Document ID: EPA-HQ-OPP-2005-0249-0009
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2005-12-28T05:00Z

Page
1
of
15
Attachment
III:
Environmental
Risk
Assessment
of
Plant
Incorporated
Protectant
(
PIP)
Inert
Ingredients
This
environmental
risk
assessment
of
PIP
inert
ingredients
is
divided
into
three
sections
which
cover
herbicide
tolerance
markers,
antibiotic
resistance
markers,
and
other
markers.

HERBICIDE
TOLERANCE
MARKERS
Phosphinothricin
acetyltransferase
(
PAT)

SUMMARY
The
Agency
has
conducted
an
environmental
risk
assessment
of
the
plant­
incorporated
protectant
(
PIP)
inert
ingredient
phosphinothricin
acetyltransferase
(
PAT),
and
the
genetic
material
necessary
for
its
production.
Topics
covered
in
this
assessment
include
mode
of
action,
ecological
effects,
endangered
species
considerations,
and
gene
flow
from
a
modified
plant
to
wild
or
weedy
relatives.
Data
cited
in
this
assessment
were
submitted
to
the
Agency
in
support
of
Dekalb's
DBT
418
and
Ciba
Seed's
Event
176
Bt
corn
registrations,
and
Syngenta's
COT
102
Bt
cotton
registration.
Ecological
data
and
published
information
on
the
biology
of
this
protein
indicate
that
this
PIP
inert
ingredient
is
not
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species.
Due
to
the
low
human
health
risks
associated
with
this
protein,
the
Agency
has
granted
an
exemption
from
the
requirement
of
a
tolerance
for
this
PIP
inert
ingredient
(
40
CFR
180.1151;
62
FR
17719,
Aug.
11,
1997).

MODE
OF
ACTION
The
PAT
enzyme
is
an
acetyltransferase
that
catalyzes
the
acetylation
the
active
ingredients
Lphosphinothricin
(
L­
PPT)
and
demethylphosphinothricin
(
DMPT),
conferring
tolerance
to
glufosinate
ammonium
herbicides.
L­
PPT
and
DMPT
inhibit
glutamine
synthetase,
resulting
in
the
accumulation
of
toxic
ammonium
ions
and
a
decrease
in
the
amount
of
glutamine,
an
essential
amino
acid
used
in
many
anabolic
processes.
In
the
presence
of
acetyl­
CoA,
the
PAT
protein
catalyses
the
acetylation
of
the
free
amino
group
of
L­
PPT
to
N­
acetyl­
L­
PPT,
a
compound
that
does
not
inhibit
glutamine
synthetase
(
OECD
2002).
The
PAT
enzyme
is
highly
specific
for
L­
PPT
and
does
not
acetylate
other
L­
amino
acids,
such
as
glutamate
(
Thompson
et
al.,
1987).

ENVIRONMENTAL
EFFECTS
The
Agency
assessed
the
toxicity
of
PAT
protein
to
representative
non­
target
organisms
that
could
be
exposed
to
this
PIP
inert
ingredient.
Toxicity
evaluations
included
the
following
Tier
I
studies1:
Mammalian
(
mouse),
Avian
(
northern
bobwhite
quail),
Freshwater
Fish
(
channel
1
Tier
I
testing
consists
of
maximum
dose
single
species
hazard
assessments
that
are
used
to
evaluate
the
potential
for
toxicity,
infectivity,
and
pathogenicity
of
a
pesticidal
agent
to
nontarget
organisms.
Page
2
of
15
catfish),
Aquatic
Invertebrate
[
cladoceran
(
Daphnia
magna)],
and
Non­
target
Arthropod
(
honey
bee
larvae,
lady
beetle,
green
lacewing,
collembola,
and
earthworm).
Tier
IV2
field
studies
of
non­
target
abundance
are
also
discussed
below.

The
wild
mammal
hazard
assessment
was
performed
on
the
basis
of
rodent
acute
oral
toxicity
data
prepared
for
human
health
risk
assessment
purposes
which
utilized
pure
PAT
protein.
Submitted
data
indicate
no
significant
adverse
effects
among
mice
dosed
with
up
to
2500
mg/
kg
body
weight
PAT
protein
(
DBT
418
MRID
439995­
02).

The
remaining
single
species
studies
were
maximum
hazard
dose
evaluations,
where
PAT
protein
toxicity
was
evaluated
by
feeding
select
non­
target
organisms
corn
pollen,
grain,
or
leaf
tissue,
which
has
been
shown
to
contain
the
PIP
inert
ingredient
PAT
and
the
active
ingredient
Cry1Ab
or
VIP3A.
Note:
Studies
submitted
in
support
of
the
COT102
registration
utilized
corn
plant
material
in
place
of
cotton
plant
material.

An
avian
study,
where
northern
bobwhite
quail
were
dosed
with
lyophilized
DBT
418
corn
leaf
tissue,
reported
no
adverse
effects
to
the
test
species
at
field
exposure
levels
(
MRID
439995­
10).
The
freshwater
fish
corn
grain
feeding
study
indicated
that
channel
catfish
diets
may
contain
up
to
50%
PAT­
containing
corn
grain
with
no
abnormalities
or
adverse
effects
on
fish
growth
(
final
report
not
submitted
as
of
9­
5­
03).
In
three
Event
176
and
four
COT102
corn
pollen
dietary
toxicity
studies,
no
adverse
effects
to
Daphnia
magna,
a
representative
of
aquatic
invertebrate
species,
honey
bee
larvae,
lady
beetle,
or
green
lacewing
were
seen
at
field
level
exposures
(
Event
176
MRIDs
433236­
10,
433396­
02,
434157­
03;
COT102
MRIDs
457921­
01,
457665­
09,
and
458358­
08).
Results
of
DBT
418
and
COT102
corn
leaf
tissue
feeding
studies
indicate
that
PAT
does
not
adversely
affect
reproduction
of
Collembola,
a
representative
plant
tissue
decomposer
(
DBT
418
MRID
439995­
12;
COT102
MRID
458358­
10).
Finally,
no
adverse
effects
were
seen
among
earthworms
exposed
for
14
days
to
soil
containing
lyophilized
DBT
418
corn
leaf
tissue
(
MRID
439995­
13).

A
semi­
field
study
of
honeybee
colonies
showed
that
the
overall
ability
of
colonies
to
produce
brood,
manage
stores
of
food
and
recruit
new
bees
was
not
affected
by
exposure
to
the
VIP3A
corn
containing
the
PAT
protein
(
MRID
458358­
13).
Field
studies
that
evaluated
arthropod
abundance
in
plots
of
stacked
VIP3A
x
Cry1Ab
Bt
corn
were
also
conducted.
To
date,
results
show
that
beneficial
arthropods
were
significantly
more
abundant
in
plots
containing
Bt
plants
than
in
plots
treated
with
conventional
chemical
insecticides
(
interim
report
MRID
458358­
07).

The
reviewed
data
showed
no
toxicity
to
non­
target
test
species.
As
a
result
of
these
findings
and
knowledge
of
the
mode
of
action
of
PAT,
the
Agency
concludes
that
no
unreasonable
adverse
effects
on
non­
target
organisms
are
expected
from
exposure
to
PAT
protein.

ENDANGERED
SPECIES
CONSIDERATIONS
2
Tier
IV
testing
refers
to
field
testing
under
simulated
or
actual
field
conditions.
These
studies
are
designed
on
a
case­
by­
case
basis
to
evaluate
specific
problems
that
cannot
be
resolved
by
lower
tier
testing.
Page
3
of
15
Because
this
PIP
inert
ingredient
is
neither
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species,
EPA
does
not
expect
that
exposure
to
the
PAT
protein
will
result
in
a
"
may
affect"
finding
for
any
endangered
or
threatened
species.

GENE
FLOW
CONSIDERATIONS
Gene
flow
may
occur
when
the
PAT
protein
is
expressed
in
a
plant
that
can
form
viable
hybrids
in
nature
with
wild
or
weedy
relatives
of
that
species.
With
the
assistance
of
the
October
2004
SAP,
EPA
has
identified
the
following
plants
as
not
having
wild
or
weedy
relatives
in
the
United
States,
its
possessions,
or
territories,
with
which
they
can
produce
viable
hybrids
in
nature:
almond
(
Prunus
communis),
apricot
(
Prunus
armeniaca),
asparagus
(
Asparagus
officinale)
avocado
(
Persea
americana),
banana
(
Musa
acuminata),
barley
(
Hordeum
vulgare),
bean
(
Phaseolus
vulgaris),
black­
eyed
pea
(
Vigna
unguiculata),
cacao
(
Theobroma
cacao),
celery
(
Apium
graveolens),
chickpea
(
Cicer
arietinum),
citrus
(
Citrus
spp.),
coffee
(
Coffea
arabicua),
corn
(
Zea
maize),
cucumber
(
Cucumis
sativus),
eggplant
(
Solanum
melongena),
guava
(
Psidium
guajava),
kiwi
(
Actinidia
spp.),
mango
(
Mangifera
indica),
nectarine
(
Prunus
persica),
okra
(
Abelmoschus
esculentus),
olive
(
Olea
europaea),
papaya
(
Carica
papaya),
parsley
(
Petroselinum
crispum),
pea
(
Pisum
sativum),
peach
(
Prunus
persica),
peanut
(
Arachis
hypogaea),
pineapple
(
Ananas
comosus),
pistachio
(
Pistacia
vera),
plum
(
Prunus
domestica),
potato
(
Solanum
tuberosum),
soybean
(
Glycine
max),
spinach
(
Spinacia
oleracea),
starfruit
(
Averrhoa
carambola),
taro
(
Colocasia
esculenta),
tomato
(
Lycopersicon
lycopersicum),
or
watermelon
(
Citrullus
lanatus).
Since
hybridization
with
wild
or
weedy
relatives
is
not
known
to
occur
among
plants
included
on
this
list,
EPA
has
concluded
that,
when
introduced
into
these
species,
PAT
presents
a
low
probability
of
risk
to
human
health
and
the
environment.

CP4
enolpyruvylshikimate­
3­
phosphate
synthase
(
CP4
EPSPS)

SUMMARY
The
Agency
has
conducted
an
environmental
risk
assessment
of
the
plant­
incorporated
protectant
(
PIP)
inert
ingredient
CP4
enolpyruvylshikimate­
3­
phosphate
synthase
(
CP4
EPSPS),
and
the
genetic
material
necessary
for
its
production.
Topics
covered
in
this
assessment
include
mode
of
action,
ecological
effects,
endangered
species
considerations,
and
gene
flow
from
a
modified
crop
to
wild
or
weedy
relatives.
Data
cited
in
this
assessment
was
submitted
to
the
Agency
in
support
of
Monsanto's
MON
810
Bt
Corn
registration.
Ecological
data
and
published
information
on
the
biology
of
this
protein
indicate
that
this
PIP
inert
ingredient
is
not
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species.
Due
to
the
low
human
health
risks
associated
with
this
protein,
the
Agency
has
granted
an
exemption
from
the
requirement
of
a
tolerance
for
this
PIP
inert
ingredient
(
40
CFR
180.1174;
61
FR
40340,
Aug.
2,
1996).

MODE
OF
ACTION
When
conventional
plants
are
treated
with
glyphosate
they
cannot
produce
the
aromatic
amino
acids
needed
to
grow.
Glyphosate
blocks
biosynthesis
of
aromatic
amino
acids
by
binding
to
5­
enolpyruvylshikimate­
3­
phosphate
synthase
(
EPSPS)
in
plants
(
Steinrucken
and
Amrhein,
1980).
Page
4
of
15
The
CP4
EPSPS
enzyme,
isolated
from
the
Agrobacterium
tumefaciens
strain
CP4,
functions
in
the
presence
of
glyphosate
and
thus
confers
tolerance
to
glyphosate
herbicides
when
expressed
in
plants.
The
EPSPS
enzyme
is
naturally
present
in
plants,
bacteria,
and
fungi
(
Levin
and
Sprinson,
1964).

ENVIRONMENTAL
EFFECTS
The
Agency
assessed
the
toxicity
of
CP4
EPSPS
protein
to
representative
non­
target
organisms
that
could
be
exposed
to
the
PIP
inert
ingredient.
Toxicity
evaluations
included
the
following
Tier
I
studies:
Mammalian
(
mouse),
Avian
(
juvenile
northern
bobwhite
quail),
and
Freshwater
Fish
(
channel
catfish).

The
wild
mammal
hazard
assessment
was
performed
on
the
basis
of
rodent
acute
oral
toxicity
data
prepared
for
human
health
risk
assessment
purposes
which
utilized
pure
CP4
EPSPS
protein.
Submitted
data
indicate
no
significant
adverse
effects
among
mice
dosed
with
up
to
572
mg/
kg
body
weight
for
CP4
EPSPS
protein
(
MRID
436433­
03).

The
avian
study
was
a
maximum
hazard
dose
evaluation,
where
CP4
EPSPS
protein
toxicity
was
evaluated
by
feeding
northern
bobwhite
quail
MON
801
corn
grain.
Results
of
this
feeding
study
showed
no
mortality
among
northern
bobwhite
quail
fed
diets
containing
MON
801
corn
grain
(
MRID
435332­
05).

Results
of
a
freshwater
fish
corn
grain
feeding
study
reported
that
no
adverse
effects
on
fish
growth
and
survival
were
seen
among
channel
catfish
fed
MON
801
corn
(
MRID
No.
438879­
01).

The
reviewed
data
showed
no
toxicity
to
non­
target
mammalian
and
avian
test
species.
As
a
result
of
these
findings,
knowledge
of
the
mode
of
action
of
CP4
EPSPS,
and
the
presence
of
a
similar
enzyme,
EPSPS,
in
all
plants,
the
Agency
concludes
that
no
unreasonable
adverse
effects
on
non­
target
organisms
are
expected
from
exposure
to
the
CP4
EPSPS
protein.

ENDANGERED
SPECIES
CONSIDERATIONS
Because
this
PIP
inert
ingredient
is
neither
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species,
EPA
does
not
expect
that
exposure
to
the
CP4
EPSPS
protein
will
result
in
a
"
may
affect"
finding
for
any
endangered
or
threatened
species.

GENE
FLOW
CONSIDERATIONS
Gene
flow
may
occur
when
the
CP4
EPSPS
protein
is
expressed
in
a
plant
that
can
form
viable
hybrids
in
nature
with
wild
or
weedy
relatives
of
that
species.
With
the
assistance
of
the
October
2004
SAP,
EPA
has
identified
the
following
plants
as
not
having
wild
or
weedy
relatives
in
the
United
States,
its
possessions,
or
territories,
with
which
they
can
produce
viable
hybrids
in
nature:
almond
(
Prunus
communis),
apricot
(
Prunus
armeniaca),
asparagus
(
Asparagus
officinale)
avocado
(
Persea
americana),
banana
(
Musa
acuminata),
barley
(
Hordeum
vulgare),
bean
(
Phaseolus
vulgaris),
black­
eyed
pea
(
Vigna
unguiculata),
cacao
(
Theobroma
cacao),
celery
(
Apium
graveolens),
chickpea
(
Cicer
arietinum),
citrus
(
Citrus
spp.),
coffee
(
Coffea
Page
5
of
15
arabicua),
corn
(
Zea
maize),
cucumber
(
Cucumis
sativus),
eggplant
(
Solanum
melongena),
guava
(
Psidium
guajava),
kiwi
(
Actinidia
spp.),
mango
(
Mangifera
indica),
nectarine
(
Prunus
persica),
okra
(
Abelmoschus
esculentus),
olive
(
Olea
europaea),
papaya
(
Carica
papaya),
parsley
(
Petroselinum
crispum),
pea
(
Pisum
sativum),
peach
(
Prunus
persica),
peanut
(
Arachis
hypogaea),
pineapple
(
Ananas
comosus),
pistachio
(
Pistacia
vera),
plum
(
Prunus
domestica),
potato
(
Solanum
tuberosum),
soybean
(
Glycine
max),
spinach
(
Spinacia
oleracea),
starfruit
(
Averrhoa
carambola),
taro
(
Colocasia
esculenta),
tomato
(
Lycopersicon
lycopersicum),
or
watermelon
(
Citrullus
lanatus).
Since
hybridization
with
wild
or
weedy
relatives
is
not
known
to
occur
among
plants
included
on
this
list,
EPA
has
concluded
that,
when
introduced
into
these
species,
CP4
EPSPS
presents
a
low
probability
of
risk
to
human
health
and
the
environment.

Glyphosate
oxidoreductase
(
GOX)

SUMMARY
The
Agency
has
conducted
an
environmental
risk
assessment
of
the
plant­
incorporated
protectant
(
PIP)
inert
ingredient
glyphosate
oxidoreductase
(
GOX),
and
the
genetic
material
necessary
for
its
production.
Topics
covered
in
this
assessment
include
mode
of
action,
ecological
effects,
endangered
species
considerations,
and
gene
flow
from
a
modified
crop
to
wild
or
weedy
relatives.
Data
cited
in
this
assessment
was
submitted
to
the
Agency
in
support
of
Monsanto's
MON
810
Bt
Corn
registration.
Ecological
data
and
published
information
on
the
biology
of
this
protein
indicate
that
this
PIP
inert
ingredient
is
not
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species.
Due
to
the
low
human
health
risks
associated
with
this
protein,
the
Agency
has
granted
an
exemption
from
the
requirement
of
a
tolerance
for
this
PIP
inert
ingredient
(
40
CFR
180.1190;
62
FR
52509,
Oct.
8,
1997).

MODE
OF
ACTION
GOX
catalyzes
the
conversion
of
glyphosate
to
aminomethylphosphonic
acid
(
AMPA)
and
glyoxylate;
by
increasing
the
speed
of
AMPA
degradation,
GOX
confers
glyphosate
tolerance
to
plants.
The
GOX
proteins
that
EPA
evaluated
for
registration
of
MON
810
Bt
Corn
and
for
the
GOX
tolerance
exemption
were
derived
from
a
gene
originally
isolated
from
Achromobacter
sp.
strain
LBAA.
Two
modified
GOX
proteins
are
specified
in
the
tolerance
exemption.
They
are
designated
GOX
and
GOXv247.
The
GOX
protein
has
the
same
amino
acid
sequence
as
the
native
protein,
with
an
additional
four
amino
acid
sequence
on
the
N­
terminus
(
remnants
of
an
added
signal
sequence).
In
GOXv247,
in
addition
to
the
four
amino
acids
at
the
N­
terminus
from
the
signal
sequence,
the
gene
sequence
was
altered
resulting
in
changes
to
three
amino
acids
in
the
protein
compared
to
the
native
protein.
These
protein
variants
are
similar
to
the
native
GOX
protein
in
terms
of
molecular
weight,
immunoreactivity,
amino
acid
sequence,
and
enzymatic
activity.

ENVIRONMENTAL
EFFECTS
The
Agency
assessed
the
toxicity
of
GOX
protein
to
mice,
a
representative
non­
target
mammalian
species
that
could
be
exposed
to
the
PIP
inert
ingredient
in
the
animal's
natural
Page
6
of
15
environment.
The
Tier
1
hazard
assessment
was
performed
on
the
basis
of
rodent
acute
oral
toxicity
data
prepared
for
human
health
risk
assessment
purposes
which
utilized
pure
GOX
protein.
Submitted
data
indicate
no
significant
adverse
effects
among
mice
dosed
with
up
to
91.3
and
104
mg/
kg
body
weight
for
GOX
and
GOXv247
proteins,
respectively
(
MRID
439037­
07
and
439037­
08).

The
reviewed
data
showed
no
toxicity
to
the
non­
target
mammalian
test
species.
As
a
result
of
this
finding,
and
knowledge
of
the
mode
of
action
of
the
enzyme,
the
Agency
concludes
that
no
unreasonable
adverse
effects
on
non­
target
organisms
are
expected
from
exposure
to
GOX
protein.

ENDANGERED
SPECIES
CONSIDERATIONS
Because
this
PIP
inert
ingredient
is
neither
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species,
EPA
does
not
expect
that
exposure
to
the
GOX
protein
will
result
in
a
"
may
affect"
finding
for
any
endangered
or
threatened
species.

GENE
FLOW
CONSIDERATIONS
Gene
flow
may
occur
when
the
GOX
protein
is
expressed
in
a
plant
that
can
form
viable
hybrids
in
nature
with
wild
or
weedy
relatives
of
that
species.
With
the
assistance
of
the
October
2004
SAP,
EPA
has
identified
the
following
plants
as
not
having
wild
or
weedy
relatives
in
the
United
States,
its
possessions,
or
territories,
with
which
they
can
produce
viable
hybrids
in
nature:
almond
(
Prunus
communis),
apricot
(
Prunus
armeniaca),
asparagus
(
Asparagus
officinale)
avocado
(
Persea
americana),
banana
(
Musa
acuminata),
barley
(
Hordeum
vulgare),
bean
(
Phaseolus
vulgaris),
black­
eyed
pea
(
Vigna
unguiculata),
cacao
(
Theobroma
cacao),
celery
(
Apium
graveolens),
chickpea
(
Cicer
arietinum),
citrus
(
Citrus
spp.),
coffee
(
Coffea
arabicua),
corn
(
Zea
maize),
cucumber
(
Cucumis
sativus),
eggplant
(
Solanum
melongena),
guava
(
Psidium
guajava),
kiwi
(
Actinidia
spp.),
mango
(
Mangifera
indica),
nectarine
(
Prunus
persica),
okra
(
Abelmoschus
esculentus),
olive
(
Olea
europaea),
papaya
(
Carica
papaya),
parsley
(
Petroselinum
crispum),
pea
(
Pisum
sativum),
peach
(
Prunus
persica),
peanut
(
Arachis
hypogaea),
pineapple
(
Ananas
comosus),
pistachio
(
Pistacia
vera),
plum
(
Prunus
domestica),
potato
(
Solanum
tuberosum),
soybean
(
Glycine
max),
spinach
(
Spinacia
oleracea),
starfruit
(
Averrhoa
carambola),
taro
(
Colocasia
esculenta),
tomato
(
Lycopersicon
lycopersicum),
or
watermelon
(
Citrullus
lanatus).
Since
hybridization
with
wild
or
weedy
relatives
is
not
known
to
occur
among
plants
included
on
this
list,
EPA
has
concluded
that,
when
introduced
into
these
species,
GOX
presents
a
low
probability
of
risk
to
human
health
and
the
environment.

ANTIBIOTIC
RESISTANCE
MARKER
Neomycin
phosphotransferase
II
(
NPTII)

SUMMARY
Page
7
of
15
The
Agency
has
conducted
an
environmental
risk
assessment
of
the
plant­
incorporated
protectant
(
PIP)
inert
ingredient
neomycin
phosphotransferase
II
(
NPTII),
and
the
genetic
material
required
for
its
production.
Topics
covered
in
this
assessment
include
mode
of
action,
ecological
effects,
endangered
species
considerations,
and
gene
flow
from
a
modified
crop
to
wild
or
weedy
relatives.
Data
cited
in
this
assessment
was
submitted
to
the
Agency
in
support
of
Monsanto's
NewLeaf
Potato
and
YieldGard
Plus
Corn
registrations
and
is
discussed
in
more
detail
in
the
Bacillus
thuringiensis
Plant­
Incorporated
Protectant
and
MON863
Biopesticide
Registration
Action
Documents.
Ecological
data
and
published
information
on
the
biology
of
this
protein
indicate
that
this
PIP
inert
ingredient
is
not
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species.
Due
to
the
low
human
health
risks
associated
with
this
protein,
the
Agency
has
granted
an
exemption
from
the
requirement
of
a
tolerance
for
this
PIP
inert
ingredient
(
40
CFR
180.1134;
59
FR
49125,
Sep.
28,
1994).

MODE
OF
ACTION
The
NPTII
enzyme
inactivates,
by
phosphorylation,
a
range
of
aminoglycoside
antibiotics,
including
neomycin
and
kanamycin.
NPTII
occurs
naturally
in
bacteria,
is
not
known
to
be
toxic,
and
degrades
rapidly
under
simulated
gastric
conditions.
Microbially­
produced
and
plantproduced
NPTII
enzymes
have
similar
molecular
weights
and
terminal
amino
acids,
indicating
that
glycosylation
and
post­
transcription
modifications
do
not
occur
with
plant­
expressed
NPTII.

ENVIRONMENTAL
EFFECTS
The
Agency
assessed
the
toxicity
of
NPTII
protein
to
representative
non­
target
organisms
that
could
be
exposed
to
the
PIP
inert
ingredient.
Toxicity
evaluations
included
the
following
Tier
I
studies:
Mammalian
(
mouse),
Avian
(
juvenile
northern
bobwhite
quail),
Freshwater
Fish
(
channel
catfish),
Aquatic
Invertebrate
[
cladoceran
(
Daphnia
magna)],
and
Non­
target
Arthropod
(
collembola,
lady
beetle,
and
monarch
butterfly).
A
Tier
IV
field
study
of
non­
target
abundance
is
also
discussed
below.

The
wild
mammal
hazard
assessment
was
performed
on
the
basis
of
rodent
acute
oral
toxicity
data
prepared
for
human
health
risk
assessment
purposes
which
utilized
pure
NPTII
protein.
Submitted
data
indicate
no
significant
adverse
effects
among
mice
dosed
with
up
to
5000
mg/
kg
body
weight
NPTII
protein
(
MRID
430547­
01).

The
remaining
single
species
studies
were
maximum
hazard
dose
evaluations,
where
NPTII
protein
toxicity
was
evaluated
by
feeding
select
non­
target
organisms
NewLeaf
or
YieldGard
Plus
plant
material,
which
has
been
shown
to
contain
the
PIP
inert
ingredient
NPTII
and
the
active
ingredient
Cry3A
(
NewLeaf)
or
Cry3Bb1
(
YieldGard
Plus).
The
avian
studies,
where
northern
bobwhite
quail
were
fed
potato
tuber
tissue
for
the
NewLeaf
and
corn
grain
for
the
YieldGard
Plus
evaluations,
reported
no
adverse
effects
to
the
test
species
at
field
exposure
levels
(
NewLeaf
MRIDs
429322­
14,
429322­
15;
MON863
MRID
449043­
15).
The
freshwater
fish
corn
grain
feeding
study
indicated
that
channel
catfish
diets
may
contain
up
to
35%
NPTII
protein­
containing
corn
grain
meal
with
no
adverse
effects
on
fish
growth,
feed
conversion
efficiency,
survival,
behavior,
or
body
composition
(
MON863
MRID
449043­
19).
In
a
corn
pollen
feeding
study,
no
adverse
effects
to
Daphnia
magna,
a
representative
of
aquatic
invertebrate
species,
were
seen
at
field
level
exposure
(
MON863
MRID
449043­
18).
In
separate
Page
8
of
15
corn
pollen
dietary
toxicity
studies,
no
adverse
effects
to
adult
and
larval
lady
beetles
were
seen
at
field
level
exposure
(
diet
composed
of
50%
pollen)
(
MON863
MRIDs
455382­
04,
453613­
02).
A
dietary
toxicity
study
with
the
monarch
butterfly
also
showed
no
adverse
effects
to
larvae
when
exposed
to
field
levels
of
corn
pollen
(
MON863
MRID
455382­
05).
Finally,
results
of
a
corn
leaf
tissue
feeding
study
indicated
that
the
NPTII
protein
does
not
adversely
affect
reproduction
of
Collembola,
a
representative
plant
tissue
decomposer
(
MON863
MRID
449043­
17).

A
field
study
that
evaluated
arthropod
abundance
in
YieldGard
Plus
fields
was
also
conducted.
Results
showed
that
beneficial
arthropods
(
i.
e.
lady
beetles,
damsel
bugs,
flower
flies,
soldier
beetles,
big
eyed
bugs,
spiders,
minute
pirate
bugs,
green
lacewings,
brown
lacewings,
stink
bugs,
and
ground
beetles)
were
significantly
more
abundant
in
plots
containing
Bt
plants
than
in
plots
treated
with
conventional
chemical
insecticides
(
MRID
456530­
03).

The
reviewed
data
showed
no
toxicity
to
non­
target
test
species.
As
a
result
of
these
findings,
and
knowledge
of
the
mode
of
action
of
the
enzyme,
the
Agency
concluded
that
no
unreasonable
adverse
effects
on
non­
target
organisms
are
expected
from
exposure
to
NPTII
protein.

ENDANGERED
SPECIES
CONSIDERATIONS
Because
this
PIP
inert
ingredient
is
neither
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species,
EPA
does
not
expect
that
exposure
to
the
NPTII
protein
will
result
in
a
"
may
affect"
finding
for
any
endangered
or
threatened
species.

GENE
FLOW
CONSIDERATIONS
EPA
anticipates
that
gene
flow
may
occur
if
NPTII
is
expressed
in
plants
that
have
wild
or
weedy
relatives
in
the
United
States,
its
possessions,
or
territories.
However,
no
unreasonable
adverse
effects
to
the
environment
are
expected
from
movement
of
this
inert
into
wild
plant
populations,
because
the
trait
is
unlikely
to
confer
selective
advantage
to
recipient
plants.

EPA
considers
the
frequency
of
horizontal
gene
transfer
(
HGT)
from
NPTII­
expressing
plants
to
microbes
to
be
very
low.
Studies
conducted
under
a
range
of
test
conditions
could
not
demonstrate
HGT
from
plants
to
microbes,
nor
is
there
a
clear
mechanism
for
such
transfer
(
Nap
et
al.,
1992;
Redenbaugh
et
al.
1994;
Schlüter
et
al.,
1995;
SAP
Report,
2001).
Nonetheless,
if
HGT
was
to
occur
it
would
not
have
a
significant
affect
on
existing
populations
of
neomycin
and
kanamycin
resistant
bacteria,
because
resistance
to
these
groups
of
antibiotics
is
widespread
in
naturally
occurring
microbes
in
humans
and
the
environment.

Due
to
the
low
probability
that
gene
flow
from
NPTII­
containing
plants
would
confer
a
selective
advantage
on
wild
or
weedy
relatives
of
that
species
and
the
low
frequency
of
HGT
from
NPTIIexpressing
plants
to
microbes,
EPA
has
concluded
that
this
inert
marker
poses
low
risk
to
human
health
and
the
environment
when
used
in
any
plant
as
part
of
a
PIP.

OTHER
MARKERS
Page
9
of
15
Beta­
D­
glucuronidase
(
GUS)
from
Escherichia
coli
SUMMARY
The
Agency
has
conducted
an
environmental
risk
assessment
of
the
Escherichia
coli­
derived
plant­
incorporated
protectant
(
PIP)
beta­
D­
glucuronidase
(
GUS),
and
the
genetic
material
necessary
for
its
production.
Topics
covered
in
this
assessment
include
mode
of
action,
ecological
effects,
endangered
species
considerations,
and
gene
flow
wild
or
weedy
relatives.
Data
cited
in
this
assessment
were
submitted
to
the
Agency
in
support
of
Monsanto's
Bollgard
II
Bt
cotton
registration
and
are
discussed
in
the
Bollgard
II
BRAD.
Ecological
data
and
published
information
on
the
biology
of
this
protein
indicate
that
this
PIP
inert
ingredient
is
not
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species.
Due
to
the
low
human
health
risks
associated
with
this
protein,
the
Agency
has
granted
an
exemption
from
the
requirement
of
a
tolerance
for
this
PIP
inert
ingredient
(
40
CFR
180.1216;
66
FR
42961,
Aug.
16,
2001).

MODE
OF
ACTION
E.
coli
 ­
glucuronidase
(
GUS),
an
enzyme
that
catalyzes
the
hydrolysis
of
glucuronides,
is
introduced
into
plants
to
serve
as
a
visual
marker
of
transformation
(
Guivarc'h
et
al.,
1996).
GUS
from
E.
coli
has
a
pH
optimum
near
7.0
and
maintains
enzymatic
activity
for
approximately
2
hours
at
55
º
C,
but
is
rapidly
inactivated
at
60
º
C.
This
enzyme
is
ubiquitous
in
the
digestive
system
of
humans
and
other
vertebrates.
Other
types
of
GUS
enzyme
are
present
in
the
liver,
spleen,
kidneys,
salivary
glands,
breast
milk
and
a
variety
of
other
tissues
in
humans,
other
vertebrates
and
a
number
of
invertebrates,
as
well
as
in
the
fruit,
seed
coat,
and
endosperm
of
various
plants.

ENVIRONMENTAL
EFFECTS
The
Agency
assessed
the
toxicity
of
GUS
protein
to
representative
non­
target
organisms
that
could
be
exposed
to
the
PIP
inert
ingredient.
Toxicity
evaluations
included
the
following
Tier
I
studies:
Mammalian
(
mouse),
Avian
(
juvenile
northern
bobwhite
quail),
Freshwater
Fish
(
channel
catfish),
and
Non­
target
Arthropod
(
collembola).

The
wild
mammal
hazard
assessment
was
performed
on
the
basis
of
rodent
acute
oral
toxicity
data
prepared
for
human
health
risk
assessment
purposes
which
utilized
pure
GUS
protein.
Submitted
data
indicate
no
significant
adverse
effects
among
mice
dosed
with
up
to
100.0
mg/
kg
body
weight
GUS
protein
(
MRID
449888­
00).

The
remaining
single
species
studies
were
maximum
hazard
dose
evaluations,
where
GUS
protein
toxicity
was
evaluated
by
feeding
select
non­
target
organisms
Bollgard
II
plant
tissue,
which
has
been
shown
to
contain
the
PIP
inert
ingredient
GUS
and
the
active
ingredients
Cry1Ac
and
Cry2Ab
in
cotton
leaf
and
seed
material.
The
avian
cottonseed
meal
feeding
study
reported
no
adverse
effects
to
northern
bobwhite
quail
at
field
exposure
levels
(
MRID
450863­
16).
The
freshwater
fish
cottonseed
meal
feeding
study
indicated
that
channel
catfish
diets
may
contain
up
to
20%
GUS
protein­
containing
cotton
seed
meal
with
no
adverse
effects
on
fish
growth,
feed
conversion
efficiency,
survival,
behavior,
or
body
composition
(
MRIDs
450863­
18
and
453371­
03).
Finally,
results
of
a
cotton
tissue
feeding
study
indicated
that
the
GUS
protein
is
non­
toxic
to
Page
10
of
15
Collembola,
a
representative
plant
tissue
decomposer,
and
did
not
adversely
affect
the
rate
of
Collembola
reproduction
(
MRID
450863­
14).

The
reviewed
data
showed
no
toxicity
to
non­
target
test
species.
As
a
result
of
these
findings,
and
the
natural
occurrence
of
this
protein
in
a
variety
of
species,
and
knowledge
of
the
mode
of
action
of
the
enzyme,
the
Agency
concluded
that
no
unreasonable
adverse
effects
on
non­
target
organisms
are
expected
from
exposure
to
GUS
protein.

ENDANGERED
SPECIES
CONSIDERATIONS
Because
this
PIP
inert
ingredient
is
neither
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species,
EPA
does
not
expect
that
exposure
to
the
GUS
protein
will
result
in
a
"
may
affect"
finding
for
any
endangered
or
threatened
species.

GENE
FLOW
CONSIDERATIONS
EPA
anticipates
that
gene
flow
may
occur
if
GUS
is
expressed
in
plants
that
have
wild
or
weedy
relatives
in
the
United
States,
its
possessions,
or
territories.
However,
due
to
the
low
probability
that
gene
flow
from
a
GUS­
containing
plant
would
confer
a
selective
advantage
on
wild
or
weedy
relatives
of
that
species,
EPA
has
concluded
that
this
inert
marker
poses
low
risk
to
human
health
and
the
environment
when
used
in
any
plant
as
part
of
a
PIP.

Phosphomannose
isomerase
(
PMI)

SUMMARY
The
Agency
has
conducted
an
environmental
risk
assessment
of
the
Escherichia
coli­
derived
plant­
incorporated
protectant
(
PIP)
phosphomannose
isomerase
(
PMI),
and
the
genetic
material
required
for
its
production.
Topics
covered
in
this
assessment
include
mode
of
action,
ecological
effects,
endangered
species
considerations,
and
gene
flow
from
a
modified
crop
to
wild
or
weedy
relatives.
Data
cited
in
this
assessment
were
submitted
to
the
Agency
in
support
of
Syngenta's
MIR604
Bt
corn
registration.
Ecological
data
and
published
information
on
the
biology
of
this
protein
indicate
that
this
PIP
inert
ingredient
is
not
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species.
Due
to
the
low
human
health
risks
associated
with
this
protein,
the
Agency
has
granted
an
exemption
from
the
requirement
of
a
tolerance
for
this
PIP
inert
ingredient
(
40
CFR
180.1252;
69
FR
26770,
May
14,
2004).

MODE
OF
ACTION
The
PMI
protein
is
a
ubiquitous
enzyme
involved
in
carbohydrate
metabolism.
Plant
cells
take
up
mannose
and
convert
it
to
mannose­
6­
phosphate,
an
inhibitor
of
glycolysis.
PMI
activity
converts
mannose­
6­
phosphate
to
fructose­
6­
phosphate,
an
intermediate
of
glycolysis,
which
positively
supports
growth
of
transformed
cells
(
Todd
and
Tague,
2001).
PMI,
or
a
highly
homologous
enzymatic
protein,
is
expressed
in
many
species
including
enteric
bacteria,
fungi,
insects,
some
species
of
nematodes,
and
mammals
including
monkeys,
mice
and
man.
Page
11
of
15
ENVIRONMENTAL
EFFECTS
The
Agency
assessed
the
toxicity
of
PMI
protein
to
representative
non­
target
organisms
that
could
be
exposed
to
the
PIP
inert
ingredient.
Toxicity
evaluations
included
the
following
Tier
I
studies:
Mammalian
(
mouse)
and
Freshwater
Fish
(
rainbow
trout).

The
wild
mammal
hazard
assessment
was
performed
on
the
basis
of
rodent
acute
oral
toxicity
data
prepared
for
human
health
risk
assessment
purposes
which
utilized
pure
PMI
protein.
Submitted
data
indicate
no
significant
adverse
effects
among
mice
dosed
with
up
to
5,050
mg/
kg
of
dosing
solution
or
3,080
mg/
kg
of
PMI
protein
(
MRID
459344­
07).

The
freshwater
fish
study
was
a
maximum
hazard
dose
evaluation,
where
PMI
protein
toxicity
was
evaluated
by
feeding
juvenile
rainbow
trout
Event
MIR604
corn
grain,
which
has
been
shown
to
contain
the
PIP
inert
ingredient
PMI
and
the
active
ingredient
Cry1Ab.
Results
of
the
feeding
study
indicate
that
rainbow
trout
diets
may
contain
up
to
50%
PMI
protein­
containing
corn
seed
meal
with
no
adverse
effects
on
fish
weight
or
length
(
MRID
461556­
17).

The
reviewed
data
showed
no
toxicity
to
non­
target
mammalian
and
freshwater
fish
test
species
used
in
the
evaluations.
As
a
result
of
these
findings,
and
the
natural
occurrence
of
this
protein
in
a
variety
of
microbe,
insect,
plant
and
mammalian
species,
the
Agency
has
concludes
that
no
unreasonable
adverse
effects
on
non­
target
organisms
are
expected
from
exposure
to
PMI
protein.

ENDANGERED
SPECIES
CONSIDERATIONS
Because
this
PIP
inert
ingredient
is
neither
a
known
toxin
and/
or
pathogen
of
plant
or
animal
species,
EPA
does
not
expect
that
exposure
to
the
PMI
protein
will
result
in
a
"
may
affect"
finding
for
any
endangered
or
threatened
species.

GENE
FLOW
CONSIDERATIONS
EPA
anticipates
that
gene
flow
may
occur
if
PMI
is
expressed
in
plants
that
have
wild
or
weedy
relatives
in
the
United
States,
its
possessions,
or
territories.
However,
due
to
the
low
probability
that
gene
flow
from
a
GUS­
containing
plant
would
confer
a
selective
advantage
on
wild
or
weedy
relatives
of
that
species,
EPA
has
concluded
that
this
inert
marker
poses
low
risk
to
human
health
and
the
environment
when
used
in
any
plant
as
part
of
a
PIP.
Page
12
of
15
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429322­
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A
Dietary
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409:
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161
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433236­
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(
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Bt
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PHO176­
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Toxicity
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Daphnids
(
Daphnia
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under
Static­
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prepared
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(
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maculata
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Bt"
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03
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Evaluation
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57
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435332­
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Campbell,
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A
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436433­
03
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(
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inert
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in
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Mice:
Lab
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Unpublished
study
prepared
by
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179
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438879­
01
Jackson,
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D.
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(
1995)
Evaluation
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Corn
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801
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Lab
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Number:
94­
01­
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39
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439037­
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Bailey,
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et
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(
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Preparation
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Study
in
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with
(
Inert
Ingredient):
Lab
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02­
30­
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447­
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42
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439037­
08
Naylor,
M.
(
1994)
Acute
Oral
Toxicity
Study
of
(
Inert
Ingredient)
in
Albino
Mice:
Lab
Project
Number:
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93­
206:
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Unpublished
study
prepared
by
Monsanto
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204
p.

439995­
02
Merriman,
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(
1996)
An
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with
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inert
ingredient):
Lab
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Number:
DGC­
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A18:
3406.2:
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prepared
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and
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50
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439995­
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Study
with
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DGC­
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439995­
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13601.1095.6100.630:
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449043­
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11231
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449043­
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Assessment
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55
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449043­
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48­
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31
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449043­
19
Meng,
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1999)
Evaluation
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859
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449888­
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450863­
14
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Assessment
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2
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Amended
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Lab
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252.6152:
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99­
063:
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16174.
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study
prepared
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49
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450863­
16
Gallagher,
S.;
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J.;
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J.
(
2000)
Insect
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2
In
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A
Dietary
Toxicity
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with
the
Northern
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Lab
Project
Number:
139­
449:
WL­
99­
065:
MSL
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study
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40
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450863­
18
Li,
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E.
(
2000)
Evaluation
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Cotton
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15813
and
15985
as
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Feed
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Final
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Lab
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XX­
99­
056:
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16179:
99­
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National
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22
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453371­
03
Li,
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E.
(
2001)
Evaluation
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Cottonseed
Meal
Derived
from
Insect
Protected
Cotton
Lines
15813
and
15985
as
a
Feed
Ingredient
for
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Lab
Project
Number:
XX­
99­
056:
MSL
16179.
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study
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National
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23
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453613­
02
Bryan,
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(
2001)
Dietary
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BT
Corn
Pollen
Expressing
a
Variant
of
Cry3Bb1
Protein
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2000­
158:
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04
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2001)
Dietary
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Expressing
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Larvae
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16907:
00­
01­
39­
26.
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Teixeira,
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FFMIR604­
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