[Federal Register: April 18, 2007 (Volume 72, Number 74)]
[Proposed Rules]
[Page 19640-19660]
[DOCID:fr18ap07-19]

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ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 174

[EPA-HQ-OPP-2006-0643; FRL-8100-5]
RIN 2070-AD49

Exemption from the Requirement of a Tolerance under the Federal
Food, Drug, and Cosmetic Act for Residues of Plant Virus Coat Proteins
that are Part of a Plant-Incorporated Protectant (PVC-Proteins);
Supplemental Proposal

AGENCY:  Environmental Protection Agency (EPA).

ACTION:  Proposed rule.

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SUMMARY:  EPA is proposing to exempt from the Federal Food, Drug, and
Cosmetic Act (FFDCA) section 408 requirement of a tolerance, residues
of coat proteins from viruses that naturally infect plants that humans
consume when such coat proteins are produced in living plants as part
of a plant-incorporated protectant (PIP) and the criteria proposed for
this exemption are met. EPA believes there is a reasonable certainty
that no harm will result from aggregate exposure to such residues,
including all anticipated dietary exposures and all other exposures for
which there is reliable information. This proposed exemption would
eliminate the need to establish a maximum permissible level in food for
these residues.

DATES:  Comments must be received on or before July 17, 2007.

ADDRESSES:  Submit your comments, identified by docket identification
(ID) number EPA-HQ-OPP-2006-0643, by one of the following methods:
      Federal eRulemaking Portal: http://www.regulations.gov.

Follow the on-line instructions for submitting comments.
      Mail: Office of Pesticide Programs (OPP) Regulatory
Public Docket (7502P), Environmental Protection Agency, 1200
Pennsylvania Ave., NW., Washington, DC 20460-0001.
      Delivery: OPP Regulatory Public Docket (7502P),
Environmental Protection Agency, Rm. S-4400, One Potomac Yard (South
Bldg.), 2777 S. Crystal Dr., Arlington, VA. Deliveries are only
accepted during the Docket's normal hours of operation (8:30 a.m. to 4
p.m., Monday through Friday, excluding legal holidays). Special
arrangements should be made for deliveries of boxed information. The
Docket Facility telephone number is<PHONE_NUMBER>.
     Instructions: Direct your comments to docket ID number
EPA-HQ-OPP-

2006-0643. EPA's policy is that all comments received will be included
in the docket without change and may be made available on-line at
http://www.regulations.gov, including any personal information

provided, unless the comment includes information claimed to be
Confidential Business Information (CBI) or other information whose
disclosure is restricted by statute. Do not submit information that you
consider to be CBI or otherwise protected through regulations.gov or e-
mail. The regulations.gov website is an ``anonymous access'' system,
which means EPA will not know your identity or contact information
unless you provide it in the body of your comment. If you send an e-
mail comment directly to EPA without going through regulations.gov,
your e-mail address will be automatically captured and included as part
of the comment that is placed in the docket and made available on the
Internet. If you submit an electronic comment, EPA recommends that you
include your name and other contact information in the body of your
comment and with any disk or CD-ROM you submit. If EPA cannot read your
comment due to technical difficulties and cannot contact you for
clarification, EPA may not be able to consider your comment. Electronic
files should avoid the use of special characters, any form of
encryption, and be free of any defects or viruses.
    Docket: All documents in the docket are listed in the docket index.
Although listed in the index, some information is not publicly
available, e.g., CBI or other information whose disclosure is
restricted by statute. Certain other material, such as copyrighted
material, is not placed on the Internet and will be publicly available
only in hard copy form. Publicly available docket materials are
available either in the electronic docket at http://www.regulations.gov
, or, if only available in hard copy, at the OPP

Regulatory Public Docket in Rm. S-4400, One Potomac Yard (South Bldg.),
2777 S. Crystal Dr., Arlington, VA. The hours of operation of this
docket facility are from 8:30 a.m. to 4 p.m., Monday through Friday,
excluding legal holidays. The Docket Facility telephone number is (703)
305-5805.

FOR FURTHER INFORMATION CONTACT:  Melissa Kramer, Hazard Assessment
Coordination and Policy Division (7202M), Office of Science
Coordination and Policy, 1200 Pennsylvania Ave., NW., Washington, DC
20460-0001; telephone number:<PHONE_NUMBER>; fax number: (202) 564-
8502; e-mail address<EMAIL_ADDRESS>
SUPPLEMENTARY INFORMATION:

I. General Information

A. Does this Document Apply to Me?

    You may be potentially affected by this action if you are a person
or company involved with agricultural biotechnology that may develop
and market PIPs. Potentially affected entities may include, but are not
limited to:
     Pesticide and other agricultural chemical manufacturing
(NAICS code 32532), e.g., establishments primarily engaged in the
formulation and preparation of agricultural and household pest control
chemicals.
     Food manufacturing (NAICS code 311), e.g., establishments
primarily engaged in the manufacturing of food or feed.
     Crop production (NAICS code 111), e.g., establishments
primarily engaged in growing crops, plants, vines, or trees and their
seeds.
     Colleges, universities, and professional schools (NAICS
code 611310), e.g., establishments of higher learning which are engaged
in development and marketing of virus-resistant plants.
     Research and development in the physical, engineering, and
life sciences (NAICS code 54171), e.g., establishments primarily
engaged in conducting research in the physical, engineering, or life
sciences, such as agriculture and biotechnology.
    This listing is not intended to be exhaustive, but rather provides
a guide for readers regarding entities likely to be affected by this
action. Other types of entities not listed in this unit could also be
affected. The North American Industrial Classification System (NAICS)
codes have been provided to assist you and others in determining
whether or not this action might apply to certain entities. To
determine whether you or your business may be affected by this action,
you should carefully examine the applicable provisions of 40 CFR part
174. If you have questions regarding the applicability of this action
to a particular entity, consult the person listed under FOR FURTHER
INFORMATION CONTACT.

B. What Should I Consider as I Prepare My Comments for EPA?

    1. Docket. EPA has established a docket for this action under
docket ID number EPA-HQ-OPP-2006-0643. Publicly available docket
materials are available either in the electronic docket at http://www.regulations.gov
, or, if only available in hard copy, at the Office

of Pesticide Programs (OPP) Regulatory Public Docket in Rm. S-4400, One
Potomac Yard (South Bldg.), 2777 S. Crystal Dr., Arlington, VA. The
hours of operation of this docket facility are from 8:30 a.m. to 4
p.m., Monday through Friday, excluding legal holidays. The Docket
Facility telephone number is<PHONE_NUMBER>.
    2. Tips for preparing your comments. When submitting comments,
remember to:
    i. Identify the document by docket ID number and other identifying
information (subject heading, Federal Register date and page number).
    ii. Follow directions. The Agency may ask you to respond to
specific questions or organize comments by referencing a Code of
Federal Regulations (CFR) part or section number.
    iii. Explain why you agree or disagree; suggest alternatives and
substitute language for your requested changes.
    iv. Describe any assumptions and provide any technical information
and/or data that you used.
    v. If you estimate potential costs or burdens, explain how you
arrived at your estimate in sufficient detail to allow for it to be
reproduced.
    vi. Provide specific examples to illustrate your concerns and
suggest alternatives.
    vii. Explain your views as clearly as possible, avoiding the use of
profanity or personal threats.
    viii. Make sure to submit your comments by the comment period
deadline identified.

II. What Action is the Agency Proposing?

    EPA is proposing to exempt the following from the FFDCA section 408
requirement of a tolerance: Residues of coat proteins from viruses that
naturally infect plants that humans consume as part of a normal diet,
including any metabolites or degradates of those coat proteins, when
such coat proteins are produced in living plants as part of a PIP and
the criteria proposed for this exemption are met. The proposed criteria
are intended to clearly identify and exempt only those residues for
which a long history of safe exposure and consumption can support
exemption. EPA believes there is a reasonable certainty that no harm
will result from aggregate exposure to such residues, including all
anticipated dietary exposures and all other exposures for which there
is reliable information. This proposed exemption would eliminate the
need to establish a maximum permissible level in food for these
residues.

III. What is the Agency's Authority for Taking this Action?

    EPA is proposing to establish this tolerance exemption on its own
initiative under sections 408(e) and (c) of FFDCA, 21 U.S.C. 346a(c)
and (e). Under FFDCA section 408, EPA regulates pesticide chemical
residues by establishing tolerances limiting the amounts of residues
that may be present in or on food or by establishing exemptions from
the requirement of a tolerance for such residues. Food includes
articles used for food or drink by humans or animals. A food containing
pesticide residues may not be moved in interstate commerce without an
appropriate tolerance or an exemption from the requirement of a
tolerance.
    Section 408 of FFDCA applies to all ``pesticide chemical
residues,'' which are defined as residues of either a ``pesticide
chemical'' or ``any other added substance that is present on or in the
commodity or food primarily as a result of the metabolism or other
degradation of a pesticide chemical'' (21 U.S.C. 321(q)(2)). FFDCA
defines ``pesticide chemical'' as: ``any substance that is a pesticide
within the meaning of the Federal Insecticide, Fungicide, and
Rodenticide Act, including all active and inert ingredients of such
pesticide'' (21 U.S.C. 321(q)(1)). The Federal Insecticide, Fungicide,
and Rodenticide Act (FIFRA) section 2(u) defines ``pesticide'' as:
``(1) any substance or mixture of substances intended for preventing,
destroying, repelling, or mitigating any pest, (2) any substance or
mixture of substances intended for use as a plant regulator, defoliant,
or desiccant, and (3) any nitrogen stabilizer. . .'' (7 U.S.C. 136(u)).
Under FIFRA section 2(t), the term ``pest'' includes: ``(1) any insect,
rodent, nematode, fungus, weed, or (2) any other form of terrestrial or
aquatic plant or animal life or virus, bacteria, or other
microorganism. . . which the Administrator declares to be a pest. . .''
subject to certain exceptions (7 U.S.C. 136(t)).
    Section 408(c)(2)(A)(i) of 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 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) of FFDCA, 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) of FFDCA, 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 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.
    Section 408(e)(1)(C) of FFDCA also grants EPA the authority to
establish ``general procedures and requirements to implement this
section'' (21 U.S.C. 346a(e)(1)(C)).

IV. Context

A. What is the Relationship of this Proposal to Other Regulatory
Requirements under FIFRA and FFDCA?

    When the genetic material that encodes an entire or a portion of a
plant virus coat protein is introduced into living plants with the
intention of preventing or mitigating viral disease in the plants, the
genetic material and any substances produced from the genetic material
constitute a type of pesticide termed a ``plant virus coat protein
plant-incorporated protectant'' or ``PVCP-PIP.'' PVCP-PIPs meet the
FIFRA section 2(u) definition of ``pesticide'' because they are
introduced into plants with the intention of ``preventing, destroying,
repelling, or mitigating any pest. . .'' (7 U.S.C. 136(u)) and plant
viruses meet the FIFRA section 2 definition of ``pest'' (7 U.S.C.
136(t)). PVCP-PIPs are considered pesticide chemicals under FFDCA which
defines a ``pesticide chemical'' as ``any substance that is a pesticide
within the meaning of the Federal Insecticide, Fungicide, and
Rodenticide Act, including all active and inert ingredients of such
pesticide.'' As such, residues of PVCP-PIPs in or on food (hereinafter
simply ``in food'') are subject to FFDCA section 408.
    Since PVCP-PIPs are a relatively newly described type of pesticide,
the discussion in this unit provides information explaining how this
FFDCA proposed action on residues of the plant virus coat protein
portion of a PVCP-PIP (called here the ``PVC-protein'') would affect
the FFDCA and FIFRA status of the complete PVCP-PIP. To this end,
several pieces of information are presented: A description of the
anticipated residues of PVCP-PIPs; a discussion of the FFDCA status,
either current or proposed, of all anticipated PVCP-PIP residues; a
discussion of what would be considered in determining the FFDCA status
of the complete PVCP-PIP; and a discussion of how the FFDCA status of
PVCP-PIP residues relates to the FIFRA status of the PVCP-PIP.
    1. What are the components of a PIP? A PIP is defined at 40 CFR
174.3 as ``a pesticidal substance that is intended to be produced and
used in a living plant, or in the produce thereof, and the genetic
material necessary for production of such a pesticidal substance. It
also includes any inert ingredient contained in the plant, or produce
thereof.''
    2. What are the anticipated residues of PVCP-PIPs? Based on the
definition of a PIP, EPA anticipates residues of a PVCP-PIP would
include residues of any PVC-protein; the nucleic acids associated with
the PVCP-PIP, e.g., the genetic material encoding the PVC-protein; and
any inert ingredient as defined for PIPs at 40 CFR 174.3. Each of these
three classes of residues will also include any metabolite and
degradate of that class in accordance with FFDCA section 201 that
defines a ``pesticide chemical residue'' as ``a residue in or on raw
agricultural commodity or processed food of (A) a pesticide chemical;
or (B) any other added substance that is present on or in the commodity
or food primarily as a result of the metabolism or other degradation of
a pesticide chemical'' (21 U.S.C. 321(q)(2)).
    3. What is the FFDCA status of each identified class of residues?
For the complete PVCP-PIP to be exempt from FFDCA section 408, all
three classes of PVCP-PIP residues listed above must be exempt, i.e.,
residues of the PVC-protein, the nucleic acids associated with the
PVCP-PIP, and any inert ingredient as defined for PIPs at 40 CFR 174.3.
The units below discuss the status of residues of the PVC-protein under
this proposed action, the status of residues of the nucleic acids
associated

with the PVCP-PIP, and the status of residues of inert ingredients.
    i. Residues of PVC-proteins. Residues in this category consist of
residues of the PVC-protein and any metabolites or degradates of that
protein. This proposal would exempt from tolerance requirements
residues of PVC-proteins that meet certain criteria.
    Coat proteins are those substances that viruses produce to
encapsulate and protect the viral nucleic acid and to perform other
important tasks for the virus, e.g., assistance in viral replication,
movement within the plant, and transmission of the virus from plant to
plant by insects (Ref. 1). Current scientific information suggests that
prevention or mitigation of disease by some PVCP-PIPs may be protein-
mediated because for certain PVCP-PIPs efficacy is correlated with the
concentration of coat protein produced by the transgene (Ref. 2). In
protein-mediated resistance, the coat protein is thought to impede the
infection cycle by interfering with the disassembly of infecting
viruses (Ref. 3). In such cases, EPA would consider the PVC-protein to
be the pesticidal substance. Residues of such PVC-proteins and their
metabolites and degradates that meet the proposed criteria would be
covered by this proposal.
    In transgenic plants employing a second mechanism of resistance
called post-transcriptional gene silencing (PTGS), prevention or
mitigation of viral disease is not correlated with the level of PVC-
protein expression. Indeed, virus resistance can occur even when a coat
protein gene expresses untranslatable RNA sequences and no PVC-protein
is detected. In PTGS, RNA fragments appear to be pesticidal substances
(Ref. 3). (See Unit II.E. of the companion document published elsewhere
in this Federal Register for a more detailed description of PTGS.) Even
when PTGS is the mechanism of resistance, any PVC-protein that might be
produced is part of the PVCP-PIP. Residues of such PVC-proteins and
their metabolites and degradates that meet the proposed criteria are
also covered by this proposal.
    ii. Residues of nucleic acids. Residues in this category include
residues of the genetic material necessary for the production of the
pesticidal substance and the genetic material for any inert ingredient
as defined at 40 CFR 174.3. Residues in this category would also
include residues of any nucleic acids effecting the pesticidal action
of the PVCP-PIP, e.g., residues of nucleic acids involved in PTGS.
     ``Nucleic acids'' are defined at 40 CFR 174.3 as ``ribosides or
deoxyribosides of adenine, thymine, guanine, cytosine, and uracil;
polymers of the deoxyribose-5'-monophosphates of thymine, cytosine,
guanine, and adenine linked by successive 3'-5' phosphodiester bonds
(also known as deoxyribonucleic acid); and polymers of the ribose-5'-
monophosphates of uracil, cytosine, guanine, and adenine linked by
successive 3'-5' phosphodiester bonds (also known as ribonucleic acid).
The term does not apply to nucleic acid analogues (e.g.,
dideoxycytidine), or polymers containing nucleic acid analogues.''
Nucleic acids are currently exempt from FFDCA tolerance requirements.
See 40 CFR 174.475 and 66 FR 37817 (July 19, 2001) (FRL-6057-5). EPA is
not proposing to amend this exemption.
    iii. Residues of any inert ingredient. Residues in this category
consist of residues of any inert ingredient that is part of a PVCP-PIP
and any metabolite or degradate of an inert ingredient. An inert
ingredient for a PIP is defined at 40 CFR 174.3 as ``any substance,
such as a selectable marker, other than the active ingredient, where
the substance is used to confirm or ensure the presence of the active
ingredient, and includes the genetic material necessary for the
production of the substance, provided that genetic material is
intentionally introduced into a living plant in addition to the active
ingredient.''
    A tolerance or tolerance exemption is required for residues of any
substance in food that meets the 40 CFR 174.3 definition of an inert
ingredient (e.g., a selectable marker intentionally introduced into the
plant as part of a PVCP-PIP such as a protein conferring resistance to
an herbicide). Part 180 and part 174, subpart W, of 40 CFR list inert
ingredients for which tolerance exemptions have been established. If an
inert ingredient is not listed at part 180 or part 174, subpart W, an
applicant would need to petition the Agency in accordance with 40 CFR
180.7 to obtain a tolerance or tolerance exemption for residues of that
particular inert ingredient in order for food containing residues of
the PVCP-PIP to move in interstate commerce--even if all other residues
of the PIP are exempt.
    4. What is the relationship between the FIFRA status of a PVCP-PIP
and the FFDCA status of its residues? A tolerance exemption does not
exempt a PVCP-PIP from FIFRA regulation. However, in order for a PVCP-
PIP in food plants to be exempted from FIFRA regulation, a tolerance
exemption must exist for all residues associated with a PVCP-PIP or
FFDCA requirements must be otherwise met. (See the general
qualification for exemption under FIFRA at 40 CFR 174.21(b).) The FIFRA
status of a PVCP-PIP is determined based on factors in addition to
FFDCA section 408 considerations because FIFRA requires the Agency to
consider additional risk and benefit issues beyond those addressed
under section 408 of FFDCA. Concurrently with this proposed FFDCA
exemption, the Agency is publishing a proposal under which PVCP-PIPs
might meet the general qualification for FIFRA exemption at 40 CFR
174.21(a) based on different criteria than the criteria in this
proposal.

B. What is the History of this Proposal?

    1. Scientific input. EPA sponsored (or cosponsored with other
Federal agencies) six conferences relevant to development of this
proposed rule: On October 19-21, 1987, a meeting on ``Regulatory
Considerations: Genetically-Engineered Plants'' at Cornell University
in Ithaca, NY; on September 8-9, 1988, a ``Transgenic Plant
Conference'' in Annapolis, MD; on November 6-7, 1990, a conference on
``Pesticidal Transgenic Plants: Product Development, Risk Assessment,
and Data Needs'' in Annapolis, MD; on April 18-19, 1994, a ``Conference
on Scientific Issues Related to Potential Allergenicity in Transgenic
Food Crops'' in Annapolis, MD; on July 17-18, 1997, a ``Plant Pesticide
Workshop'' in Washington, DC; and on December 10-12, 2001, a conference
on ``Assessment of the Allergenic Potential of Genetically Modified
Foods'' in Chapel Hill, NC. Information from these conferences has been
incorporated as appropriate in development of this proposed rule.
    EPA has requested the advice of two scientific advisory groups at
five meetings while developing its approach to PIPs. On December 18,
1992, EPA convened the FIFRA Scientific Advisory Panel (SAP) to review
a draft policy on PIPs (then called plant-pesticides) and to respond to
a series of related questions posed by the Agency dealing primarily
with EPA's approach under FIFRA. On July 13, 1993, EPA requested the
advice of a Subcommittee of the EPA Biotechnology Science Advisory
Committee (BSAC) on a series of scientific questions dealing with EPA's
approach to PIPs under FFDCA. On January 21, 1994, EPA asked for advice
on the Agency's approach to PIPs under both statutes at a joint meeting
of the SAP and the BSAC. To evaluate more recent scientific advances,
EPA again brought these issues to the SAP on October 13-14, 2004. On
December 6-8, 2005, EPA requested the SAP to respond to a series of
scientific

questions related to this proposal. EPA carefully considered advice
from all five meetings in the development of this proposed rule.
    2. Federal Register documents. The history of this proposal
consists of the original proposed exemption that appeared in the
November 23, 1994 Federal Register (59 FR 60545) (FRL-4755-4), a
supplemental document that appeared in the May 16, 1997 Federal
Register (62 FR 27149) (FRL-5716-6), and a supplemental document which
appeared in the July 19, 2001Federal Register (66 FR 37855) (FRL-6760-
4).
    i. November 23, 1994. EPA published a package of five separate
documents in the November 23, 1994 Federal Register which described
EPA's policy and proposals for PIPs under FIFRA and FFDCA (59 FR 60496,
60519, 60535, 60542, and 60545). In one of these documents (59 FR
60545), EPA proposed to exempt from the requirement of a tolerance,
residues of plant virus coat proteins produced and used in living
plants as a plant-incorporated protectant (then called a plant-
pesticide). The proposed exemption from the requirement of a tolerance
read as follows:
     ``Residues of coat proteins from plant viruses, or segments of the
coat proteins, produced in living plants as plant-pesticides are exempt
from the requirement of a tolerance'' (59 FR 60547).
    ii. May 16, 1997. In August of 1996, Congress enacted the Food
Quality Protection Act (FQPA), which amended FFDCA and FIFRA. On May
16, 1997, EPA published a supplemental document in the Federal Register
(62 FR 27149) to provide the public with an opportunity to comment on
EPA's analysis of how certain FQPA amendments to FFDCA and FIFRA apply
to the proposed exemption from the requirement of a tolerance for
residues of PVC-proteins.
    In that supplemental document, EPA explained how most of the
substantive factors that the amended FFDCA requires EPA to consider in
evaluating pesticide chemical residues had been considered in the
Agency's 1994 proposed tolerance exemption. Even though the Agency may
not have used the terminology specified in FQPA, EPA did take into
account most of the same factors in issuing its 1994 proposal to exempt
residues of PVC-proteins, or segments of such proteins, from FFDCA
tolerance requirements. EPA therefore sought comment on the
requirements imposed by FQPA that the Agency had not addressed in its
1994 proposal, specifically:
    a. EPA's conclusion that there are no substances outside of the
food supply that may have a cumulative toxic effect with residues of
PVC-proteins,
    b. EPA's conclusion that there are no substances outside of the
food supply to which humans might be exposed through non-occupational
routes of exposure that are related via a common mechanism of toxicity
to residues of PVC-proteins,
    c. Any available information on PVC-proteins causing estrogenic
effects,
    d. EPA's rationale, described in greater detail, for concluding
that PIPs are likely to present a limited exposure of pesticidal
substances to humans in which the predominant, if not the only, route
of exposure will be dietary, and
    e. EPA's rationale, described in greater detail, for concluding
that the Agency's analysis concerning the dietary safety of food
containing PVC-proteins applies to infants and children as well as
adults.
    iii. July 19, 2001. In July of 2001, EPA published a supplemental
document in the Federal Register (66 FR 37855) to provide the public
with additional opportunity to comment on the FIFRA and FFDCA
exemptions for PIPs that the Agency proposed in 1994 but had not yet
finalized by 2001. EPA also requested comment on the information,
analyses, and conclusions pertaining to PVCP-PIPs contained in the NRC
report entitled ``Genetically Modified Pest-Protected Plants: Science
and Regulation'' (Ref. 4). In addition, the public was given an
opportunity to comment on a clarification of the language in the
original 1994 proposal on PVCP-PIPs that EPA was considering in
response to public comment. The purpose of the clarification was to
circumscribe more clearly those residues proposed for exemption.
    The documents, including associated public comments, and the
reports of the meetings described above are available in the public
dockets established for each of the associated rulemakings as described
in Unit XII.B.
    This proposed rule completely supersedes these previous proposals.
EPA does not intend to respond to comments submitted on those
proposals. Thus, individuals who believe that any comments submitted on
any of the earlier proposals remain germane to this proposal should
submit them (or relevant portions) again during this comment period.

C. Rationale Supporting the Proposed FFDCA Tolerance Exemption

    EPA's base of experience with viruses infecting food plants has led
the Agency to draw three conclusions on which it is relying to support
this proposed tolerance exemption for residues of PVC-proteins in food.
First, virus-infected plants have always been a part of the human and
domestic animal food supply. Most crops are frequently infected with
plant viruses, and food from these crops has been and is being consumed
without adverse human or animal health effects. Second, plant viruses
are not infectious to humans, including children and infants, or to
other mammals. Third, plant virus coat proteins, while widespread in
food, have not been associated with toxic or allergenic effects to
animals or humans. These conclusions are derived from a base of
experience and information sufficient to support this proposed
tolerance exemption.
    1. 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 (Refs. 5, 6, 7, 8, 9, and 10). Most plants are
infected by at least one virus, and components of plant viruses,
including coat proteins, are often found in the produce of crop plants.
For example, at the beginning of this century virtually every
commercial cultivar of potatoes grown in the United States and Europe
was infected with either one or a complex of potato viruses (Ref. 10).
Even plants that show no disease symptoms are often found to be
infected with viruses (Refs. 9 and 11). In addition, a common
agricultural practice used since the 1920s for protection against viral
disease involves intentionally inoculating healthy plants with a mild
form of a virus in order to prevent infection by a more virulent form
(Ref. 11). A recent analysis of viral sequences isolated from fecal
samples of healthy humans showed the presence of large quantities of
plant pathogenic viruses from 35 different plant virus species with
evidence suggesting dietary origins for the most prevalent (Ref. 12). A
great deal of information supports the ubiquitous appearance of plant
viruses in foods, and to date there have been no reports of adverse
human or animal health effects 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 nontransgenic types of food'' (Ref. 4). The
FIFRA SAP addressed the issue of dietary risk at its December 18, 1992
meeting (Ref. 13). The SAP stated, ``Since 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 PVC-
proteins 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'' (Ref. 14). The 2005 SAP also agreed
that ``[h]istorically, virus infected plants have been a part of the
human and domestic animal food supply without adverse human or animal
health effects'' (Ref. 15).
    In general, EPA anticipates that dietary exposure through human and
animal consumption of plants containing residues of PVC-proteins that
would qualify for the proposed exemption will be similar to or less
than the dietary exposure to plant virus coat proteins currently found
in food plants naturally infected with viruses. Experiments have shown
the amount of PVC-protein found in plants containing a PVCP-PIP to be
as much as one hundred- to one thousand-fold lower than the amount of
plant virus coat protein found naturally in virus-infected plants, even
when the resistance is believed to be mediated by the PVC-protein
itself (Refs. 8 and 16). The difference in amount of PVC-protein
present is even more marked for virus-resistant plants employing
resistance mediated by RNA. In such cases, little to no detectable coat
protein is produced in a plant containing a PVCP-PIP (Refs. 3 and 17).
Such information conforms to information EPA has received from the
scientific advisory groups the Agency has consulted (see Unit IV.B.1.).
Although the Agency believes that the PVC-proteins which qualify for
this proposed tolerance exemption are safe at any level given the long
history of human dietary exposure to high levels of such proteins, the
anticipated low levels of exposure to PVC-proteins in food lend
additional support to this proposed exemption.
    2. Not infectious to humans. Any virus/host relationship is
characterized by a high degree of specificity (Ref. 8). Plant viruses
usually infect plants only within a certain taxonomic group and are
unable to infect humans or other vertebrates (Refs. 18 and 19).
Cellular machinery for processing genetic material is highly specific.
For example, plant viruses are unable to recognize and attach to the
specific sites on mammalian cells needed to penetrate the cell
membrane, and plant viruses cannot be processed by mammalian cellular
machinery. Plant viruses therefore do not and cannot infect mammals and
other vertebrates. In addition, multiple virus components in addition
to the coat protein have a role in and are necessary for plant
infection. Plant viral coat proteins alone are not infectious to
plants, and whole, intact plant viruses are not infectious to humans.
Therefore, it is reasonable to assume that a single component of plant
viruses, e.g., the PVC-protein, will not be infectious to humans.
    3. No toxic or allergenic effects to animals or humans. Humans and
domestic animals have been and are exposed to plant viruses in the food
supply because most crops are frequently infected with plant viruses.
Food from these crops has been and is being consumed with no indication
of human or animal toxicity related to plant virus infections.
Additionally, in experiments where purified plant virus preparations
have been injected into laboratory animals, no adverse effects have
been reported (Ref. 17). Furthermore, the Agency is not aware of any
coat protein from a virus that naturally infects plants that has been
identified as a food allergen for humans. Finally, the amount of PVC-
protein likely to be found in food is anticipated to generally be lower
than the amount of virus coat protein found in food naturally infected
with plant viruses (as discussed in Unit IV.C.1.).The 2005 SAP
questioned whether an increased propensity for allergies in humans
affects the relevance of the history of safe use to the current safety
of virus coat proteins. Several studies have documented a general
increase in atopy in human populations; these studies show that over
the last several decades there has been an increasing proportion of
human populations that have an allergic sensitization to particular
allergens (Refs. 20, 21, and 22). However, there is no reason to
believe that PVC-proteins in the environment would have any impact on
this phenomenon. EPA is aware of no evidence that previously
nonallergenic substances are now able to elicit an immune response, and
no plant virus coat proteins have ever been identified as allergens.
Moreover, the amount of plant virus coat protein in the environment is
not expected to increase due to the use of PVCP-PIPs. On the contrary,
PVCP-PIPs generally express PVC-protein at levels below that found in
natural virus infections, and the virus-resistant phenotype conferred
by PVCP-PIPs should significantly reduce levels of natural virus
infection in plants, thereby decreasing the amount of plant virus coat
protein in the environment where PVCP-PIPs are deployed.

D. Key Issue: Determination of Natural Virus Variation

    A key issue facing EPA in developing this exemption is how to
clearly describe for regulatory purposes those PVC-proteins that are
within the range of naturally occurring plant virus coat proteins and
to which the rationale discussed in Unit IV.C. therefore applies. If a
plant virus coat protein gene is isolated in nature and not modified,
the PVC-protein would clearly be within the range of natural variation.
However, many coat protein genes are modified in creating a PVCP-PIP,
e.g., to increase product efficacy or allow appropriate expression in
the plant. Some of these modifications may affect a PVC-protein,
although most of these variations would not be expected to differ
significantly (e.g., in terms of toxicity or allergenicity) from the
naturally occurring coat protein. In fact, given the considerable
variation in naturally occurring viral coat proteins, it is also
possible that naturally occurring plant viruses exist with some of the
minor modifications that could conceivably be introduced into PVC-
proteins.
    However, EPA's task of defining this variation is complicated by
the variable nature of plant virus genomes and the fact that the full
extent of variation for even a single plant virus is currently unknown.
Sequencing of plant virus genomes has revealed that a large number of
variants exist within most populations of both RNA and DNA viruses. Due
to this inherent heterogeneity in virus populations, they are often
described as ``quasispecies'' that exist as a pool of different
sequences varying around a consensus sequence (Refs. 23, 24, and 25).
    Genetic variation in virus populations arises due to several
processes including mutation, recombination, and reassortment. Mutation
is a change in the genetic material that most commonly occurs when
replication errors lead to incorporation of an incorrect nucleotide
into the daughter sequence (Ref. 26). New virus variants are also
generated by recombination, the natural process that occurs during
replication of DNA or RNA whereby new combinations of genes are
produced. Recombination is more likely to occur the more closely
related viruses are, but recombination between different viral species
is also believed to occur (Refs. 27 and 28). Evidence of past
recombination having led to the creation of new DNA and RNA viruses has
been

found in a number of different groups including bromoviruses (Ref. 29),
caulimoviruses (Ref. 30), luteoviruses (Ref. 31), nepoviruses (Ref.
32), cucumoviruses (Ref. 33), and geminiviruses (Refs. 27 and 34).
Sequence analysis of viruses from the family Luteoviridae indicated
that this family has evolved via both intra- and inter-familial
recombination (Ref. 35). In viruses with segmented genomes, variation
may also be caused by reassortment whereby entire segments are
exchanged between viruses (Ref. 36).
    Attempts to describe the range of variation for naturally occurring
plant virus coat proteins are complicated not only by variation within
species but also by variation among species (See Ref. 37 for review).
For example, cucumber mosaic cucumovirus (CMV) has a relatively high
degree of variation (Ref. 38) compared to tobacco mild green mosaic
tobamovirus (Ref. 39). The greater variability in CMV would be expected
based on the relatively wide host range and relatively high
recombination rate of this virus. Such wide-ranging, inherent
variability confounds attempts to establish meaningful estimates of
normal variability for coat proteins of plant viruses as a group.
    A large number of viral coat protein sequences are currently
available in the literature and in public sequence repositories, e.g.,
the National Center for Biotechnology Information. However, EPA has
concluded that no single standard could capture the degree of variation
across all viruses, and hundreds of plant viruses have been identified
to date (Ref. 40). It would be at best impractical for EPA to describe
individually for all virus groups all potential modifications that
would produce a PVC-protein that falls within the range of natural
variation given the vast (and yet still incomplete) amount of data that
currently exists. The 2005 SAP concurred with these conclusions:
``Currently, it is extremely difficult to identify modifications that
would be expected to be `within the range of natural variation for all
virus families'. This would require prior knowledge of the natural
variation limits of the individual PVC proteins, which is not
available. Specific modifications can be identified that would raise
potential concerns, but it is not clear that it is possible to create a
comprehensive list of these changes for all virus families'' (Ref. 15).
    At the present time, insufficient information exists to develop a
standard that would describe a priori the degree to which a PVC-protein
could be modified and yet still remain within the natural variability
of plant virus coat proteins found in virus populations either
generally or for any species in particular. In light of this, and
relying extensively on the advice of the 2005 FIFRA SAP meeting (Ref.
15), EPA has developed two proposals to exempt PVC-protein residues
from the requirement of a tolerance:
    1. A categorical exemption for a subset of PVC-proteins based on
developer self-determination that the encoded PVC-protein is virtually
unmodified when compared to an entire unmodified coat protein from a
virus that naturally infects plants that humans consume in toto or in
part, and
    2. An exemption for more extensively modified proteins that is
conditional on an Agency determination after review that the encoded
PVC-protein is minimally modified when compared to an unmodified coat
protein from a virus that naturally infects plants that humans consume
in toto or in part.

E. Structure of the Proposed FFDCA Tolerance Exemption

    1. Proposed categorical exemption. Under the proposed exemption at
Sec.  174.477(a), when the encoded PVC-protein is virtually unmodified
when compared to an entire unmodified coat protein from a virus that
naturally infects plants that humans consume in toto or in part, the
residues of the PVC-protein would be exempt from the requirement of a
tolerance without Agency review. If the PVC-protein is expressed from a
plant virus coat protein gene that was isolated from a virus found
naturally in a food plant in the United States and was not modified,
the PVC-protein would meet this criterion. Additionally, a PVC-protein
would meet this criterion if the developer has evidence showing it has
an amino acid sequence that is virtually unmodified when compared to an
unmodified plant virus coat protein sequence from a virus that
naturally infects plants that humans consume, e.g., as found in a
database. Although EPA cannot a priori identify all existing natural
coat protein variants, the requirement of being virtually unmodified
when compared to an entire unmodified coat protein ensures that the
exempted PVC-protein falls within the existing base of experience on
which the proposed exemption relies.
    EPA intends, with the requirement that the PVC-protein be virtually
unmodified when compared to ``an entire unmodified coat protein,'' to
exclude from the categorical exemption residues of modified PVC-
proteins, e.g., PVC-proteins containing insertions, deletions, or amino
acid substitutions (except as described below by the definition of
virtually unmodified), as well as chimeric PVC-proteins that are
encoded by a sequence constructed by fusing portions of two or more
plant virus coat protein genes. EPA is proposing to exclude such PVC-
proteins from the categorical exemption because of advice from the 2005
SAP that insufficient information exists at this time to allow EPA to
describe a priori a single standard articulating which of these types
of changes would be consistently expected to fall within the natural
range of variation of viruses and/or which types of changes could be
determined not to affect toxicity or allergenicity without any EPA
review (see Unit IV.D.).
    The Agency proposes to define the term ``unmodified'' to mean,
``having or coding for an amino acid sequence that is identical to an
entire coat protein of a naturally occurring plant virus.'' The Agency
is considering several options for defining the term virtually
unmodified. Under this proposal, any virtually unmodified PVC-protein
would qualify for a tolerance exemption without Agency review. Under
one option, this term would mean, ``having or coding for an amino acid
sequence that is identical to an entire coat protein of a naturally
occurring plant virus, except for the addition of one or two amino
acids at the N- and/or C-terminus other than cysteine, asparagine,
serine, and threonine and/or the deletion of one or two amino acids at
the N- and/or C-terminus.'' As noted by the 2005 SAP, the terminal ends
of a protein ``are the least structurally constrained regions of a
protein. As such, the ends can be thought of as being essentially
`unstructured,' and therefore unlikely to serve as allergenic epitopes
or to make major contributions to the overall structure of the
molecule. Addition (or deletion) of one or two amino acids is unlikely
to change this.'' However, the SAP also noted the possibility that the
addition of amino acids such as cysteine with side chains that could
promote cross-linking or aggregation between molecules or other amino
acids that can serve as sites for post-translational modifications
should be evaluated on a case-by-case basis (Ref. 15). EPA has
identified cysteine, asparagine, serine, and threonine as the amino
acids containing side chains that could promote cross-linking or serve
as sites for post-translational modifications. EPA therefore excludes
the addition of these amino acids from the proposed definition of
virtually unmodified. The 2005 SAP report mentioned alanine as an amino
acid involved in

glycosylation; however, EPA has found no evidence that alanine is
involved in glycosylation or promotes cross-linking. The Agency has
therefore not excluded the addition of alanine under the definition of
virtually unmodified.
    The Agency is also considering two possible changes to the above
definition of virtually unmodified. The first change would remove the
restriction that cysteine, asparagine, serine, or threonine may not be
added to the naturally occurring protein. Under this alternative, a
PVC-protein would qualify for the tolerance exemption without Agency
review if it has an amino acid sequence that is identical to an entire
coat protein of a naturally occurring plant virus except for the
addition, substitution, or deletion of one or two amino acids at the N-
and/or C-terminus. The rationale underlying such an alternative would
be that addition of any amino acid to the N- or C-terminus, e.g.,
including those that could be glycosylated, is unlikely to introduce
any concern. In order for an amino acid to be glycosylated, a protein
must also have a specific enzyme recognition site. The creation of such
a recognition site by the addition, substitution, or deletion of one or
two amino acids, particularly at the end of the protein, is expected to
be extremely rare because it would involve randomly producing a set of
amino acids involved in a specific interaction. The addition of an
amino acid with a side group that is capable of forming a covalent
bond, e.g., cysteine, is likewise unlikely to alter the safety of the
expressed protein. Such amino acid residues would typically be
unavailable due to interactions that occur within the protein's normal
folding conformation. A plant virus coat protein is large enough that
protein functionality or chemistry would not be dramatically different
from a PVC-protein that is identical except for its possessing two
additional amino acids at the N- and/or C-terminus. As previously
stated, the 2005 SAP said the terminal ends of a protein ``are the
least structurally constrained regions of a protein'' (Ref. 15). In
addition, virus coat proteins are self-assembling, structural proteins
that contain elements necessary for continual infection and replication
of the entire virus particle. As a structural element of a virus
particle, one important function of the coat protein is the ability to
interact with itself to form stable particles. Most if not all plant
virus coat proteins will naturally aggregate (Refs. 41 and 42), so the
addition of amino acids that could promote cross-linking or aggregation
would not fundamentally change the nature of the PVC-protein.
    The second change to the above definition of virtually unmodified
that the Agency is considering would allow truncated proteins to fall
under the definition. Under this alternative, a PVC-protein would be
exempt without Agency review if it has an amino acid sequence that is
identical to a single contiguous portion of a coat protein of a
naturally occurring plant virus, except for the addition or
substitution of one or two amino acids at the N- and/or C-terminus of
the single contiguous portion other than cysteine, asparagine, serine,
and threonine. EPA intends that ``identical to a single contiguous
portion'' would exclude proteins with internal modifications. The
rationale underlying such an alternative would be that truncated PVC
proteins have been reported to occur in nature (Ref. 43), as pointed
out by the 2005 SAP. ``Naturally occurring truncated forms of the PVCs
could be generated by post-transcriptional and translational events,
including incomplete translation due to routine errors causing a
ribosome to dissociate from an mRNA, post-translational processing, the
presence of a mutation that introduces a premature stop codon, or by
infrequent translation initiation at downstream AUGs. . . . Whether the
truncation is at the N- or C-terminus is not relevant to allergenicity
or toxicity'' (Ref. 15). The SAP also said, ``Determining whether PVC-
proteins containing terminal deletions, or any other modifications, are
within the range of natural variation would require the development of
a database of the natural variation and truncated forms of PVC-proteins
that occur naturally. If a truncated PVC-protein does fall within the
range of natural variation, the likelihood of increased toxicity and
allergenicity would be low'' (Ref. 15). However, such a database may
not be necessary because the potential for toxicity and allergenicity
of a whole plant virus coat protein is low enough that the likelihood
of a truncated form of such a protein being toxic or allergenic would
not rise to the level requiring regulation. Such a change in toxicity
or allergenicity would require the truncation to expose new allergenic
epitopes or specific recognition/binding sites in the protein that
could make the protein toxic, but there is no indication that plant
virus coat proteins possess such regions. The 2000 SAP indicated that
``[i]n general, peptide fragments that result from the breakdown of
proteins are less toxic than the intact protein'' (Ref. 44).
    Either of the changes discussed above could be adopted alone, or
both could be adopted together. If EPA adopts both changes, a PVC-
protein would be exempt from the requirement of a tolerance without
Agency review if it has an amino acid sequence that is identical to a
single contiguous portion of a coat protein of a naturally occurring
plant virus; except for the addition or substitution of one or two
amino acids at the N- and/or C-terminus of the single contiguous
portion.
    EPA is proposing to require that the virus used as the source of
the coat protein sequence ``naturally infects plants that humans
consume'' as an additional means of ensuring the proposed exemption is
limited to PVCP-PIPs that fall within the base of experience discussed
previously in this unit. This phrase is intended to limit the proposed
exemption to residues of PVC-proteins that are already part of the
normal human diet as naturally occurring plant virus coat proteins or
are minimally modified from such proteins (see Unit IV.C.1.). The
exemption would not extend to PVC-proteins encoded in part by sequences
from animal or human viruses.
    EPA proposes to define the term ``naturally infect'' to mean
``infect by transmission to a plant through direct plant-to-plant
contact (e.g., pollen or seed), an inanimate object (e.g., farm
machinery), or vector (e.g., arthropod, nematode, or fungus). It does
not include infection by transmission that occurs only through
intentional human intervention, e.g., manual infection in a laboratory
or greenhouse setting.'' The Agency is proposing this definition
specifically to exclude transmission that occurs only through
intentional human intervention because such transmission would have
little relevance to normal human dietary exposure. Viruses that may be
able to infect plant species in a laboratory or greenhouse setting
through manual infection may not ever infect such species in nature.
EPA intends to include within this definition viruses that are likely
to have been part of the human diet due to their ability to spread
without intentional human intervention. EPA recognizes that humans may
play an inadvertent role in infection (e.g., by transmitting the virus
on farm machinery). Such unintentional (and often unavoidable)
transmission can be an important means of virus transmission, leading
to the presence of natural virus coat proteins in food plants that
humans consume. EPA therefore includes this mode of transmission in the
definition of naturally infect to encompass those viruses that would be
expected to be at

least occasionally found in the plant and therefore be a normal
constituent of the human diet. To further clarify that the proposed
exemption applies only to coat proteins from plant viruses, EPA is
specifically including the word ``plant'' as an adjective in the name,
i.e., ``PVC-proteins'' are ``plant virus coat proteins.''
    EPA has considered whether to limit the proposed exemption to PVC-
proteins from PVCP-PIPs based on viruses that naturally infect the
particular food plant in which the PVC-protein is expressed. EPA must
address whether there would be any safety issues raised from exposure
to PVC-proteins if the virus used to create the PVCP-PIP does not
naturally infect the particular plant species into which the PVCP-PIP
is inserted. A PVC-protein may be expressed in a food plant that the
virus does not naturally infect when heterologous resistance to a
particular virus is conferred through a different virus' coat protein
gene (e.g., Ref. 45). However, the Agency believes such PVC-proteins
could be safely exempted from tolerance requirements because these
proteins would still reasonably be expected to be part of the normal
diet as long as they naturally infect plants used as food. Based on
their broad host range, plant viruses are known generally to infect a
wide variety of plants that humans consume. People generally eat a
broad range of food plants through which they would reasonably be
expected to be exposed to a wide variety of plant virus coat proteins
(Ref. 12). In addition, EPA is not aware that any plant viral coat
proteins have been identified as allergens, so it is unlikely that a
person with food allergies avoids a particular food plant because of an
allergic reaction to a viral coat protein. Based on this rationale and
in the absence of contravening evidence, EPA concludes that a PVC-
protein expressed in a plant that is not normally infected by the virus
from which the PVC-protein was derived would raise no safety issues as
long as the corresponding virus infects other plants that are consumed
by humans.
    When EPA asked the 2005 SAP to comment on this issue, the Panel
``expressed some disagreement as to whether the level of risk
associated with human exposure to any protein is solely dependent on
the protein itself. One Panel member concluded that the host producing
the protein is of secondary importance. Others expressed concern
related to expression of PVC-proteins in plants that are known to be
highly allergenic such as peanut'' (Ref. 15). The Panel did not
elaborate on the rationale for such concerns at this point in the SAP
report. EPA's interpretation of this issue is that the concern is due
to the possibility, articulated elsewhere in the Panel report, that
``the changed infectivity status of the plant may also induce changes
in the overall protein expression pattern of the plant. Thus, in
various tissues of the plant, natural plant proteins that have been
identified as allergens may be expressed to a different, and in some
cases, higher extent compared to a non-infected or a virus-infected
plant without PVCP-PIP. In particular, pathogenesis-related (PR)
proteins are known to be very inducible, and their expression levels
may vary many-fold. Several pathogenesis-related proteins have been
described as allergens (Breiteneder et al. 2000 and 2004), most notably
the major birch pollen protein Bet v1 (Breiteneder et al. 1989). An
increased expression of PR-proteins in pollen could increase both the
risk of sensitization and the risk of elicitation of allergic
reactions'' (Refs. 15, 46, 47, and 48). This concern is distinct from
the concern that EPA addressed above, namely that the PVC-protein
itself may introduce an allergen into a food source where it is not
anticipated to be found. The issue the SAP raised would generally be
addressed by the Food and Drug Administration (FDA) in evaluating food
composition. However, EPA has not found evidence that introduction of a
PVCP-PIP would affect induction of PR proteins per se. PR proteins are
a normal constituent of plants because plants express such proteins in
response to environmental stresses, including virus infection, exposure
to certain chemicals, and wounding. Some plant tissues even
constitutively express such proteins, e.g., those likely to be attacked
by pests or exposed to environmental stresses such as ultra-violet (UV)
irradiation (Ref. 49). Moreover, given the large number and variety of
pathogens (including viruses) encountered by plants in the field, and
given differences in the virus-infectivity status of plants that occur
naturally, humans consume varying amounts of PR proteins as part of the
normal diet. The level found in plants containing a PVCP-PIP is
therefore expected to be within the range of natural variation.
    EPA has also considered whether a geographic limitation on this
proposed categorical exemption would be necessary to ensure that the
exemption extends only to residues that are part of the U.S. diet;
i.e., that the proposed exemption would only extend to PVC-proteins
that are part of a PVCP-PIP constructed from a virus that occurs
naturally in the United States. EPA believes that such a limitation is
unnecessary to ensure that the PVC-proteins proposed for exemption fall
within the base of experience supporting the proposal. Humans have long
consumed viruses infecting food plants with no adverse effects. Given
the extent of modern market practices in which food is shipped globally
for human consumption, human dietary exposure to all viruses that
infect food plants is likely to occur broadly. The lack of any known
adverse effects attributable to plant viruses suggests that plant virus
coat proteins in the diet are safe to humans.
    EPA has also considered whether additional conditions are necessary
to ensure that the expression level of virtually unmodified PVC-
proteins found in plants is no greater than the level of plant virus
coat protein generally found in a natural virus infection. The 2005 SAP
suggested that ``for both modified and unmodified proteins, the Agency
might wish to consider. . . expression levels'' when determining
whether to exempt a PVC-protein from tolerance requirements (Ref. 15).
The SAP apparently based this suggestion on the assumption that EPA
considered exposure level to be an important component of a PVC-protein
risk assessment given that the Agency's background material for the
Panel indicated that the dietary exposure to PVC-proteins is
anticipated to be similar to or less than the dietary exposure to plant
virus coat proteins currently found in food plants naturally infected
with viruses. However, even though EPA addresses exposure level in
evaluating safety (e.g., see Unit IV.C.1.), the Agency also believes
that the PVC-proteins that qualify for this proposed exemption are safe
at any level that could be produced in a plant. Humans have been
exposed to plant virus coat proteins over long periods of time at
varying and sometimes high levels, and to date there is no indication
that any plant virus coat protein is an allergen or a toxin. The Agency
therefore believes that the hazard associated with PVC-proteins that
are virtually unmodified from natural plant viral coat proteins is
sufficiently low that it does not rise to the level warranting
regulation, even if in some cases exposure to a PVC-protein might be
greater than the exposure to the corresponding natural plant virus coat
protein. Nevertheless, the Agency regards the anticipated low levels of
exposure through food to the PVC-proteins covered by this proposal as
additional support for this proposed categorical exemption. According
to the 2005 SAP, ``On a per cell basis, it is

almost certain that all viral gene products are expressed at higher
levels in virus-infected than transgenic plants'' (Ref. 15).
    2. Proposed exemption conditional on Agency determination. The
Agency recognizes that product developers frequently modify the genetic
material of a PVCP-PIP, e.g., in order to achieve greater efficacy
(Ref. 50) and that most of these changes would be unlikely to result in
proteins affecting potential dietary risk. However, the Agency cannot
at this time articulate a criterion that would ensure all PVC-proteins
with such modifications fall within the base of experience supporting
the proposed exemption.
    The question of how to objectively define criteria on which the
regulated community may rely to determine a priori how much a virus
coat protein may be modified and still fall within the range of natural
variation is a key challenge. EPA first considered the question of how
to describe residues that fall within the base of experience supporting
exemption when the Agency issued its proposal on November 23, 1994 (59
FR 60539). In the July 19, 2001 supplemental notice (66 FR 37865), EPA
again addressed the question of how to describe PVCP-PIPs that fall
within the recognized base of experience supporting the proposed
categorical exemption.
    In October 2004, the FIFRA SAP was asked to consider the degree and
ways a plant virus coat protein gene might be modified while still
retaining scientific support for the idea that humans have consumed the
products of such genes for generations and that such products therefore
present no new dietary exposures (Ref. 14). They responded, ``There was
no clear consensus on how much change would be necessary to invalidate
this assumption, although there was general agreement that the
appropriate comparison is to the range of natural variation in the
virus population.'' The 2005 SAP also addressed this question. They
concurred that, ``it is extremely difficult to identify modifications
that would be expected to be `within the range of natural variation for
all virus families'. . . . Given the possible range of natural
variations for PVC proteins, it would be appropriate to assess whether
specific modifications are within natural variation limits of the PVC
protein on a case-by-case basis'' (Ref. 15).
    EPA believes that developing objectively defined criteria on which
the regulated community could rely to determine whether a modified PVC-
protein falls within the natural range of variation for a particular
virus is not currently feasible because the Agency knows of no
generally applicable, established baseline for what constitutes the
range of natural variation of a virus. EPA thus does not believe that
proposing an exemption that would allow developers to self-determine
eligibility of modified PVC-proteins would be supportable. Rather, EPA
is proposing that under proposed Sec.  174.477(b), the residues of such
a PVC-protein would be exempt only if the Agency determines after
review that the encoded PVC-protein is minimally modified when compared
to an unmodified coat protein from a virus that naturally infects
plants that humans consume in toto or in part.
    In determining whether a PVC-protein is minimally modified from a
natural viral coat protein, EPA will consider first how similar the
PVC-protein is to a natural viral coat protein by evaluating
information on the PVCP-PIP genetic construct, PVC-protein deduced
amino acid sequence, and biochemical characterization of the PVC-
protein as expressed in the plant (e.g., molecular weight to evaluate
potential post-translational modifications). EPA might also evaluate
developer-submitted analyses that characterize the PVC-protein sequence
relative to the range of natural coat protein variation found in public
sequence databases. Those PVC-proteins determined to be similar to a
natural viral coat protein would be further evaluated to determine
whether the modified PVC-protein is as safe as an unmodified protein by
considering information from an amino acid sequence comparison with
known protein toxins and allergens. The type and extent of information
that would need to be provided in order for EPA to determine whether a
PVC-protein is minimally modified and therefore qualifies for the
exemption would be determined on a case-by-case basis.
    The 2005 SAP identified certain modifications that might raise
potential concerns when considering if a protein is minimally modified,
including ``the addition or removal of protease recognition sites, the
addition or removal of cysteine residues involved in internal cross-
links, the addition or removal of proline residues that act as
secondary structure `break points,' and the addition or removal of
asparagines and alanines involved in glycosylation'' (Ref. 37). By
contrast, the report identified ``[m]odifications such as single amino
acid substitutions with biochemically similar amino acids that do not
affect secondary or tertiary structure'' as potentially being of
relatively little concern (Ref. 37). EPA would consider this guidance
as appropriate in evaluating individual exemption petitions to
determine whether a protein is minimally modified.
    Regarding the 2005 SAP suggestion that EPA might wish to consider
expression levels in determining whether to exempt a PVC-protein from
tolerance requirements, the Agency believes that such an evaluation is
not necessary to determine whether a PVC-protein is minimally modified.
EPA would necessarily have to find such proteins to be similar to a
natural viral coat protein in order for them to qualify for this
proposed exemption. EPA believes that minimally modified PVC-proteins
are safe at any level for the same reasons discussed above for
virtually unmodified proteins (Unit IV.E.1.). In both cases, the hazard
associated with PVC-proteins qualifying for this proposed tolerance
exemption is sufficiently low that it does not rise to the level
warranting regulation, even if in some cases exposure to a PVC-protein
might be greater than the exposure to the corresponding natural plant
virus coat protein. (However, see Unit XI. for a discussion of how
exposure level could possibly be considered under the proposed
exemption structure when reviewing minimally modified proteins.)
    Under proposed Sec.  174.477(b), the procedures for obtaining a
determination that a PVC-protein fits under the tolerance exemption
would be no different than those currently provided under the statute
for obtaining a tolerance exemption. A person can file a submission
requesting a determination (21 U.S.C. 346a(d)) of whether a particular
PVC-protein fits under the tolerance exemption, or the Agency can
initiate an action to issue a determination (21 U.S.C. 346a(e)). After
a person files a submission under FFDCA section 408(d)(1) proposing
that a particular PVC-protein falls under this exemption because it is
minimally modified from a natural plant virus coat protein, FFDCA
section 408(d)(3) requires that the Administrator determine whether a
petition meets the requirements of the statute and publish a summary of
the petition and other required information in the Federal Register
within 30 days of making that determination. Alternatively, the
Administrator may publish a notice of proposed rulemaking and provide a
period of generally not less than 60 days for public comment. In either
case, EPA will publish any final rule exempting a PVC-protein from the
requirement of a tolerance in the Federal Register and allow 60 days
for any person to file objections thereto (21 U.S.C. 346a(g)(2)).

Currently no fees would be associated with either the proposed
categorical exemption under Sec.  174.477(a) or the Agency's
determination under proposed Sec.  174.477(b) that a particular PVC-
protein fits under the tolerance exemption.
    For residues of a PVC-protein that would not qualify for this
proposed exemption under either Sec.  174.477(a) or (b) because the
Agency cannot determine that the encoded PVC-protein is minimally
modified from an unmodified coat protein from a virus that naturally
infects food plants, an applicant may petition the Agency for an
individual tolerance exemption under FFDCA section 408 (see also 40 CFR
180.7).

F. Tolerance Issues Associated with Unintended Protein Production when
Virus Resistance is Mediated through Post-Transcriptional Gene
Silencing

    Section 408 of the FFDCA does not require a tolerance or tolerance
exemption if residues will not be present in food moving in interstate
commerce. However, with the exception of residues that meet the
requirements proposed at Sec.  174.477(a), the mere fact that a
developer may not detect residues during product development will not
protect the food from seizure if residues are subsequently found
following commercialization, either because detection techniques
improve or because the protein is unexpectedly produced. If such an
event occurs and no tolerance exemption exists for residues of that
PVC-protein (regardless of its safety), any food containing the PVC-
protein residues would be adulterated and subject to seizure. In
addition, any FIFRA exemption that may have been applicable for the
PVCP-PIP would no longer be valid because 40 CFR 174.21(b) would no
longer be satisfied. Any sale or distribution of such a PVCP-PIP would
constitute sale and distribution of an unregistered pesticide, in
violation of FIFRA section 12(a)(1).
    The 2005 SAP suggested that the construction of certain PVCP-PIPs
may offer a reasonable level of assurance that PVC-protein production
would not occur, i.e., transgene insertions where the transcribed
segment lacks an initiator codon or insertions of transcribed inverted
repeat constructs that constitutively produce transcripts that are
folded into double-stranded RNA as the immediate product of transgene
transcription (Ref. 15). However, for other types of constructs,
questions remain about circumstances under which PVC-protein might be
detected and/or produced in food at some point after commercialization
even though PVC-protein may not have been detected and/or produced
during product development. For example, it is known that in some cases
PTGS must be triggered before transgene RNA production can be
effectively suppressed. Lindbo et al. (Ref. 51) used tobacco etch virus
(TEV) to infect transgenic tobacco plants containing a TEV coat protein
gene. Plants temporarily developed symptoms but were able to recover
from infection. Recovered transgenic plant tissue showed significantly
reduced levels of transgene mRNA, and PVC-protein was undetectable.
However, plant tissues unchallenged with virus did express PVC-protein,
suggesting that in at least some cases of PTGS-induced virus
resistance, PVC-protein may be produced until virus infection occurs.
B[eacute]clin et al. (Ref. 52) showed that in transgenic tobacco lines
expressing a [beta]-glucuronidase (uidA) transgene, suppression of
transgene expression always occurs but is initiated at different plant
developmental stages: Either 15 days after germination or 2 months
post-germination. Prior to PTGS initiation, transgenic protein is
expressed, suggesting that in at least some cases lack of protein
production may only occur after a certain developmental stage is
reached. Likewise, Pang et al. (Ref. 53) found that plant developmental
stage plays an important role in the timing of PTGS initiation.
    Experiments demonstrating that plant developmental stage determines
PTGS initiation suggest that any environmental factors influencing
plant growth would also affect the amount of time before RNA and
protein production is effectively suppressed. At least one experiment
has looked more directly at the influence of environmental factors on
PTGS. Szittya et al. (Ref. 54) demonstrated that cold temperatures
inhibited transgene-induced RNA silencing leading to increased levels
of transgene mRNA, although they did not report on the level of
transgenic protein.
    In addition to temporal changes in protein production that may be
influenced by varying environmental conditions, PTGS may also be
associated with variation in protein expression across different plant
tissues. Plant lines expressing a nitrate reductase transgene were
found to display PTGS in leaves and stem tissue but not in shoot apical
or axillary meristems (Ref. 52). As in other experiments (Ref. 51),
transgene protein was not detectable and transgene mRNA levels were
significantly reduced in plant tissue displaying PTGS. However, plant
tissue in which gene silencing does not occur showed normal levels of
transgene mRNA, and transgenic protein was produced.
    It has been shown that PTGS can be suppressed by viruses that
encode certain suppressor proteins leading to loss of the virus-
resistant phenotype conferred by a PVCP-PIP. For example, Savenkov and
Valkonen (Ref. 55) showed that resistance to Potato virus A (PVA) in
Nicotiana benthiana could be overcome when plants were challenged with
Potato virus Y (PVY). Although levels of transgene mRNA in healthy
transgenic plants were extremely low or below the detection limit,
transgene mRNA was readily detectable in PVY-infected plants where
suppression of gene silencing had apparently occurred. The study did
not report whether PVC-protein was produced from the transgene mRNA.
    The 2005 SAP was asked to comment on issues associated with protein
production in the case of plants containing a PVCP-PIP that confers
resistance through an RNA-mediated mechanism. The Panel responded that
``[g]iven the wide variety of conditions that can modulate the
transition from PTGS to no PTGS for non-[inverted repeat (IR)]
transgenes. . .it is likely that a non-IR transgene insertion that
retains an initiation codon for protein synthesis will make at least a
low level of protein in at least some plant tissues over the course of
its development, especially in the field where there is exposure to
environmental extremes and virus infections. Thus, these PVCP-PIP
plants may accumulate virus-derived mRNA and proteins in these
situations'' (Ref. 15). EPA notes that the Panel further concluded that
``[b]ecause of low levels of accumulation and sequence identity to the
natural viral pathotypes. . .these PVCP-PIPs pose similarly low risks''
as PVCP-PIPs that produce no protein (Ref. 15). However, any PVC-
protein residue in food that is not covered by a tolerance or tolerance
exemption would constitute an adulterant of the food supply
irrespective of the protein's safety or the level at which it is
detected.
    The above considerations suggest that many factors should be
considered in making a determination of whether residues of a PVC-
protein will be present in food derived from a crop containing a PVCP-
PIP. Due to the serious consequences of having an unapproved residue in
the food supply (as discussed earlier in this unit), EPA strongly
recommends that developers consult with the Agency before determining
that no tolerance or tolerance exemption for the PVC-protein

would be necessary based solely on the premise that no residues of the
protein are anticipated to be present. EPA expects that the Agency
would conclude no PVC-protein tolerance exemption would be necessary
for insertion events where the transgene either lacks an initiation
codon for protein synthesis or is inserted in an inverted-repeat
orientation, provided that evidence is given to the Agency to verify
the characteristics of the insertion event. For such constructs, the
2005 SAP indicated the PVCP-PIP ``could be safely determined to have no
[PVC-protein] expression regardless of plant tissue, developmental
stage, environmental conditions, or exposure to virally-encoded
suppressors of PTGS'' (Ref. 15).
    For all other types of PVCP-PIP insertion events, EPA is
considering several approaches under FFDCA for PVC-proteins that are
not readily detectable, but which the SAP indicated would likely be
produced under some circumstances (Ref. 15), some of which might result
in the PVC-protein being in food. EPA does not currently have a
preferred approach and presents several options to promote full
consideration of the issues. These options are not necessarily mutually
exclusive, and the approach pursued may vary depending on the
characteristics of the PVCP-PIP under consideration. The discussion
below relates only to proteins that EPA review would determine to be
minimally modified, i.e., proteins that are similar, but not identical
to natural plant virus coat proteins. Virtually unmodified PVC-proteins
would be covered under the proposed tolerance exemption without any
Agency action. The discussion is not relevant to proteins that would
not be able to qualify under this proposal as either virtually
unmodified or minimally modified because the proposed tolerance
exemption would not cover such proteins regardless of how EPA
implements the exemption.
    Under one approach, when no PVC-protein is detected during product
development, EPA would not issue a determination of whether the PVC-
protein is minimally modified (and therefore falls under this proposed
tolerance exemption). Section 408 of FFDCA does not require a tolerance
or tolerance exemption for foods that do not bear any residues, and
such an approach would be consistent with current EPA practice
regarding chemical pesticide residues in that tolerance determinations
are not generally issued for substances when residue studies
demonstrate that detectable residues will not be present in food.
However, if food is subsequently found bearing residues of the PVC-
protein, that food would be adulterated and subject to seizure unless
and until EPA could make a determination that the PVC-protein is
minimally modified and is therefore covered by this proposed tolerance
exemption.
    Any adulterant in the food supply would likely cause public concern
and great expense--whether or not the PVC-protein were subsequently
determined to be safe. The Agency also notes that these costs are not
necessarily borne by the product developer, but rather may
disproportionately affect farmers and/or food producers because any
adulterated food would be subject to seizure or recall. The Agency is
considering this approach under the assumption that the absence of
detectable protein using rigorous testing could give reasonable
assurance that PVC-protein residues would not be found in food and
therefore a tolerance determination would be unnecessary to prevent
adulteration of the food supply. EPA would expect developers to provide
the Agency with data acquired during product development that
demonstrates no PVC-protein residues in food would be reasonably
anticipated during the commercial life of the PVCP-PIP. For example,
such data could be obtained by testing for protein and/or mRNA
production in all plant tissues and all developmental stages that are
harvested for food production under a variety of circumstances and
environmental conditions representative of those that the plant may
experience during its commercial cultivation. Challenge with a known
PTGS suppressor protein introduced by a replicating virus vector,
genetic crosses, or agro-infiltration (Ref. 56) may also in some cases
be a sufficient and less burdensome technique to show that no PVC-
protein is able to be translated from the PVCP-PIP. The potential to
elicit protein production from silenced transgenes has been shown by
studies investigating whether particular proteins are able to suppress
such silencing (Ref. 56). The 2005 SAP discussed such a technique,
indicating that ``[t]o determine if PTGS-based PVCP-PIP plants have the
potential to produce proteins, the most effective test is to use viral
suppression of PTGS. In this type of assay, the PVCP-PIP plants are
infected with viruses from the potyvirus, cucumovirus, and tombusvirus
genera. These viruses encode different classes of PTGS suppressor
proteins. . . Protein and RNA are then extracted from the infected
plant tissue and assayed for the presence of the PVCP-PIP accumulated
full-length RNA and protein. Standard tests for protein detection are
ELISA and immunoblot (`Western' blot) analyses with specific
antibodies. Triplicate experiments should be sufficient to determine
that the results of these tests are reproducible'' (Ref. 15). Given
that FFDCA does not require a developer to demonstrate that no
tolerance exemption is necessary, EPA would require such testing as a
condition of either registering or exempting the PVCP-PIP under FIFRA.
    If the developer detects a PVC-protein during the course of
investigating potential PVC-protein production, e.g., through challenge
with a suppressor protein, this protein would only be covered under the
proposed categorical tolerance exemption, i.e., without any Agency
action, if the protein falls within the definition of a virtually
unmodified PVC-protein. Therefore, unless the protein is virtually
unmodified from a natural plant virus coat protein, EPA would expect a
developer to provide the Agency with information for a determination of
whether the PVC-protein qualifies as minimally modified and meets the
proposed conditional tolerance exemption. (See Unit IV.E.2. for a
discussion of the factors EPA intends to consider in making this
determination.)
    When possible, EPA would expect to see biochemical characterization
of the PVC-protein. However, EPA recognizes that such characterization
may be difficult or even impossible in some cases. For example, when
only very low levels of protein are produced, it may be difficult to
obtain sufficient amounts of protein for biochemical characterization.
In addition, EPA recognizes the cost and burden of producing sufficient
protein for such characterization may not be warranted for PVC-proteins
given that an evaluation based on the construct sequence alone could
consider most of the issues EPA intends to evaluate when determining
whether a PVC-protein is minimally modified (see Unit IV.E.2.).
    EPA is therefore also considering a second approach to addressing
PVC-proteins that are not detected during product development but whose
presence as residues in food cannot be ruled out for the commercial
life of the PVCP-PIP. Under this approach, EPA would evaluate the PVC-
protein to determine whether it qualifies as minimally modified from a
natural plant virus coat protein and is thus eligible for this proposed
tolerance exemption based only on its amino acid sequence as deduced
from the sequence of the inserted gene. EPA notes the advice of the
2005 SAP that ``[i]t is critical to evaluate the protein as expressed
in the

host, including factors such as post-translational modifications''
(Ref. 15). Nevertheless, EPA considers evaluating the protein as
expressed in the host to be less important for minimally modified PVC-
proteins than for many other types of proteins. A PVC-protein would not
be expected to have significantly different post-translational
modifications than a plant virus coat protein produced in a virus-
infected plant. Because plant viruses replicate in plant cells as part
of their normal life cycle, any post-translational modifications are
expected to be the same for a PVC-protein expressed from a plant
transgene as for a plant virus coat protein expressed from a viral
genome in a virus-infected plant.
    As a third alternative, EPA is considering whether the Agency could
expand this proposed tolerance exemption to cover all PVC-proteins that
would be produced from constructs where resistance is demonstrated to
EPA to be mediated through PTGS, e.g., those that confer virus
resistance in the absence of detectable protein production for at least
some period of time. The rationale for this alternative would be, as
indicated by the 2005 SAP, that ``PTGS-based virus resistance requires
greater than 90% RNA sequence homology between the PVCP-PIP transgene
and the target virus, indicating that the viral mRNA and protein
produced in PVCP-PIP plants will be nearly identical to the viral
pathotype that occurs in the United States'' (Ref. 15). To implement
this alternative, the Agency would have to be able to conclude, without
any case-by-case examination, that any PVC-protein produced from a
PVCP-PIP that mediates resistance through PTGS would be safe. Even if a
PVC-protein were detected before product deployment, such a protein
would not need any evaluation by the Agency in order to be covered by
this tolerance exemption. The rationale for this approach would be that
any such PVC-protein would meet the conditions of a minimally modified
protein (as discussed in Unit IV.E.2.) given the necessity for
transgene transcript sequence similarity to natural plant virus coat
protein sequences in order for PTGS to effectively function. Although
EPA does not believe it could identify a priori which modifications
would be within the range of natural variation for the protein, under
this rationale the induction of PTGS would be an a priori indicator
that such a PVC-protein is within the range of natural variation of the
protein. The 2005 SAP suggested that all PTGS-based PVCP-PIPs would
``pose similarly low risks'' as those that would have no protein
expression under any circumstances (Ref. 15), giving scientific support
for this option. However, the Agency notes that this advice is not
entirely consistent with advice regarding PVC-protein safety received
by the Panel. For one, both the 2004 and 2005 SAPs were unable to
endorse a tolerance exemption for PVC-proteins other than those that
are virtually unmodified from a natural plant virus coat protein unless
the Agency performed a case-by-case review of some nature. PVC-proteins
could be encoded for by a nucleic acid sequence that meets the 90%
similarity required for PTGS to function but fail to be virtually
unmodified from a natural virus coat protein (see Unit IV.E.1.).
Moreover, the 2005 SAP recommended that ``[d]etermining whether PVC-
proteins containing terminal deletions, or any other modifications, are
within the range of natural variation would require the development of
a database of the natural variation and truncated forms of PVC-proteins
that occur naturally'' (Ref. 15). While PTGS requires a relatively high
sequence similarity with natural virus coat proteins to function, only
a portion of the coat protein gene is necessary, suggesting that many
truncated proteins would be encompassed in this exemption without any
review of whether they occur naturally. (See, however, EPA's discussion
of whether truncated proteins could be determined to be exempt without
Agency review in Unit IV.E.2.) The 2005 SAP also suggested that a low
level of protein expression would indicate low risk, but prior SAPs and
other scientific experts have been unable to establish a threshold
below which the level of protein would not present concerns with
respect to food allergenicity (Refs. 57 and 58).

V. Toxicological Profile

    Consistent with section 408(b)(2)(D) of FFDCA, EPA has reviewed the
available scientific data and other relevant information in support of
this proposed 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.
    EPA's risk assessment was based primarily on an analysis of human
experiences with the breeding and cultivation of agricultural plants as
well as food preparation and consumption. EPA combined human experience
in consuming food containing coat proteins from viruses that naturally
infect plants with knowledge of plant genetics, plant physiology,
phytopathology, microbial ecology, ecology, biochemistry, and plant
breeding to evaluate the potential risks of the residues of PVC-
proteins qualifying for this proposed exemption.
    EPA considered the nature of any toxic effects that might be caused
by residues of PVC-proteins proposed for exemption. As mentioned above,
coat proteins from plant viruses that naturally infect plants are
widespread in foods (Refs. 6, 7, and 10) and are not associated with
toxic or pathogenic effects in humans or vertebrates (Refs. 18 and 19).
Residues of PVC-proteins qualifying for this proposed exemption are
virtually unmodified or minimally modified from other coat proteins
from viruses that naturally infect food plants and that have been
safely consumed for hundreds if not thousands of years. Given this long
history of safe use and the fact that toxicity is an unusual property
among proteins in general (Ref. 59), consumption of food containing
residues of PVC-proteins qualifying for this proposed exemption is not
expected to present a toxic effect on humans or animals.
    EPA considered the available information on the various dietary
consumption patterns of consumers and major identifiable consumer
subgroups as it pertains to residues of PVC-proteins in food. Plant
virus coat proteins are, and always have been, widespread in all food
from crop plants since most plants are susceptible to infection by one
or more viruses. Thus, all consumers and all major identifiable
consumer subgroups are, and have been, exposed to plant virus coat
proteins. Implementation of this proposed exemption is not expected to
alter the current consumption patterns of plant virus coat proteins
except perhaps to reduce exposure through a decrease in virus-infected
plants. Therefore, EPA does not expect any special sensitivities to
arise due to the consumption of residues of PVC-proteins that are
proposed to be exempted.

VI. Aggregate Exposures

    In examining aggregate exposure, section 408 of FFDCA directs EPA
to consider available information concerning 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).

    EPA considered the available information on the likely aggregate
exposure level of consumers to PVC-proteins qualifying for this
proposed exemption and to other related substances, including exposures
to plant virus coat proteins occurring through natural processes such
as viral infection of a food plant. This analysis included a
consideration of exposures from dietary sources as well as from other
non-occupational sources.
    The PVC-proteins qualifying for this proposed exemption and plant
virus coat proteins that occur naturally are both produced in living
plants and are subject to the natural processes of degradation and
decay that all biological materials undergo. They are broken down by
enzymatic processes of living organisms into constituent parts that are
used as building blocks for other biological substances (Ref. 60).
Because of their biodegradable nature, neither PVC-proteins nor
naturally occurring plant virus coat proteins bioaccumulate (i.e.,
build up in tissues because the body is unable to either break the
substance down or eliminate it) or biomagnify (i.e., progressively
build up in successive trophic levels because it bioaccumulates in the
bodies of organisms lower in the food chain). Humans ingesting
naturally occurring plant virus coat proteins and residues of PVC-
proteins qualifying for this proposed exemption in food are likely to
quickly degrade them and use their constituent elements as nutrients.
    Because of these characteristics, there is limited potential for
exposures to PVC-proteins qualifying for this proposed exemption beyond
direct physical exposure to a plant. In most cases, the predominant
exposure route will be dietary. In general, EPA anticipates that
dietary exposure to PVC-proteins qualifying for this proposed exemption
through human and animal consumption of plants expressing PVC-proteins
will be similar to, or less than the amounts of plant virus coat
proteins currently consumed through food plants that are infected
naturally with viruses (see Unit IV.C.1.). Exposure through other
routes is unlikely because the substances are in the plant tissue and
thus are found either within the plant or in close proximity to the
plant. EPA expects non-dietary exposure (i.e., non-food oral, dermal,
and inhalation) in non-occupational settings to be negligible.

A. Dietary Exposure

    EPA considered the consequences of dietary exposure to PVC-proteins
that are the subject of this proposed exemption. A large base of
experience exists, including information on human dietary exposure, for
foods that contain coat proteins from viruses that naturally infect
plants. As plant virus coat proteins are ubiquitous in food, EPA
concluded that all humans are exposed to plant virus coat proteins
throughout their lives as part of their diet. Neither naturally
occurring plant virus coat proteins nor the PVC-proteins qualifying for
this exemption are toxic, and there is no evidence that consumption in
food of residues of PVC-proteins qualifying for this proposed exemption
would lead to any harm.
    1. Food. As mentioned in Unit IV.C.1., the Agency has concluded
that dietary exposures to PVC-proteins qualifying for this proposed
exemption will be similar to or less than the amounts of plant virus
coat proteins currently found and consumed in food plants that have
been naturally infected by viruses. Even if there were notable exposure
to PVC-proteins, there is no evidence that PVC-proteins are toxic to
humans. Moreover, the Agency is not aware of any coat protein from a
virus that naturally infects plants that has been identified as a food
allergen for humans. The residues that are proposed to be exempted by
this Federal Register document would not differ substantially from
residues of naturally occurring plant virus coat proteins.
    2. Drinking water exposure. EPA also evaluated potential non-
occupational exposures in drinking water. Residues of PVC-proteins that
qualify for this proposed exemption are produced inside the plant
itself. When the plant dies or a part is removed from the plant,
microorganisms colonizing the tissue immediately begin to degrade it
using the components of the plant tissue (including residues of PVC-
proteins) as building blocks for making their own cellular components
or for fueling their own metabolisms. PVC-proteins and naturally
occurring plant virus coat proteins are subject to the same processes
of biodegradation and decay that all biological materials undergo and
are not known to either bioaccumulate or biomagnify (Ref. 60). Even if
they were to reach surface waters (e.g., through plant parts or pollen
falling into bodies of water), they are unlikely to present anything
other than a very negligible exposure in drinking water drawn either
from surface water or ground water sources due to biodegradation of
these residues.

B. Other Non-Occupational Exposure

    Residential exposure to PVC-proteins qualifying for this proposed
exemption would be limited. Residential exposure could occur through
use of PVCP-PIPs in ornamental plants or in plants grown in home
gardens. Such exposure to PVC-proteins is expected to be negligible on
a per-person basis compared to exposure to PVC-proteins and natural
plant virus coat proteins in the diet. Furthermore, PVC-proteins
qualifying for this exemption would not be toxic, and there is no
evidence that exposure to such PVC-proteins would lead to any harm.
    1. Dermal exposure. Residues of PVC-proteins qualifying for this
proposed exemption may be present in sap or other plant exudates and
thus may present some limited opportunity for dermal exposure to
persons coming physically into contact with the plant or raw
agricultural food from the plant. Individuals preparing meals are those
most likely to experience dermal contact with the residues on a non-
occupational basis. As noted by the 2005 SAP, PVC-proteins' ``natural
exposure route may be via oral ingestion. However, genetically modified
expression of PVCP-PIPs would lead to the presence of [PVC-proteins] in
other plant compartments such as pollen grains which lead to other
sites of exposure including respiratory and cutaneous surfaces'' (Ref.
15). However, the potential amount involved in such exposure on a per
person basis is likely to be negligible in comparison to potential
exposure through the dietary route to PVC-proteins and natural plant
virus coat proteins (Ref. 61). Moreover, PVC-proteins qualifying for
this proposed exemption or naturally occurring plant virus coat
proteins that occur in food are unlikely to cross the barrier provided
by the skin (Ref. 62).
    2. Inhalation exposure. Pollen could potentially contain residues
of PVC-proteins qualifying for this proposed exemption. Individuals
(e.g., those visiting, living, or working near enough to farms,
nurseries, or other plant-growing areas to be exposed to wind-blown
pollen) may be exposed to the pollen through inhalation. On a per
person basis, the potential amount of pollen involved in these
exposures is likely to be negligible in comparison to potential
exposure through the dietary route (Ref. 61). Some members of the 2005
SAP indicated that ``[i]ntroduction of new proteins to pollens and
other plant materials may have the potential to cause problems, and
consideration by the Agency is warranted'' (Ref. 15). As the Panel
explained, ``While plant viruses systemically infect plant tissues,
there is tissue specific regionalization of viruses. Therefore [plant
virus coat proteins] would be restricted within certain compartments.
Transgenic expression of some PVC-PIPs would

promote [PVC-protein] expression in different plant tissues relative to
what would naturally occur (i.e., all cells). This could lead to
heightened levels of [PVC-proteins] in certain tissues (i.e., pollen
grains) and the effects (specifically to allergenicity) are not yet
known. This has implications for non-dietary exposure of plant
proteins. In some instances, [plant virus coat protein's] natural
exposure route may be via oral ingestion. However, genetically modified
expression of PVCP-PIPs would lead to the presence of [PVC-proteins] in
other plant compartments such as pollen grains which lead to other
sites of exposure including respiratory and cutaneous surfaces'' (Ref.
15). However, other Panel members felt that ``unless there is evidence
that PCVP-PIPs are expressed on the surface of pollen grains in a
manner different from expression in wild-type plants, the risk of
increased allergy from exposure to pollen is non-existent'' (Ref. 15).
The Agency also notes that in order for expression of a PVC-protein to
be a concern, the protein would have to be expressed on the surface of
the pollen grain, it would have to actually be an antigenic protein,
and it would have to elicit an allergic response through secondary
exposure. The Agency considers that this sequence of events is very
unlikely to occur, in part because no plant virus coat proteins have
been identified as being allergenic, and PVC-proteins qualifying for
this exemption are virtually unmodified or minimally modified from
natural plant virus coat proteins. Therefore, it is unlikely that
inhalation exposure to PVC-proteins in pollen would result in adverse
effects.

VII. Cumulative Effects

    EPA examined the available information on residues of PVC-proteins
qualifying for this proposed exemption for cumulative effects with
other substances, including natural plant virus coat proteins. Plant
virus coat proteins are nontoxic proteins that are widespread in food
from plants. They have not been associated with toxic effects to
animals or humans (see Unit IV.C.3.). EPA is therefore not aware of any
other substances that could have a common mechanism of human toxicity
with residues of PVC-proteins qualifying for this exemption and cannot
identify any cumulative effects of such residues with any other
substances.

VIII. Safety Factor for Infants and Children

A. In General

    Section 408(b)(2)(C) of FFDCA 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 information base on toxicity and
exposure unless EPA determines that a different margin of safety will
be safe for infants and children. Margins of safety are incorporated
into EPA risk assessments either directly through use of a MOE analysis
or through using uncertainty (safety) factors in calculating a dose
level that poses no appreciable risk to humans.

B. Prenatal and Postnatal Sensitivity

    EPA considered available information on the dietary consumption
patterns of infants and children as it pertains to residues in food of
PVC-proteins qualifying for this proposed exemption. The range of foods
consumed by infants and children is in general more limited than the
range of foods consumed by adults. Most newborns rely on breast milk or
formula-based products for nutrition, although some infants are fed
soy-based products. Infants may begin as early as 4 months of age to
consume solid foods that are based on foods consumed by the general
adult population albeit in different proportions and with processing to
facilitate swallowing. As infants and children mature, more and more of
the foods normally consumed by adults become part of their diets, and
the relative proportions of the different types of food consumed change
to more closely resemble an adult diet. Because plant viruses are
ubiquitous in plant foods, EPA concluded that infants and children are
exposed to plant virus coat proteins from the time they begin to eat
food of plant origin. As the diets of humans change from infancy
through childhood and into adulthood, there is some possibility that
the amount of plant virus coat proteins being consumed may change, with
those consuming the greatest amounts of food of plant origin most
likely exposed to the most plant virus coat protein. However, there is
no evidence that such changes are likely to result in
disproportionately high consumption of foods containing plant virus
coat proteins among infants and children in comparison to the general
population. Furthermore, PVC-proteins qualifying for this proposed
exemption are not toxic, and there is no evidence that any amount of
exposure to such PVC-proteins in food would lead to any harm.
    EPA considered available information on the potential for special
susceptibility of infants and children, including prenatal and
postnatal toxicity, to residues of PVC-proteins qualifying for this
proposed exemption. PVC-proteins in food are not toxic. There is no
scientific evidence that residues of such PVC-proteins in food would
have a different effect on infants and children than adults due to
neurological differences between infants, children, and adults.
    The Agency's consideration of cumulative effects of the residues of
PVC-proteins qualifying for this proposed exemption on the general
population also included consideration of effects for infants and
children. Neither naturally occurring plant virus coat proteins nor
PVC-proteins qualifying for this proposed exemption are toxic when
consumed as part of the diet, and EPA is not aware of any substances
that might have a common mechanism of toxicity with these PVC-proteins.
There is no scientific evidence indicating any potential for adverse
effects on infants and children due to cumulative exposure to residues
of such PVC-proteins. EPA concludes that there is no evidence of a
common mechanism of toxicity between PVC-proteins qualifying for this
proposed exemption and any other substances, and therefore, no
cumulative effects of these PVC-proteins would reasonably be
anticipated.

C. Conclusion

    There is a complete toxicity base of information for PVC-proteins
that are the subject of this proposed exemption, and exposure data are
estimated based on data that reasonably account for potential
exposures. For residues of PVC-proteins qualifying for this proposed
exemption, EPA has determined that a tenfold margin of safety is not
necessary to protect infants and children. As noted in Unit IV.C., EPA
based its assessment of exposure and toxicity on the long history of
safe human and animal consumption of food containing plant virus coat
proteins. EPA also relied upon information from the disciplines of
plant genetics, plant physiology, plant virology, microbial ecology,
ecology, biochemistry, molecular biology, and plant breeding. Based on
all of this information, EPA concludes that PVC-proteins qualifying for
this proposed exemption in food are not toxic and may be safely
consumed, including by infants and children. There is no evidence that
exposure to such

PVC-proteins in food, including changes in exposure because of changes
in the relative proportions of the different types of food consumed
from infancy through childhood and into adulthood, leads to any harm.
Thus, on the basis of valid, complete, and reliable information, EPA
has concluded that residues in food of PVC-proteins qualifying for this
proposed exemption are safe for infants and children and that an
additional margin of safety need not be applied.

IX. Other Considerations

A. Endocrine Disruptors

    Based on available information that plant virus coat proteins are
ubiquitous in foods and have no known adverse effects when consumed as
part of the diet (see Unit IV.C.), EPA does not expect residues of PVC-
proteins qualifying for this proposed exemption to cause estrogenic or
other endocrine effects. In the May 16, 1997 supplemental document, EPA
specifically requested comment on PVC-proteins causing estrogenic
effects. No information was received indicating that either naturally
occurring plant virus coat proteins or PVC-proteins that qualify for
this proposed exemption might cause estrogenic or other endocrine
effects. If EPA becomes aware of a potential for estrogenic or
endocrine effects from exposure to residues of such PVC-proteins, the
Agency will reexamine this proposed tolerance exemption in light of
that information.

B. Analytical Method(s)

    EPA has concluded that even though methodology exists to detect
residues of PVC-proteins (Refs. 63, 64, and 65), there is no need to
employ a practical method for detecting and measuring the level of
residues of PVC-proteins qualifying for this exemption. There is no
reason to believe that the residues of PVC-proteins proposed to be
exempted in this Federal Register document would behave any differently
than naturally occurring plant virus coat proteins in food. There is a
reasonable certainty that no harm will result from exposure to any
amount of residues in food of such PVC-proteins. Because these residues
may be present in food at any level without causing harm, EPA has
concluded that an analytical method is not required for detecting and
measuring the level of residues of these PVC-proteins in food. EPA
consulted with the Department of Health and Human Services (HHS) in
making this determination.

C. Codex Maximum Residue Level

    There are no Codex maximum residue levels established for PVC-
proteins.

X. Preliminary Determination of Safety for U.S. Population, Infants,
and Children

    Based on the information discussed in this document and that
discussed in the 1994 Federal Register documents, the supplemental
documents, and the associated record as described in Unit XII.B., EPA
preliminarily concludes that there is a reasonable certainty that no
harm will result to the U.S. population, infants, and children from
aggregate exposures to residues of PVC-proteins that qualify for this
proposed exemption. Many years of experience with growing, preparing,
and consuming food from plants containing plant virus coat proteins and
information generated through years of study of the food supply (Refs.
6, 7, 8, 9, 10, and 66) indicate that adverse effects due to aggregate
exposure to PVC-proteins qualifying for this proposed exemption through
dietary, non-food oral, dermal, and inhalation routes are highly
unlikely.

XI. Request for Comment

    EPA requests comment on whether this proposed tolerance exemption
identifies those PVC-proteins that are unlikely to result in new
dietary exposures. When commenting, please use the terminology
conventions adopted in this document, i.e., use ``plant virus coat
protein'' when referring to the protein produced naturally from a plant
virus, and use ``PVC-protein'' when referring to the protein component
of a PVCP-PIP. The Agency requests comment on the following specific
issues:
    1. EPA requests comment on the options discussed in Unit IV.E.1.
for defining virtually unmodified. Under the Agency's proposed rule,
virtually unmodified proteins would be exempt from the requirement of a
tolerance without Agency review. Under one option, virtually unmodified
would be defined as having or coding for an amino acid sequence that is
identical to an entire coat protein of a naturally occurring plant
virus; except for the addition of one or two amino acids at the N- and/
or C-terminus other than cysteine, asparagine, serine, and threonine
and/or the deletion of one or two amino acids at the N- and/or C-
terminus. However, the Agency is considering removing the limitations
on which amino acids may be added and on the number of amino acids that
may be truncated from either end of a PVC-protein.
    2. In addition to the types of changes discussed in the paragraph
above, EPA requests comment on whether any other class of potential
PVC-protein modifications (e.g., a particular number of amino acid
substitutions) would always be expected to produce a PVC-protein as
safe as an unmodified plant virus coat protein such that the protein
would not warrant a case-by-case Agency review for a tolerance
exemption. The Agency also requests that commenters indicate whether
the number and combination of such modifications has any relevance to
the product's safety. In October 2004, the FIFRA SAP was asked to
consider the degree and ways a plant virus coat protein gene might be
modified while still retaining scientific support for the idea that
humans have consumed the products of such genes for generations and
that such products therefore present no new dietary exposures (Ref.
14). They responded that ``[t]here was no clear consensus on how much
change would be necessary to invalidate this assumption, although there
was general agreement that the appropriate comparison is to the range
of natural variation in the virus population.'' This question was also
addressed by the 2005 SAP which concurred that ``it is extremely
difficult to identify modifications that would be expected to be
`within the range of natural variation for all virus families'. . .
Given the possible range of natural variations for PVC proteins, it
would be appropriate to assess whether specific modifications are
within natural variation limits of the PVC protein on a case-by-case
basis'' (Ref. 15). Commenters should specifically address this advice
when formulating comments.
    3. EPA requests comment on whether there would be any safety issues
associated with exposure to PVC-proteins if the virus used to create
the PVCP-PIP does not naturally infect the particular plant species
into which the PVCP-PIP is inserted. A PVC-protein may be expressed in
a food plant that the virus does not naturally infect when heterologous
resistance to a particular virus is conferred through a different
virus' coat protein gene (e.g., Ref. 45). Such PVC-proteins could be
safely exempted from tolerance requirements if these proteins are
reasonably expected to be part of the current diet, as discussed in
Unit IV.E.1. In light of the uncertainty surrounding the SAP's remarks
concerning this issue (see Unit IV.E.1.), EPA requests comment on
whether there would be any safety issues associated with exposure tothe

PVC-proteins themselves if the virus used to create a PVCP-PIP does not
naturally infect the particular plant species into which the PVCP-PIP
is inserted.
    4. EPA requests comment on whether the Agency should consider the
level of PVC-protein expression in determining whether a PVC-protein is
virtually unmodified or minimally modified and thus exempt from
tolerance requirements. EPA concurs with the 2005 SAP that ``exposure
level is an important component of an allergenicity risk assessment''
(Ref. 15). However, it can be argued that PVC-proteins that are
virtually unmodified or minimally modified when compared to natural
plant viral coat proteins are of sufficiently low hazard that the
potential risk does not rise to the level warranting regulation, even
in the rare case that exposure to a PVC-protein might be greater than
the exposure to the corresponding natural plant virus coat protein.
Although EPA's review of PVC-proteins to determine if they are
minimally modified could allow the Agency to consider PVC-protein
expression level relative to natural levels of plant virus coat
proteins, the Agency is unsure how this factor could be readily
incorporated into the criteria for a developer-determined tolerance
exemption; EPA anticipates needing to consider the appropriateness of
data designed to address these questions on a case-by-case basis.
Therefore, if protein expression level is considered a necessary factor
in evaluating whether to exempt a virtually unmodified PVC-protein from
tolerance requirements, EPA seeks comment on how such considerations
could be articulated in a clear, unambiguous criterion.
    5. EPA requests comment on the Agency's options for how to view a
PVC-protein that would not meet the definition of virtually unmodified
and is not detected during product development if the construct
suggests that its production is likely to occur in at least some plant
tissue at some point in time (see Unit IV.F.). Specifically, EPA
requests comment on the relative costs and benefits of allowing a PVCP-
PIP that does not produce detectable PVC-protein residues in food
during product development to be sold or distributed without a PVC-
protein tolerance exemption in place. EPA is particularly interested in
information about the likelihood that protein would fail to be detected
during product development but subsequently be detected in food. The
Agency is also interested in comments on conditions under which protein
detection protocols could be conducted to provide adequate assurance
that such events would not occur, e.g., any key environmental
parameters that should be varied during testing.
    EPA also requests comment on whether obtaining characterization
data of a plant-produced PVC-protein for a tolerance review is
scientifically feasible in all cases where the PVCP-PIP insertion event
contains a translation initiation codon and is not present in an
inverted repeat orientation. The Agency would like to know for any
given crop how technically difficult it would be to attempt to induce
protein production through challenge with a known PTGS suppressor
protein, e.g., through introduction by a replicating virus vector,
genetic crosses, or agro-infiltration (Ref. 56). In addition, EPA would
like to know how likely it is that such techniques could yield
sufficient quantities of PVC-protein for analysis (e.g., mass
spectrometry or glycosylation analysis). The Agency would also be
interested in hearing of additional techniques that could be employed
to obtain plant-produced PVC-protein in cases where PTGS normally
prevents accumulation of protein but is not expected to be consistently
activated, thereby leading to PVC-protein production.
    Regarding the second alternative presented for PVC-proteins
associated with PTGS, EPA requests comment on the value of the
additional information gained by analyzing an actual PVC-protein as
produced in the plant where the inserted nucleotide sequence suggests
it would be minimally modified from a natural plant virus coat protein,
e.g., to consider potential post-translational modifications, relative
to the reduced burden and cost of analyzing safety based on the deduced
amino acid sequence from the insert alone.
    Regarding the third alternative presented, EPA requests comment on
the rationale that would be used to support expanding this tolerance
exemption to cover all PVC-proteins produced by a PVCP-PIP that
mediates resistance through PTGS, i.e., that any such protein would
meet the conditions of a minimally modified protein as discussed in
this document given the necessity for transgene transcript sequence
similarity to natural plant virus coat protein sequences in order for
PTGS to effectively function. In particular, EPA requests comment on
how to reconcile this option with prior advice of the SAP (as discussed
in Unit IV.F.).
    6. EPA requests comment on whether PVC-proteins that the Agency has
reviewed and has determined are minimally modified and therefore are
exempt from the requirement of a tolerance under proposed Sec.
174.477(b) should be listed in the CFR as is the current practice for
individual tolerance exemptions associated with other types of PIPs. If
so, EPA requests comment on whether the listing should indicate the
specific modifications of the reviewed proteins, given that each
determination would apply only to proteins with those modifications.
EPA is aware that in the past, developers have found such listings to
be useful for international trade reasons, as governments rely on EPA
tolerances to support import decisions.

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