Document ID: EPA-HQ-OPP-2010-0554-0068
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2010-06-30T04:00Z

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

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	

Satisfaction of the Conditions of Registration				14

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

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

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

	

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

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-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 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.  [ HYPERLINK
"http://www.apsnet.org/online/feature/PlumPox/plumpoxfs.pdf"
http://www.apsnet.org/online/feature/PlumPox/plumpoxfs.pdf ].  Various
other non-Prunus dicotyledonous plants have been infected under
experimental conditions [ HYPERLINK
"http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/00.057.0.01.054.htm"
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 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).	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
Service-Appalachian 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.

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,” (  HYPERLINK
"http://www.aphis.usda.gov/brs/aphisdocs/04_26401p.pdf" 
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, mirror-image 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.

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 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.  

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   HYPERLINK
"http://www.epa.gov/compliance/environmentaljustice/index.html" 
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 plant-incorporated 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 (  HYPERLINK
"http://www.heinonline.org/HOL/Page?public=false&handle=hein.fedreg/0510
43&men_hide=false&men_tab=citnav&collection=fedreg&page=7628"  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. (  HYPERLINK
"http://usda.mannlib.cornell.edu/usda/current/NoncFruiNu/NoncFruiNu-01-2
2-2010_revision.pdf" 
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 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
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).

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).

	

	         

APPENDIX A:  PRODUCT CHARACTERIZATION (  HYPERLINK
"http://ecfr.gpoaccess.gov/cgi/t/text/text-idx?c=ecfr&sid=f25d5426f66f63
ffeb9c986db92bf243&rgn=div8&view=text&node=40:23.0.1.1.9.16.1.3&idno=40"
 40 CFR § 158.2120 )

I.	Manufacturing Process

μg/mL  kanamycin and GUS at 37oC) 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 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/PPV-CP-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.  

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 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 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.

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 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.

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 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 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.

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
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. 

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 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.

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 CPG-PPV 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.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

	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.

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.

 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.

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.

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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. Post-Transcriptional 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.

Coat Protein Gene of Plum Pox Virus

Biopesticides Registration Action Document 

Proposed Decision

March 29, 2010

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