with purified plant virus preparations to generate antibodies used for analytical tests (for 

example, to identify and confirm plant virus infections), without any adverse effects on the 

animals. 
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C. Toxicity and Allergenicity Assessment

The Agency is not aware of any PVCPs that have been identified as human food allergens. To 

determine whether the PPV coat protein or the gene insert would potentially cause toxicity or 

hypersensitivity, they were sequenced and analyzed 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 hypersensitivity 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

consumption. 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. Aggregate 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 surface 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 included dietary exposure under the proposed tolerance 

exemption for the Coat Protein of Plum Pox Virus, all other tolerances or exemptions in effect 

for residues of virus 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 Virus 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 evidenced by the lack of hypersensitivity in researchers handling C5 plum 

trees for eighteen years, discussed previously. 

Exposure of infants and children to the Coat Protein of Plum Pox Virus from residential or lawn 

use is not expected, because the use site for the C5 plum is agricultural. In the unlikely event 

that the C5 plum expresses any PPV coat protein, oral exposure could occur from 

eating fresh and processed C5 plum products. The level of the coat protein would be extremely

low, as indicated by the mode of action of the PIP and the lack of detection of the coat protein in 
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C5 plums, discussed previously. The same evidence supports the Agency’s conclusion that oral 

exposure from drinking water would be highly 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 common 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 by the Food Quality Protection Act (FQPA) of 1996, 

provides that 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 postnatal 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 addition 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 any recognized toxins, novel 

proteins, anti-nutrients, virulence factors or enzymes 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 Disruptors

The active ingredient, CPG-PPV is composed of nucleic acids (RNA), and is not known to exert 

any influence on the endocrine system. To date, there is no evidence to suggest that the CPGPPV affects the immune system, functions in a manner similar to any known hormone, or that it 

acts as an endocrine disruptor. It is unlikely that this PIP will have estrogenic or endocrine 

effects. Therefore, the Agency is not requiring information on endocrine effects at this time.
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APPENDIX C: ENVIRONMENTAL ASSESSMENT (40 CFR § 158.2150)

I. Non-Target Species Effects

All possible and likely effects on mammalian, avian, fish, terrestrial and aquatic invertebrate 

non-target species were considered. The Agency determined that any 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 for 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. 

TABLE 3: Non-Target Organisms and Environmental Fate Tier I Data Requirements

and Associated OPPTS 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 Waivers - Justification

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 not required for the registration of C5 

plum. The decision to waive the data requirements for all non-target organisms is based upon 

the following justifications
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i. Non-Target Plants

The waiver of non-target plant data requirement was requested by the applicant, based upon 

information from references dating from the 19th-21st centuries on breeding in Prunus species. 

The following summarizes the information that supported the Agency’s acceptance of the

applicant’s requested waiver for conducting a non-target plant test study.

 P. domestica is naturally incompatible with most other Prunus species. The genomes of 

native species are mainly diploid, while the genome for C5 is hexaploid.

 Forced or artificial hybridation between other Prunus species with C5 results in very low 

percentages of fruit set.

 There is a greater tendency of hybrids to be produced when P. domestica is the female 

parent, rather than as a male (pollen-producing) parent in 

 The extremely low hybridization rate with self-incompatible P. domestica

(‘Honeysweet’ is self-incompatible);

 The low vigor of hybrid seedlings; and

 The low fertility of surviving hybrids 

Specifically, viable crosses made with P. domestica are unlikely and are often not vigorous or 

fertile (artificial crosses with success rates up to 1.3% are reported). The 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 incompatibility (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 absence of a PPV infection no benefit or adverse effects from the resistance 

gene is expected. However, in 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 incidence 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 by aphids. 

ii. Other Non-Target Organisms

During a meeting with the applicant prior to submission of the application for registration of the 

C5 HoneySweet 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 viral coat proteins, would provide justification to waive the requirements of all 

non-target species testing, except for non-target plants. The following summarizes the 

information that supported the Agency’s acceptance of the applicant’s requested waiver for 

conducting a non-target plant test study.
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 There is a long history of consumption of plant viruses (including PVCPs) in foods 

consumed by animals, including birds, mammals, and other vertebrates, without 

deleterious effects or evidence of toxicity.

 No exposure to aquatic species (including vertebrate and invertebrate species) is expected 

since gene is only 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 any 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. Endangered Species 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 

exposed 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 expected. 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.
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BIBLIOGRAPHY

I. Data Submissions Received and Reviewed 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 Honeysweet Plum. Transmittal of 

3 Studies.

47157301 Braverman, M. (2007) Group A - Product Analysis Test Guidelines: C5 Honeysweet Plum. 

Project Number: 0377B. Unpublished study prepared by IR-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 Honeysweet Plum Resistant to Plum Pox Virus (Plum 

Pox Viral Coat Protein Gene). Project Number: 0377B. Unpublished 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 HoneySweet Plum and the Petition for Tolerance of 

Plum Pox Virus Resistance Gene. Transmittal of 2 Studies.

47474902

Scorza, R.; Braverman, M. (2008) C5 HoneySweet 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 HoneySweet Plum: C5 HoneySweet Plum. Project Number: 0377B. 

Unpublished study prepared by 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 HoneySweet Plum. Transmittal of 1

Study.

47490601

Braverman, M. (2008) Group A-Product Analysis Test Guidelines: Amendment Number 1 

to MRID 471573-01: C5 HoneySweet 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 Potyvirus. Memorandum dated 

March 19, 2009.

Gagliardi, J. V. Environmental Risk Assessment for C5 HoneySweet 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 'HoneySweet' 

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 Hybridization in Prunus. 

Proc. Int. Symp. On Molecular Markers (Dore, Dosba and Baril, Eds.). Acta Hort. 546, ISHS 

2001.

Ravelonandro, M., P. Briard and R. Scorza. 2001. Significant Resistance of Transgenic Plums 

Against the Four Serotypes of Plum Pox Potyvirus. Proc. 18th Int. Symp. On Fruit Tree Virus 

Diseases (M.F. Clark ed.), Acta Hort. 550, ISHS 2001.

Scorza, R. M. Ravelonandro, A.M. Callhan, J. Cordts, M. Fuchs, J. Dunez and D. Gonsalves. 

1994. Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene. 

Plant Cell Reports 14: 18-22.

Scorza, R., A.M. Callahan, L. Levy, V. Damsteegt and M. Ravelonandro. 1998. Transferring 

Potyvirus Coat Protein Genes through Hybridization of Transgenic Plants to Produce Plum pox 

virus Resistant plums (Prunus domestica L.). Acta Hort 472: 421-427.

Scorza, R., A. Callahan, L. Levy, V. Damsteegt, K. Webb and M. Ravelonandro. 2001. PostTranscriptional Gene Silencing in Plum Pox Virus Resistant Transgenic European Plum 

Containing the Plum Pox Potyvirus Coat Protein Gene. Transgenic Research 10: 201-209.

Zhang, T., M. Breitbart, W.H. Lee1, J.-Q. Run, C.L. Wei, S.W.L. Soh, M.L. Hibberd, E.T. Liu, 

F. Rohwer, Y. Ruan. 2006. RNA Viral Community in Human Feces: Prevalence of Plant 

Pathogenic Viruses. PLOS Biology 4(a): 108-118.