Page
1
of
11
Attachment
II:
Draft
Approach
to
Exempting
Certain
PVC­
Proteins
from
the
Requirement
of
a
Tolerance
under
FFDCA
I.
What
Action
Does
this
Paper
Discuss?

EPA
is
considering
whether
to
establish
a
tolerance
exemption
under
section
408
of
the
Federal
Food,
Drug,
and
Cosmetic
Act
(
FFDCA)
for
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
and
when
certain
criteria
are
met.
The
criteria
EPA
is
considering
are
intended
to
clearly
identify
and
exempt
only
those
residues
for
which
a
long
history
of
safe
exposure
and
consumption
can
support
exemption.
EPA
is
still
considering
several
scientific
issues
that
would
affect
the
exact
formulation
of
these
criteria,
and
these
issues
are
discussed
below
in
this
document.
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.
A
tolerance
exemption
would
eliminate
the
need
to
establish
a
maximum
permissible
level
in
food
for
these
residues.

II.
Background
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
PVCPPIPs
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
portion
of
the
PVCP­
PIP
(
hereafter
the
"
PVC­
protein")
is
thought
to
impede
the
infection
cycle
by
interfering
with
the
disassembly
of
infecting
viruses
(
Ref.
3).
In
such
cases,
the
PVC­
protein
appears
to
be
the
active
ingredient
directly
effecting
the
pesticidal
action.
Residues
of
such
PVC­
proteins
and
their
metabolites
and
degradates
would
be
the
subject
of
this
action
if
they
met
the
specified
criteria.

In
transgenic
plants
where
post­
transcriptional
gene
silencing
(
PTGS)
has
been
activated,
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
the
active
ingredient
directly
effecting
the
pesticidal
action
(
Ref.
3).
Even
when
PTGS
is
the
mechanism
of
resistance,
should
any
PVC­
protein
be
produced,
this
PVC­
protein
is
part
of
the
PVCP­
PIP.
Residues
of
such
PVC­
proteins
and
their
metabolites
and
degradates
would
also
be
the
subject
of
this
action
if
they
met
the
specified
criteria.

A.
Potential
Rationale
Supporting
an
FFDCA
Tolerance
Exemption
Page
2
of
11
EPA's
base
of
experience
with
viruses
infecting
food
plants
has
led
the
Agency
to
draw
three
conclusions
on
which
it
would
rely
to
support
any
tolerance
exemption
for
residues
of
PVCproteins
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
effects
to
animals
or
humans.

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.
4,
5,
6,
7,
8,
9).
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.
9).
Even
plants
that
show
no
disease
symptoms
are
often
found
to
be
infected
with
viruses
(
Refs.
8,
10).
In
addition,
a
common
agricultural
practice
used
since
the
1920s
for
protection
against
viruses
involves
intentionally
inoculating
healthy
plants
with
a
mild
form
of
a
virus
in
order
to
prevent
infection
by
a
more
virulent
form
(
Ref.
10).
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.
11).
The
FIFRA
SAP
at
its
December
18,
1992
meeting
(
Ref.
12),
also
addressed
the
issue
of
dietary
risk.
The
SAP
stated
that
"[
s]
ince
viruses
are
ubiquitous
in
the
agricultural
environment
at
levels
higher
than
will
be
present
in
transgenic
plants,
and
there
has
been
a
long
history
of
`
contamination'
of
the
food
supply
by
virus
coat
protein,
there
is
scientific
rationale
for
exempting
transgenic
plants
expressing
virus
coat
protein
from
the
requirement
of
a
tolerance."
The
FIFRA
SAP
again
discussed
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.
13).

In
general,
EPA
anticipates
that
dietary
exposure
through
human
and
animal
consumption
of
plants
containing
residues
of
PVC­
proteins
that
EPA
is
considering
exempting
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
that
PVC­
protein
levels
in
plants
resistant
to
a
virus
because
of
a
PVCP­
PIP,
even
when
the
resistance
is
mediated
by
the
PVC­
protein
itself,
can
be
up
to
one
hundred­
to
one
thousand­
fold
lower
in
concentration
than
the
level
of
coat
protein
found
in
plants
naturally
infected
by
viruses
(
Refs.
7,
14).
The
difference
in
amount
of
PVCprotein
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
Page
3
of
11
PIP
(
Refs.
3,
15).
Such
information
conforms
to
information
EPA
has
received
from
the
scientific
advisory
groups
the
Agency
has
consulted.

2.
Not
infectious
to
humans
Any
virus/
host
relationship
is
characterized
by
a
high
degree
of
specificity
(
Ref.
7).
Plant
viruses
usually
infect
plants
only
within
a
certain
taxonomic
group
and
are
unable
to
infect
humans
or
other
vertebrates
(
Refs.
16,
17).
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
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
without
human
or
animal
toxicity
related
to
plant
virus
infections.
Additional
evidence
of
a
lack
of
toxicity
can
be
deduced
from
the
common
practice
of
injecting
laboratory
animals
with
purified
plant
virus
preparations
without
any
adverse
effects
on
the
animals
(
Ref.
15).
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
be
lower
than
the
amount
of
virus
coat
protein
found
in
food
naturally
infected
with
plant
viruses
(
as
discussed
in
Unit
II.
A.
1).

B.
Key
Issue:
Determination
of
Natural
Virus
Variation
The
key
issue
facing
EPA
in
developing
an
exemption
is
how
to
describe
clearly
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
II.
A
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
PVCPPIP
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
Page
4
of
11
possible
that
naturally
occurring
plant
viruses
exist
with
some
of
the
minor
modifications
that
could
reasonably
be
anticipated.

EPA's
task
of
defining
this
variation
is
complicated
by
the
variable
nature
of
plant
virus
populations
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.
18,
19,
20).

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.
21).
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.
However,
recombination
between
different
viral
species
is
also
believed
to
occur
and
to
have
generated
new
viruses
(
Refs.
22,
23).
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.
24),
caulimoviruses
(
Ref.
25),
luteoviruses
(
Ref.
26),
nepoviruses
(
Ref.
27),
cucumoviruses
(
Ref.
28),
and
geminiviruses
(
22,
Refs.
29).
Sequence
analysis
of
viruses
from
the
family
Luteoviridae
indicated
that
this
family
has
evolved
via
both
intra­
and
interfamilial
recombination
(
Ref.
30).
In
viruses
with
segmented
genomes,
variation
may
also
be
caused
by
reassortment
whereby
entire
segments
are
exchanged
between
viruses
(
Ref.
31).

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.
32
for
review).
For
example,
cucumber
mosaic
cucumovirus
(
CMV)
has
a
relatively
high
degree
of
variation
(
Ref.
33)
compared
to
tobacco
mild
green
mosaic
tobomovirus
(
Ref.
34).
The
greater
variability
in
CMV
would
be
expected
based
on
the
relatively
wide
host
range
and
relatively
high
recombination
rate
of
this
virus.

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
it
is
not
possible
to
use
this
information
to
establish
a
regulatory
standard.
One
possibility
that
EPA
considered
but
ultimately
rejected
was
to
determine
an
idealized
sequence
for
each
virus
species
in
which
each
position
of
the
sequence
represents
the
amino
acid
most
often
found
when
all
available
sequences
are
compared.
A
selection
procedure
would
be
used
to
determine
which
amino
acid
is
placed
at
a
given
position
in
the
event
that
not
all
of
the
sequences
have
the
identical
amino
acid
at
that
position.
The
percentage
of
positions
that
each
sequence
deviates
from
this
idealized
sequence
could
be
calculated
to
establish
a
maximum
deviation
found
in
nature.
A
percent
deviation
could
likewise
be
calculated
for
a
PVC­
protein
sequence
to
determine
if
it
differed
by
more
or
less
than
the
maximum
found
in
nature.
One
problem
with
such
an
approach
is
that
focusing
only
on
a
percent
deviation
does
not
take
into
account
important
information
about
which
regions
of
the
coat
protein
vary
and
which
regions
are
highly
conserved.
For
example,
a
particular
sequence
constructed
in
a
laboratory
by
modifying
a
Page
5
of
11
natural
variant
could
differ
from
the
idealized
sequence
by
less
than
the
prior
determined
percent
variability
for
natural
variants,
but
the
particular
sequence
change
could
still
be
outside
the
range
of
natural
variation
depending
on
the
region
that
was
modified.
In
addition,
the
maximum
variation
is
likely
to
change
over
time
as
additional
sequences
are
determined
and
as
viruses
evolve.
EPA
does
not
believe
it
could
develop
a
standard
that
would
take
this
sort
of
information
into
account.
Moreover,
no
single
standard
could
capture
the
degree
of
variation
across
all
viruses,
and
hundreds
of
plant
viruses
have
been
identified
to
date
(
Ref.
35).
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.

Consequently,
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,
EPA
is
considering
an
approach
to
exempt
PVC­
protein
residues
from
the
requirement
of
a
tolerance
by:
(
1)
a
categorical
exemption
for
a
subset
of
PVCproteins
based
on
developer
self­
determination
that
the
encoded
PVC­
protein
is
identical
to
any
single
contiguous
portion
of
an
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
PVCprotein
is
minimally
modified
from
an
unmodified
coat
protein
from
a
virus
that
naturally
infects
plants
that
humans
consume
in
toto
or
in
part.

C.
Potential
Exemption
Structure
1.
Categorical
exemption
When
the
encoded
PVC­
protein
is
identical
to
any
single
contiguous
portion
of
an
unmodified
coat
protein
from
a
virus
that
naturally
infects
plants
that
humans
consume
in
toto
or
in
part,
the
developer
may
determine
that
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
in
nature
from
a
food
plant
and
was
not
modified,
the
PVC­
protein
would
meet
this
criterion.
Additionally,
if
the
genetic
material
encoding
the
PVCprotein
has
been
modified
but
its
amino
acid
sequence
nevertheless
exactly
matches
a
database
sequence
from
an
unmodified
plant
virus
coat
protein
that
naturally
infects
plants
that
humans
consume,
the
PVC­
protein
would
meet
this
criterion.
Although
EPA
cannot
a
priori
identify
all
existing
natural
coat
protein
variants,
the
requirement
of
identity
with
any
single
contiguous
portion
of
an
unmodified
coat
protein
would
ensure
that
the
exempted
PVC­
protein
falls
within
the
existing
base
of
experience
on
which
any
exemption
would
rely.

Under
this
approach,
EPA
intends
to
exclude
from
the
categorical
exemption
residues
of
modified
PVC­
proteins,
e.
g.,
PVC­
proteins
containing
insertions,
internal
deletions,
or
amino
acid
substitutions,
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
considering
excluding
such
PVC­
proteins
from
the
categorical
exemption
because
insufficient
information
exists
at
this
time
Page
6
of
11
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
of
the
protein
and/
or
construct.

Segments
of
PVC­
proteins
that
are
identical
to
segments
of
an
unmodified
coat
protein
would
be
exempted
categorically
under
this
approach,
i.
e.,
without
Agency
review.
EPA
believes
the
exemption
of
segments
would
be
supported
by
the
experience
base
EPA
discussed
above
to
support
an
exemption.
It
is
probable
that
segments
of
coat
proteins
exist
in
nature
due
to
processes
such
as
incomplete
translation
of
transcripts
and
partial
degradation
of
proteins.
Incomplete
translation
may
occur
due
to
routine
replication
errors
causing
a
ribosome
to
dissociate
from
an
RNA
transcript
or
if
mutation
introduces
a
premature
stop
codon,
i.
e.,
a
nonsense
mutation.
Truncated
plant
virus
coat
proteins
are
indeed
known
to
occur
in
nature
(
Ref.
36).
Thus,
PVC­
proteins
that
are
truncated
forms
of
naturally
occurring
plant
virus
coat
proteins
would
not
significantly
increase
the
likelihood
of
exposure
to
a
toxic
or
allergenic
protein
since
humans
are
currently
exposed
to
them
in
the
diet
along
with
complete
plant
virus
coat
proteins.

The
Agency
is
considering
whether
also
to
include
in
the
categorical
exemption,
i.
e.,
without
Agency
review,
amino
acid
sequences
containing
terminal
deletion(
s)
and/
or
an
additional
Nterminal
methionine
residue.
The
AUG
codon
for
methionine
initiates
translation
in
eukaryotes
(
Ref.
37).
Among
certain
viruses
such
as
the
Potyviridae,
the
coat
protein
is
produced
as
part
of
a
polyprotein,
so
the
coding
region
for
the
coat
protein
is
excised
from
the
genetic
material
encoding
the
polyprotein
to
create
a
PVCP­
PIP
and
thus
normally
lacks
a
start
codon.
Insertion
of
an
AUG
codon
allows
for
PVC­
protein
expression,
which
may
be
needed
to
confer
virus
resistance.
EPA
believes
the
addition
of
a
single,
N­
terminal
methionine
residue
would
be
unlikely
to
affect
a
PVC­
protein's
toxicity
or
allergenicity
relative
to
a
naturally
occurring
plant
virus
coat
protein.

Under
this
approach
EPA
would
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
that
any
exemption
is
limited
to
PVCP­
PIPs
that
fall
within
the
base
of
experience
discussed
previously
in
this
paper.
EPA
would
limit
the
proposed
exemption
to
residues
of
PVC­
proteins
that
are
already
part
of
the
human
diet
as
naturally
occurring
plant
virus
coat
proteins
or
because
they
are
minimally
modified
from
such
proteins.
For
example,
the
exemption
would
not
extend
to
PVCproteins
encoded
in
part
by
sequences
from
animal
or
human
viruses.
EPA
means
by
the
phrase
"
naturally
infect"
to
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.
The
Agency
wants
specifically
to
exclude
transmission
that
occurs
only
through
intentional
human
intervention,
e.
g.,
manual
infection
in
a
laboratory
or
greenhouse
setting,
because
such
transmission
would
have
little
relevance
to
normal
human
dietary
exposure.
EPA
intends
to
include
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,
and
this
mode
of
transmission
would
be
included
under
"
naturally
infects."
Page
7
of
11
EPA
is
considering
whether
to
limit
any
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.
38).
Such
situations
may
also
arise
when
a
small
segment
of
a
plant
virus
coat
protein
gene
is
used
to
achieve
expression
of
a
coat
protein
gene
from
a
different
virus
(
e.
g.,
Ref.
39).
However,
such
PVC­
proteins
could
be
safely
exempted
from
tolerance
requirements
because
these
proteins
could
be
reasonably
expected
to
be
part
of
the
current
diet.
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.
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.
Therefore,
a
PVC­
protein
expressed
in
a
plant
that
is
not
normally
infected
by
the
corresponding
virus
would
raise
no
safety
issues
as
long
as
the
corresponding
virus
infects
plants
consumed
by
humans.

EPA
is
also
considering
whether
a
geographic
limitation
on
any
categorical
exemption
would
also
be
necessary
to
ensure
that
the
exemption
extends
only
to
residues
that
are
part
of
the
U.
S.
diet;
i.
e.,
that
any
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.
Such
a
limitation
would
not
be
necessary
to
ensure
that
any
exempted
PVC­
proteins
fall
within
the
base
of
experience
supporting
the
exemption.
Humans
have
long
consumed
viruses
infecting
food
plants
with
no
adverse
effects.
Given
modern
market
practices
in
which
food
is
shipped
globally
for
consumption
by
people
of
diverse
nationalities,
broad,
transnational
human
dietary
exposure
to
all
viruses
that
infect
plants
humans
consume
is
likely
and
has
been
associated
with
no
known
adverse
effects.

2.
Exemption
conditional
on
Agency
determination
Product
developers
frequently
modify
the
genetic
material
of
a
PVCP­
PIP,
e.
g.,
in
order
to
achieve
greater
efficacy
(
Ref.
40).
Some
of
these
modifications
may
affect
the
PVC­
protein,
although
many
of
these
protein
changes
may
be
so
minor
that
they
are
unlikely
to
significantly
affect
potential
dietary
risk.
However,
at
this
time
the
Agency
cannot
articulate
a
criterion
that
would
ensure
all
PVC­
proteins
with
such
modifications
fall
within
the
base
of
experience
discussed
above
in
Unit
II.
A.

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
at
60539).
In
the
July
19,
2001
supplemental
notice
(
66
FR
Page
8
of
11
37865),
EPA
again
addressed
the
question
of
how
to
describe
PVCP­
PIPs
that
fall
within
the
recognized
base
of
experience
supporting
an
exemption.

In
October
2004,
the
FIFRA
SAP
was
asked
to
consider
the
degree
and
ways
a
PVCP
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.
13).
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."

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
may
not
be
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
an
exemption
that
would
allow
developers
to
self­
determine
eligibility
of
modified
PVCproteins
would
be
supportable.
Rather,
EPA
is
considering
an
option
under
which
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
from
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
would
consider
first
whether
the
protein
is
substantially
similar
to
a
natural
viral
coat
protein
by
evaluating
information
on
the
genetic
construct,
amino
acid
sequence,
and
molecular
weight
of
the
PVC­
protein.
EPA
might
also
evaluate
information
developed
by
the
submitter
from
public
sequence
databases
on
where
the
PVC­
protein
sequence
falls
relative
to
the
range
of
natural
variation.
Those
PVC­
proteins
that
are
determined
to
be
substantially
similar
would
be
further
evaluated
to
determine
whether
the
modified
PVC­
protein
is
as
safe
as
an
unmodified
protein
by
considering
information
on
the
expression
level
of
the
PVC­
protein
relative
to
levels
generally
found
in
plants
humans
consume
and
information
from
amino
acid
sequence
comparisons
with
known
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"
would
be
determined
on
a
case­
by­
case
basis.

For
residues
of
PVC­
proteins
that
would
not
qualify
for
either
the
categorical
or
conditional
exemption
discussed
above,
an
applicant
would
be
able
to
petition
the
Agency
for
an
individual
tolerance
exemption
under
FFDCA
section
408
(
See
also
40
CFR
180.7).

D.
Tolerance
Issues
Associated
with
Post­
Transcriptional
Gene
Silencing
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.
41)
used
tobacco
etch
virus
(
TEV)
to
infect
transgenic
tobacco
plants
containing
a
TEV
coat
protein
gene.
Plants
temporarily
developed
symptoms
but
were
able
Page
9
of
11
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.
Béclin
et
al.
(
Ref.
42)
showed
that
in
transgenic
tobacco
lines
expressing
a
 ­
glucuronidase
(
uidA)
transgene,
suppression
of
transgene
expression
always
occurs
but
is
initiated
at
different
plant
developmental
stages:
either
15
days
after
germination
or
two
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.
43)
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
possibly
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.
44)
demonstrated
that
cold
temperatures
inhibited
transgene­
induced
RNA
silencing
leading
to
increased
levels
of
transgene
mRNA,
although
the
level
of
transgenic
protein
was
not
reported.

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.
42).
As
in
other
experiments
(
Ref.
41),
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
is
known
that
PTGS
can
be
suppressed
leading
to
loss
of
the
virus­
resistant
phenotype
conferred
by
a
PVCP­
PIP.
For
example,
Savenkov
and
Valkonen
(
Ref.
45)
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
was
extremely
low
or
below
the
detection
limit,
it
was
readily
detectable
in
PVY­
infected
plants
where
suppression
of
gene
silencing
had
apparently
occurred.
The
study
did
not
report
whether
PVCprotein
was
produced
from
the
transgene
mRNA.

Such
experiments
suggest
that
many
factors
should
be
considered
in
making
a
determination
of
whether
a
PVC­
protein
might
be
produced
after
commercialization.
Some
characteristics
of
the
PVCP­
PIP,
e.
g.,
one
that
produces
untranslatable
or
antisense
coat
protein
transcripts,
may
offer
a
reasonable
level
of
assurance
that
PVC­
protein
production
would
not
occur.

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