October
2,
2003
Memorandum
Subject:
Review
of
Monsanto's
Application
for
a
Section
3
Registration
of
Bacillus
thuringiensis
Cry1Ab
and
Cry3Bb1
Proteins
and
the
Genetic
Material
Necessary
for
their
Production
in
YieldGard
®
Plus
Corn.
DP
Barcode
No.
D287602;
Case
No.
067359;
Submission
No.
S627345;
Chemical
No.
006498;
EPA
Registration
No.
524­
LUL;
MRID
Nos.
457917­
00
and
460697­
01.

To:
Mike
Mendelsohn,
Regulatory
Action
Leader
Biopesticides
and
Pollution
Prevention
Division,
7511C
From:
Robyn
Rose,
Entomologist
Biopesticides
and
Pollution
Prevention
Division,
7511C
Through:
Zigfridas
Vaituzis,
PhD,
Microbiologist
Biopesticides
and
Pollution
Prevention
Division,
7511C
Classification:
Supplemental
to:

1.
Submission
and
review
of
two
acceptable
non­
target
beneficial
insect
studies
with
the
Cry3Bb1
protein.
Recommended
insects
include
the
minute
pirate
bug
(
Orius
insidiosus)
and
a
Carabid
beetle
(
e.
g.,
Pterostichus
spp.).
2.
Small
and
large­
scale
field
studies
should
be
conducted
with
YieldGard
®
Plus
Corn
to
verify
there
are
no
adverse
ecological
effects
to
non­
target
invertebrate
populations.
3.
Developing
and
implementing
a
monitoring
for
resistance
plan,
remedial
action
plan,
grower
education
and
compliance
assurance
program
that
is
applicable
for
the
European
corn
borer
and
corn
rootworm.
Results
of
the
IRM
grower
agreement,
grower
education
and
compliance
assurance
programs
should
be
submitted
to
EPA
annually
by
January
31.
4.
Submit
field
degradation
studies
evaluating
accumulation
and
persistence
of
Cry1Ab
and
Cry3Bb1
from
YieldGard
®
Plus
Corn
in
several
different
soils
in
various
strata.
Representative
fields
must
have
been
planted
with
YieldGard
®
Plus
Corn
for
at
least
three
consecutive
years
and
include
both
conventional
tillage
and
no­
till
samples
and
be
harvested
under
typical
agronomic
conditions.
Sampling
must
continue
until
the
limit
of
detection
is
reached.
Studies
should
include
soils
with
high
levels
of
a
variety
of
2
clays.
Both
ELISA
and
insect
bioassays
need
to
be
conducted
and
compared
to
determine
if
Cry1Ab
and
Cry3Bb1
are
accumulating
or
persisting
in
soil
samples.

Background:
Monsanto
is
requesting
a
full
and
unrestricted
FIFRA
Section
3
registration
for
commercialization
of
YieldGard
®
Plus
Corn.
Materials
supporting
a
request
to
register
Bacillus
thuringiensis
Cry1Ab
and
Cry3Bb1
Proteins
and
the
Genetic
Material
Necessary
for
their
Production
in
YieldGard
®
Plus
Corn
(
EPA
Registration
No.
524­
LUL)
were
submitted
by
Monsanto
to
EPA
on
August
20,
2002
and
on
September
4,
2003.
On
April
1,
2003,
an
amendment
to
the
application
for
registration
was
submitted
that
included
a
revised
insect
resistant
management
plan
(
IRM).
Cry1Ab
(
EPA
Reg.
No.
524­
489)
and
Cry3Bb1
(
EPA
Reg.
No.
524­
528)
are
currently
registered
pesticidal
active
ingredients
with
the
EPA
that
have
been
granted
an
exemption
from
the
requirement
of
a
tolerance.
Corn
events
MON
810
and
MON
863
were
developed
from
recombinant
DNA
techniques.
YieldGard
®
Plus
Corn
is
the
brand
name
Monsanto
is
proposing
to
use
for
a
new
end­
use
product
containing
the
Cry1Ab
and
Cry3Bb1
active
ingredients
stacked.
YieldGard
®
Plus
Corn
was
developed
by
crossing
inbred
lines
of
event
MON
810
lepidopteranactive
corn
with
inbred
lines
of
event
MON
863
coleopteran­
active
corn.

Monsanto
suggests
that
there
is
a
need
for
corn
containing
Cry1Ab
and
Cry3Bb1
because
"
there
is
an
overlap
in
the
geographical
distribution
of
corn
borer
and
corn
rootworm
pests."
Areas
of
the
western
Corn
Belt
such
as
Nebraska,
Kansas
and
Colorado
as
well
as
the
Texas
Panhandle,
Oklahoma,
southeast
Missouri,
Tennessee
and
Kentucky
will
potentially
have
high
densities
of
corn
borers
and
corn
rootworms,
thus
leading
to
the
need
for
active
ingredients
against
both
pests.
According
to
Monsanto,
YieldGard
®
Plus
Corn
provides
root
and
stalk
protection
against
corn
borers
and
corn
rootworms
respectively
"
at
levels
comparable
to
their
single
trait
parental
lines.

This
review
includes
a
Data
Evaluation
Report
(
DER)
of
Volume
1
of
4
of
Monsanto's
August
20
submission
to
register
YieldGard
®
Plus
Corn
and
a
DER
of
the
September
4
submission
that
shows
a
lack
of
interaction
between
the
Cry1Ab
and
Cry3Bb1
proteins.
Volume
1
includes
the
Administrative
Materials
for
the
registration
application.
The
product
label,
product
analysis,
residue
data,
non­
target
organism
data,
toxicology
data,
efficacy
data
and
insect
resistance
management
(
IRM)
data
submitted
by
Monsanto
in
Volume
1
are
summarized
in
the
DER.

Conclusions:

Product
Analysis
The
product
analysis
data
found
in
Volumes
2
(
MRID
No.
457917­
01)
and
3
(
MRID
No.
457917­
02)
of
Monsanto's
submission
requesting
the
registration
of
YieldGard
®
Plus
Corn
will
be
reviewed
by
EPA
to
verify
that
adequate
data
has
been
submitted
to
support
this
registration.
3
Residue
Data
Cry1Ab,
Cry3Bb1,
nptII
and
the
genetic
material
necessary
for
their
production
in
corn
have
each
been
granted
an
exemption
from
the
requirement
of
a
tolerance.
However,
the
Cry3Bb1
tolerance
exemption
is
currently
time­
limited.
EPA
is
currently
reviewing
Monsanto's
request
for
a
permanent
exemption
from
the
requirement
of
a
tolerance
for
Cry3Bb1.
The
Cry1Ab
and
Cry3Bb1
tolerance
exemptions
are
applicable
to
YieldGard
®
Plus
Corn.

Non­
target
Organism
Data
Monsanto
notes
that
no
synergistic,
additive
or
antagonistic
effects
of
Bt
toxins
have
been
found
against
lepidopteran
insects.
However,
studies
conducted
thus
far
include
microbial
Bt
formulations
and
not
plant
incorporated
protectants
(
PIPs).
Pleiotropic
or
other
effects
of
inserting
Bt
genes
into
corn
plants
may
affect
the
concentration
level
in
the
plant
and
toxicity
to
target
or
non­
target
organisms.
Nevertheless,
non­
target
beneficial
insect
data
may
be
waived
provided
the
susceptibility
of
target
pests
to
YieldGard
®
Plus
Corn
is
comparable
to
their
susceptibility
to
the
single
trait
Cry1Ab
and
Cry3Bb1
corn.
If
there
is
no
change
in
susceptibility
among
susceptible
insects,
then
it
is
unlikely
that
there
will
be
a
difference
in
effects
of
the
stacked
versus
single
trait
hybrids
on
non­
target
insects.
According
to
data
collected
by
Monsanto,
susceptibility
of
the
European
corn
borer,
western
corn
rootworm
and
fall
armyworm
was
comparable
among
YieldGard
®
Plus
Corn
and
the
single
trait
Cry1Ab
and
Cry3Bb1
hybrids.

Mammalian
Wildlife.
Mammalian
toxicology
information
gathered
to
date
on
Bt
Cry
proteins
does
not
show
a
hazard
to
wild
or
domesticated
mammals.
The
data
submitted
to
EPA
for
Cry1Ab
and
Cry3Bb1
indicate
that
there
is
no
significant
toxicity
to
rodents
from
acute
oral
testing
at
the
maximum
hazard
dose.
In
addition,
there
are
no
reports
of
adverse
effects
on
livestock
after
several
years
of
feeding
with
Bt
corn.
Mammalian
wildlife
exposure
to
the
Bt
Cry
proteins
is
considered
likely;
however,
the
mammalian
toxicology
information
gathered
to
date
does
not
show
a
hazard
to
wild
or
domesticated
mammals.
Therefore,
no
hazard
to
mammalian
wildlife
is
anticipated.

Avian.
Cry1Ab
administered
by
oral
gavage
at
a
dosage
up
to
2,000
mg
protein/
kg
body
weight,
resulted
in
no
apparent
effect
upon
bobwhite
quail
after
14
days.
A
study
with
a
non­
commercial
line
of
MON
80187
showed
no
mortality
or
differences
in
food
consumption,
body
weight,
or
behavior
when
bobwhite
quail
were
fed
50,000
or
100,000
ppm
Cry1Ab
in
corn
meal.
In
addition,
there
are
no
reports
of
adverse
effects
from
the
commercial
poultry
industry
after
several
years
of
using
Bt
corn
in
poultry
feeds.

The
dietary
LC
50
value
for
Cry3Bb1
corn
grain
(
MON
853,
MON
854
and
MON
855)
when
fed
to
juvenile
Northern
Bobwhite
for
5
days
was
reported
to
be
greater
than
70,000
ppm
(
10%
of
the
diet),
the
only
concentration
tested.
No
adverse
effects
on
bobwhite
quail
were
seen
in
eight
days.
These
data
show
that
there
will
be
no
hazard
to
avian
wildlife
from
incidental
field
exposure
to
Cry3Bb1
corn.
These
data
are,
however,
not
sufficient
to
make
a
hazard
assessment
from
4
repeated
exposure
to
higher
doses
of
Cry3Bb1
corn.
This
study
is
classified
as
supplemental.
The
concentration
tested
(
10%
corn
in
the
diet)
is
too
low.
The
Agency
has
requested
a
six
week
broiler
study
with
60%
­
70%
MON
863
corn
in
the
diet
to
assess
hazard
to
non­
target
birds
from
continuous
exposure
to
high
levels
of
Cry3Bb1
protein.

Data
collected
thus
far
indicate
that
the
Cry1Ab
and
Cry3Bb1
protein
produced
in
corn
does
not
show
a
hazard
to
birds.
However,
these
data
are
not
sufficient
to
make
a
final
hazard
assessment
from
repeated
exposure(
s)
to
higher
doses
of
Bt
corn.
A
six
week
broiler
study
with
60
to
70%
corn
in
the
diet
is
necessary
to
assess
hazards
from
chronic
exposure
of
wild
and
domesticated
fowl.

Aquatic
Species.
There
is
no
evidence
for
sensitivity
of
aquatic
(
including
endangered)
species
to
Cry
proteins.
Toxicity
studies
with
aquatic
organisms
show
very
limited
hazard
for
fish
or
invertebrates
exposed
to
either
corn
pollen
or
to
bacterially
expressed
Cry
protein.
In
addition,
aquatic
exposure
from
Bt
crops
is
extremely
small.
A
simple
standard
pond
scenario
(
1
hectare
pond,
2
meters
deep
draining
a
10
hectare
watershed
planted
with
corn)
was
used
to
develop
a
worst
case
EEC
for
Cry1Ab
and
Cry3Bb1
protein
(
high
protein
expression
level)
on
the
basis
of
corn
pollen
loadings
from
airborne
pollen
deposition
and
agricultural
runoff
from
corn
plant
tissue
left
in
the
field
at
the
end
of
harvest
(
assuming
that
no
degradation
of
the
protein
takes
place).
Airborne
pollen
deposition
will
result
in
water
concentrations
less
than
78
ng
Cry1Ab
protein/
L
when
based
on
conservative
estimates
for
pollen
dispersal.
The
contribution
of
Cry1Ab
to
the
pond
through
agricultural
runoff
is
comparable
(
66
ng
L­
1
based
on
GENECC).
Thus,
total
water
concentration
of
less
than
144
ng
Cry1Ab
protein/
L
is
projected
under
worst
case
conditions
(
Wolt,
2000).
Airborne
and
agricultural
runoff
for
Cry3Bb1
is
calculated
to
be
3.9
ng
protein/
mL.
Thus,
total
water
concentration
of
less
than
3.9
ng
Cry3Bb1
protein/
mL
is
projected
under
worst
case
conditions.

Aquatic
Invertebrate.
The
major
source
of
Bt
Cry
proteins
in
fresh
water
would
be
corn
pollen.
Toxicity
studies
with
corn
pollen
containing
Cry1Ab
proteins
conducted
using
Daphnia
magna
show
an
acute
EC
50
was
greater
than
100
mg/
L
in
one
study
and
in
another150
mg/
L.
The
LOEC
(
lowest
observed
effect
concentration)
was
found
to
be
150
mg/
L.
The
amount
of
pollen
was
considered
to
well
exceed
the
144
ng
Cry1Ab
protein
/
L
projected
aquatic
exposure
in
the
fields
under
worst
case
conditions.

The
test
material
for
Cry3Bb1
consisted
of
corn
pollen
from
corn
plants,
line
MON
858.
The
Cry3Bb1
content
was
estimated
to
be
18.8

g/
g
fresh
weight
pollen.
No
treatment
mortality
or
behavior
change
was
reported
between
the
dosed
and
control
replicates
for
the
48­
hr
exposure
period.
Since
the
Cry3Bb1
protein
is
confined
to
corn
tissue,
and
the
worst
case
aquatic
EEC
is
calculated
to
be
3.9
ng
Cry3Bb1
protein/
mL,
there
is
no
substantial
exposure
to
aquatic
invertebrates,
and
therefore
no
hazard
from
the
registered
use
of
Cry3Bb1
containing
corn
is
anticipated.

Freshwater
Fish.
The
requirement
for
a
fresh
water
fish
static
renewal
toxicity
study
for
Cry1Ab
5
protein
has
been
waived
based
on
a
lack
of
any
substantial
exposure
of
fish
to
the
Bt
Cry
proteins
produced
in
Bt
crops
(
Wolt,
2000).
Farm
fish
diets
made
with
corn
containing
the
Cry
proteins
do
not
adversely
affect
susceptible
target
insect
larvae,
as
determined
through
bioassay
testing
and
analyses
using
ELISA
indicate
that
Cry
protein
is
not
detectable
in
the
fish
feed
samples.
Therefore,
farm
fish
eating
a
food
mix
made
from
corn
containing
the
Bt
delta
endotoxin
would
not
be
exposed
to
detectable
active
Bt
Cry
protein.

Channel
catfish
were
fed
diets
containing
finely
ground
corn
grain
from
two
insect­
protected
Cry3Bb1
corn
lines
(
MON
853
and
MON
859)
for
eight
weeks.
The
results
indicate
that
corn
grain
derived
from
the
two
transgenic
lines
producing
Cry3Bb1
can
be
used
as
a
feed
ingredient
in
channel
catfish
diets
at
levels
of
up
to
35%
without
adverse
effect
on
fish
growth,
feed
conversion
efficiency,
survival,
behavior,
or
body
composition.
Significant
differences
were
observed
only
as
lower
percentage
fillet
moisture
among
fish
fed
corn
grain
of
the
line
MON
859;
however,
these
are
relatively
unremarkable
and
are
unlikely
related
to
the
different
diets.
There
were
no
significant
differences
noted
in
feed
consumption,
weight
gain,
feed
conversion
ratio,
survival,
percentage
visceral
fat,
or
percentages
fat,
protein,
or
ash
in
fillets
of
channel
catfish
fed
the
different
test
diets.
No
abnormal
fish
behavior
was
observed
in
the
study.
In
view
of
the
lack
of
demonstrated
toxicity
to
channel
catfish
and
minimal
aquatic
exposure,
no
fresh
water
fish
hazard
is
expected
from
the
uses
of
Cry3Bb1
protein
in
corn
crops.

Plants.
Since
the
Cry1Ab
and
Cry3Bb1
active
ingredient
in
YieldGard
®
Plus
Corn
is
an
insect
toxin
(
Bt
endotoxin)
that
has
never
shown
any
toxicity
to
plants,
the
plant
toxicity
studies
have
been
waived.

Honey
Bees.
Feeding
tests
were
conducted
on
both
honey
bee
larvae
and
adults
for
Cry1Ab
proteins.
At
a
single
dose
of
Cry1Ab,
20
ppm
showed
no
adverse
effects
to
larval
honey
bees
under
the
test
conditions.
The
LC
50
for
Cry1Ab
was
determined
to
be
greater
than
20
ppm.
In
adult
honey
bees,
no
statistically
significant
differences
were
seen
among
the
various
treatment
and
control
groups.

Testing
was
conducted
with
Bacillus
thuringiensis
Cry
3B2.11231
protein
(
purity
96%;
1.79
mg
active
protein/
mL
water;
current
nomenclature
refers
to
this
protein
as
Cry3Bb1)
inoculated
directly
into
larval
brood
cells
prior
to
capping.
Within
18
days
after
treatments
were
administered,
all
larvae
emerged
from
capped
brood
cells.
All
of
the
larvae
(
100%)
treated
with
Cry3Bb1
protein
survived
to
pupation
or
"
capping";
whereas,
97.5%
(
2.5%
mortality)
of
the
honey
bee
larvae
in
the
control
group
survived
to
pupation.
There
was
no
statistical
difference
(
p=
0.05)
in
total
percent
mortality
during
the
larval
development
or
adult
emergence
stages
between
treated
and
control
groups.
Based
on
the
results
presented
in
the
study,
it
can
be
concluded
that
honey
bee
development
and
survival
are
not
affected
by
exposure
to
the
Cry3Bb1
protein.
There
was
88.8%
mortality
of
larvae
treated
with
the
reference
substance
potassium
arsenate
which
indicated
that
bees
were
exposed
to
the
treatments.
The
LC
50
for
honey
bee
larvae
was
determined
to
be
>
1,790
ppm
Cry3Bb1
protein.
6
Adult
honey
bee
testing
consisted
of
a
control
group
fed
30%
sucrose
in
deionized
water,
a
reference
group
fed
100

g/
mL
potassium
arsenate,
and
a
test
group
fed
360

g/
mL
of
Cry3Bb1
protein
and
a
water
only
group.
The
study
concluded
that
360

g/
mL
Cry3Bb1
protein
did
not
affect
survival
or
behavior
of
adult
honey
bees.
The
maximum
hazard
dose
LC
50
of
>
360

g/
mL
is
20X
the
concentration
found
in
pollen.
Therefore,
no
hazard
to
adult
honey
bees
is
expected
from
exposure
to
the
Cry3Bb1
protein
in
corn
pollen.

Lady
Beetles.
Lady
beetle
(
Hippodamia
convergens)
predator
toxicity
studies
submitted
at
the
time
of
Cry1Ab
registration
demonstrate
that
corn
pollen
containing
the
anti­
lepidopteran
Cry
proteins
do
not
cause
detectable
adverse
effects
to
lady
beetles.
Purified
Cry1Ab
protein
at
20
ppm
also
showed
no
adverse
effects
or
behavior
changes.
The
test
insects
were
exposed
to
the
active
ingredient
at
approximately
the
dose
that
would
be
ingested
by
the
beetles
feeding
on
aphids
under
field
conditions.

Since
the
Cry3Bb1
protein
specifically
targets
coleopteran
(
beetle)
insects,
particular
attention
is
warranted
regarding
potential
effects
of
Cry3Bb1
on
lady
beetles.
A
diet
containing
purified
Cry3Bb1
protein
and
honey
was
fed
to
the
adult
lady
beetle
(
H.
convergens)
at
rates
one
and
20
times
the
maximum
protein
concentration
found
in
corn
leaf
tissue.
When
the
negative
control
group
reached
20%
mortality
on
day
10
,
the
results
showed
no
significant
differences
in
the
mortality
rate
between
lady
beetles
fed
400
and
8,000

g
Cry3Bb1/
mL
of
diet.
Results
from
this
study
showed
that
the
LC
50
for
Cry3Bb1
when
incorporated
in
diet
and
fed
to
H.
convergens
is
>
8,000

g
Cry3Bb1
protein/
mL
diet.
Mortality
for
the
1,000
and
10,000

g
potassium
arsenate/
mL
diet
groups
were
55%
and
95%
respectively
at
day
10.
This
demonstrates
that
toxicity
can
be
measured
by
mixing
a
test
substance
in
the
lady
beetle
diet.
Lady
beetles
do
not
feed
on
corn
plant
tissue.
They
do,
however,
feed
on
corn
pollen
and
prey
on
pest
insects
that
may
feed
on
corn
tissue
and
contain
Cry3Bb1
in
their
gut,
thus
exposing
lady
beetles
to
the
Bt
protein.
There
is
approximately
390

g
Cry3Bb1/
g
fresh
weight
corn
tissue.
Lady
beetle
exposure
is
expected
to
be
significantly
lower
than
this
since
the
corn
tissue
would
be
metabolized,
eliminated,
or
otherwise
degraded
within
the
prey
species.
Since
the
maximum
hazard
dose
LC
50
was
found
to
be
8,000

g
Cry3Bb1/
mL
diet
which
is
20
times
higher
than
maximum
expected
exposure
levels.

At
certain
times
corn
pollen
may
comprise
up
to
50%
of
lady
beetle
larvae's
diet.
Therefore
the
effects
of
corn
pollen
containing
event
MON
863
Cry3Bb1
protein
on
lady
beetle
larvae
(
Coleomegilla
maculata)
was
evaluated.
Pollen
was
fed
to
lady
beetle
larvae
in
a
diet
consisting
of
equal
amounts
of
lyophilized
tephritid
fruit
fly
eggs
and
bee
pollen.
Diets
contained
50%
pollen
(
93

g
Cry3Bb1/
g
fresh
pollen
weight)
since
this
is
the
potential
level
of
field
exposure
and
an
equal
amount
of
the
tephritid
fruit
fly
diet.
First
instar
lady
beetle
larvae
were
individually
placed
in
test
arenas
to
avoid
cannibalism.
There
was
not
a
statistically
significant
difference
between
developmental
time
of
larvae
to
pupae
and/
or
adults;
nor
was
there
a
difference
in
adult
weight
survival
between
larvae
fed
bee
pollen
or
corn
pollen
nor
was
there
a
difference
between
larvae
fed
Bt
and
non­
Bt
pollen.
There
was
a
significant
difference
between
the
reference
group
(
potassium
arsenate)
and
other
test
groups
since
no
larvae
survived
in
the
reference
group.
The
7
100%
mortality
observed
in
the
reference
group
verified
that
the
lady
beetles
were
ingesting
the
diet.
This
test
was
conducted
with
pollen
levels
greater
than
or
equal
to
levels
lady
beetle
larvae
are
expected
to
be
exposed
to
in
the
field.
Therefore,
the
LD
50
for
Cry3Bb1
expressed
in
corn
pollen
is
>
93

g/
g
fresh
pollen
weight.
This
study
demonstrates
that
lady
beetle
larvae
will
not
be
adversely
affected
by
Cry3Bb1
field
corn.

Coleomegilla
maculata
lady
beetle
adults
were
fed
diets
of
transgenic
corn
pollen
mixed
with
fruit
fly
eggs
to
determine
the
potential
effects
of
transgenic
pollen
to
beetles
(
MRID
No.
453613­
01).
The
corn
(
MON
863)
test
pollen
(
assayed
at
the
time
of
testing)
contained
the
Cry3Bb1
protein
at
a
concentration
of
37.4

g/
g
pollen.
After
30
days
of
diet
exposure,
83.3
and
80.0%
of
adult
C.
maculata
survived
in
the
test
and
control
pollen
groups,
respectively.
While
these
survival
rates
were
significantly
less
than
that
in
the
assay
control
group
(
bee
pollen
which
exhibited
100%
survival),
there
were
no
significant
differences
between
the
test
and
control
pollen
groups.
All
adults
in
the
positive
control
(
arsenate
treated
corn
pollen)
died
in
less
than
8
days.
Results
indicated
that
transgenic
Bt
corn
pollen
expressing
the
variant
Cry3Bb1
protein
have
no
significant
negative
effects
on
the
survival
of
Coleomegilla
maculata
adults.

Hippodamia
convergens
adults
were
fed
diets
of
transgenic
corn
pollen
in
honey
to
determine
the
potential
effects
of
transgenic
pollen
to
non­
target
beetles.
The
corn
(
MON
863)
test
pollen
(
assayed
at
the
time
of
testing)
contained
the
Cry3Bb1
protein
at
a
concentration
of
37.4

g/
g
pollen.
After
15
days
of
diet
exposure,
84%
and
81%
of
adult
Hippodamia
convergens
survived
in
the
test
pollen
and
control
pollen
groups,
respectively.
There
were
no
significant
differences
in
survival
among
the
test
pollen,
control
pollen
and
the
assay
control
(
honey
only)
treatment
groups.
Only
5%
of
beetles
exposed
to
the
positive
control
(
arsenate
treated
corn
pollen)
survived.
Results
demonstrate
that
transgenic
Bt
corn
pollen
expressing
the
variant
Cry3Bb1
protein
had
no
significant
negative
effects
on
the
survival
of
Hippodamia
convergens
adults
from
dietary
exposure.

No
adverse
effects
were
detected
when
Coleomegilla
maculata
and
Hippodamia
convergens
were
fed
MON
863
pollen
in
diet
in
the
laboratory.
Pollen
levels
fed
on
by
the
lady
beetles
in
this
study
exceeded
concentrations
that
are
expected
to
be
encountered
in
the
field.
Therefore,
it
can
be
concluded
the
MON
863
will
not
pose
a
hazard
to
lady
beetle
adults
in
the
field.

Parasitic
Hymenoptera.
No
adverse
effects
were
observed
when
a
maximum
hazard
dose
of
20
ppm
Cry
1Ab
was
tested
on
Brachymeria
intermedia.
Therefore
the
LC
50
is
greater
than
20
ppm
and
no
adverse
effect
to
parasitic
Hymenoptera
are
expected
from
exposure
to
Cry1Ab
protein
in
the
field.

A
dietary
toxicity
study
with
the
parasitic
Hymenoptera
(
Nasonia
vitripennis)
was
conducted
with
Bacillus
thuringiensis
Cry
3B2.11231
(
Cry3Bb1)
protein
(
purity
96%;
34.5
mg
active
protein/
mL
water).
Wasps
were
tested
at
rates
of
400
and
8,000
ppm
Cry3Bb1
protein
which
is
approximately
equivalent
to
1X
and
20X
the
maximum
protein
concentration
in
MON
863
plant
tissue.
The
LC
50
for
parasitic
Hymenoptera
was
determined
to
be
>
8,000
ppm
Cry3Bb1
protein.
8
When
an
adjustment
for
mortality
in
the
control
group
is
considered,
mortality
in
the
8,000
ppm
treatment
group
is
45%.
Although
differences
in
mortality
between
the
control
and
treatment
groups
were
not
significantly
different
(
p>
0.05),
a
treatment
effect
at
20X
EEC
could
not
be
precluded
in
this
study.
At
test
termination
mortality
for
the
100
ppm
potassium
arsenate
reference
group
was
33%
(
24
of
73)
and
100%
mortality
(
70
of
70)
in
the
1,000
ppm
reference
group.

Based
on
this
test,
the
LC
50
for
adult
parasitic
Hymenoptera
exposed
to
dietary
Cry3Bb1
is
>
8,000
ppm.
The
hazard
assessment
is
based
on
4000
ppm
Cry3Bb1
protein
which
is
10X
the
field
concentration
in
plants.
However,
because
parasitic
Hymenoptera
do
not
feed
directly
on
corn
plant
tissues,
minimal
exposure
of
parasitic
Hymenoptera
to
Cry3Bb1
protein
is
expected.
As
a
result,
no
hazard
to
Nasonia
vitripennis
is
expected
from
exposure
to
MON
863
Cry3Bb1
corn.

The
preliminary
review
of
the
Nasonia
vitripennis
study
was
initially
found
acceptable
by
the
Agency
(
memorandum
from
Robyn
Rose
to
Mike
Mendelsohn
dated
May
20,
2002).
However
the
August
27,
2002
SAP
concluded
that
the
parasitic
Hymenoptera
(
Nasonia
vitripennis)
testing
was
not
appropriate.
The
SAP
concluded
that
"[
the
levels
of
exposure
of
...
Nasonia
to
active
protein
were
not,
for
example,
determined
throughout
their
respective
tests.
The
test
protein
...
within
a
diet
broth
...
could
have
degraded
considerably."
Not
only
were
the
procedures
in
this
study
questioned
by
the
SAP,
the
appropriateness
of
testing
this
organism
is
questionable.
N.
vitripennis
is
a
dipteran
parasitoid
that
does
not
occur
in
corn
fields.
A
more
appropriate
parasitoid
that
occurs
in
corn
fields
(
e.
g.
Tricogramma
or
Macrocentrus
grandii)
should
be
considered.
Since
Tricogramma
and
Macrocentrus
are
lepidopteran
parasitoids,
testing
another
beneficial
organism
rather
than
a
parasitoid
is
appropriate.
Therefore,
the
Agency
recommends
additional
maximum
hazard
dose
laboratory
testing
of
a
beneficial
coleopteran
such
as
a
carabid
(
ground
beetle).

Green
Lacewing.
The
studies
submitted
to
support
the
initial
MON
810
registration
showed
no
significant
adverse
effects
to
green
lacewing
larvae
at
a
maximum
hazard
dose
of
16.7
ppm
Cry1Ab
protein
in
a
7
day
feeding
study.
Therefore,
the
LC
50
is
greater
than
16.7
ppm
and
no
adverse
effect
to
green
lacewing
was
expected
as
a
result
of
exposure
to
Cry1Ab
protein
at
field
concentrations.

Green
lacewing
larvae
were
fed
the
Cry
3Bb1
protein
in
a
moth
egg
(
Sitotroga
sp.)
and
water
meal
diet
at
rates
of
400
and
8,000
ppm
which
is
approximately
equivalent
to
1X
and
20X
the
maximum
protein
concentration
in
plant
tissue.
There
was
20%
mortality
in
the
negative
control
group
on
day
10.
Compared
to
the
negative
control,
at
day
10,
there
was
no
significant
increase
in
green
lacewing
larval
mortality
when
fed
1X
(
400
ppm)
and
20X
(
8,000
ppm)
the
maximum
Cry3Bb1
protein
concentration
found
in
plant
tissue.
At
test
termination
mortality
for
the
1,000
ppm
reference
group
(
potassium
arsenate)
was
43%
(
13
of
30)
and
100%
mortality
in
the
10,000
ppm
reference
group.
The
data
show
that
the
LD
50
for
green
lacewing
larvae
exposed
to
Cry3Bb1
in
diet
is
>
8,000
ppm.
Based
on
these
results
it
is
not
expected
that
the
green
lacewing
9
will
be
adversely
affected
when
exposed
to
Cry3Bb1
in
the
field.

The
preliminary
review
of
the
green
lacewing
larva
study
was
initially
found
acceptable
by
the
Agency.
However
the
August
27,
2002
SAP
concluded
that
the
green
lacewing
(
Chrysoperla
carnea)
testing
was
not
appropriate.
Several
public
comments
also
addressed
this
issue.
The
SAP
concluded
that
"[
the
levels
of
exposure
of
Chrysoperla
to
active
protein
were
not,
for
example,
determined
throughout
their
respective
tests.
The
test
protein
was
held
for
a
week
within
a
diet
broth
in
the
Chrysoperla
test
chamber,
and
could
have
degraded
considerably."

Additional
problems
were
recognized
with
the
Chrysoperla
laboratory
study.
Green
lacewing
are
difficult
to
test
in
the
laboratory
because
of
a
high
rate
of
mortality.
In
this
study,
the
test
was
terminated
after
10
days
because
there
was
>
20%
mortality
in
the
positive
control.
In
addition,
it
is
questionable
whether
the
green
lacewings
are
ingesting
the
Cry3Bb1
protein
that
is
coated
around
moth
eggs
in
a
diet.
Since
green
lacewing
have
piercing­
sucking
mouthparts,
they
may
not
be
exposed
to
the
protein
on
the
external
surface
of
the
egg
diet.
Therefore,
Monsanto
should
conduct
a
laboratory
insect
toxicity
test
on
an
alternate
organism.
The
minute
pirate
bug
(
Orius
insidiosus)
would
be
a
more
appropriate
species
to
test
than
the
green
lacewing.
Orius
typically
occur
in
corn
fields
as
egg
predators
and
they
typically
feed
on
pollen.
Therefore,
a
laboratory
study
should
be
conducted
feeding
O.
insidiosus
both
pollen
and
purified
protein
in
diet.
Feeding
O.
insidiosus
Cry3Bb1
protein
in
diet
will
allow
for
a
test
at
the
maximum
hazard
dose;
whereas,
feeding
O.
insidiosus
pollen
expressing
the
Cry3Bb1
protein
will
provide
an
evaluation
of
potential
effects
from
actual
exposure
scenarios.

Non­
target
Invertebrate
Waiver.
It
can
be
concluded
from
the
leaf
disk,
whole
plant
and
in
vitro
studies
with
purified
Bt
protein
that
there
are
no
interactive
effects
on
susceptible
insect
pests
when
the
Cry1Ab
and
Cry3Bb1
proteins
are
combined
in
YieldGard
®
Plus
Corn.
Since
combining
these
proteins
in
YieldGard
®
Plus
Corn
does
not
change
the
level
of
susceptibility
of
susceptible
pests
compared
to
single
trait
MON
810
and
MON
863
corn,
it
can
be
concluded
that
there
will
not
be
a
difference
for
non­
target
insects
that
are
not
susceptible
to
the
Cry1Ab
or
Cry3Bb1
proteins.
The
level
of
Cry1Ab
and
Cry
3Bb1
expression
in
YieldGard
®
Plus
Corn
is
comparable
to
single
trait
MON
810
and
MON
863
hybrids
so
changes
in
insect
susceptibility
are
not
expected.
Therefore,
non­
target
beneficial
insect
data
for
YieldGard
®
Plus
Corn
may
be
bridged
to
data
submitted
for
Cry1Ab
and
Cry3Bb1
corn.

Collembola.
Purified
Bt
insecticidal
proteins
derived
from
E.
coli
(
200
ppm),
including
Cry1Ab
protein,
had
no
observable
toxicological
effect
on
two
species
of
Collembola:
Folsomia
candida
and
Xenylla
grisea.
The
Agency
also
required
a
Collembola
study
using
leaf
material
rather
than
bacterially­
derived
Cry1Ab.

Collembola
(
Folsomia
candida)
were
fed
diets
consisting
of
transgenic
corn
leaf
tissue
containing
Cry3Bb1
protein
mixed
with
dry
granulated
Brewer's
yeast.
Diets
contained
a
ratio
of
0.50,
5.0
and
50%
corn
leaf
tissue
in
Brewer's
yeast
which
was
equivalent
to
8.73,
87.3
and
872.5

g
Cry
protein/
gram
diet
respectively.
The
corn
leaf
tissue
contained
1,745

g
Cry3Bb1
protein/
g
dried
10
leaf
tissue.
These
results
show
a
LD
50
>
872.5

g/
g
diet
of
Cry3Bb1
protein.
The
study
also
noted
that
a
diet
containing
50%
corn
leaf
tissue
expressing
the
Cry3Bb1
Bt
protein
(
a
maximum
hazard
dose)
did
not
adversely
affect
reproduction
of
Collembola.
This
test
was
conducted
at
concentration
levels
much
greater
than
Collembola
are
expected
to
be
exposed
to
in
the
field.
The
primary
route
Collembola
would
be
exposed
to
Cry3Bb1
in
the
field
is
from
decaying
root
tissue
(
and
possibly
from
pollen
to
a
much
lesser
degree).
MON
863
is
expressed
in
corn
roots
in
the
range
of
3­
66

g/
g
which
is
significantly
lower
than
the
levels
used
in
this
test.

Earthworms.
Earthworm
feeding
studies
submitted
to
the
Agency
for
all
of
the
registered
Cry
proteins
demonstrate
that
the
Cry
proteins
are
not
toxic
at
the
expected
environmental
concentration.
.
Saxena
and
Stotzky
(
2001)
report
that
earthworms
do
ingest
Bt
Cry
proteins
with
the
soil
without
harmful
effects.
In
this
study,
there
were
no
significant
differences
in
the
percent
mortality
and
weight
of
earthworms
after
40
days
in
soil
planted
with
Bt
or
non­
Bt
corn
or
not
planted,
or
after
45
days
in
soil
amended
with
biomass
of
Bt
or
non­
Bt
corn
or
not
amended.
However,
the
toxin
was
present
in
both
the
casts
and
guts
of
the
worms
in
these
tests.

The
14­
Day
LC
50
value
for
earthworms
exposed
to
Cry1Ab
insecticidal
protein
derived
from
E.
coli
in
an
artificial
soil
substrate
was
determined
to
be
greater
than
200
mg/
kg
(
ppm),
which
was
the
single
concentration
tested.
There
were
no
statistically
significant
effects
at
the
single
dose
tested.
Although
this
study
was
graded
supplemental,
Bt
Cry1Ab
proteins
expressed
in
the
corn
plant
are
not
expected
to
generate
a
toxic
effect
in
the
earthworm;
therefore,
in
light
of
recent
recommendations
by
the
FIFRA
Scientific
Advisory
Panel
(
USEPA,
2001)
that
invertebrates
known
not
to
be
affected
by
the
Cry
proteins
specific
for
insects
not
be
tested,
no
additional
follow­
up
of
this
study
is
required.

The
14­
day
LC
50
for
earthworms
exposed
to
Cry3Bb1
protein
11231
in
an
artificial
soil
substrate
was
determined
to
be
greater
than
570
mg
Cry3Bb1
protein/
kg
dry
soil.
However,
the
percent
mortality
reported
was
38%.
The
mortality
in
the
57.0
mg/
kg
group
was
8%.
It
was
noted
in
the
study
design
that
the
levels
of
buffer
salt
in
the
test
groups
were
higher
than
expected
because
of
a
miscalculation.
The
actual
concentration
of
sodium
bicarbonate
salt
in
the
57.0
and
570
mg
Cry3Bb1
protein/
kg
treatment
groups
was
70
and
699
mg/
kg,
respectively.
The
higher
concentrations
did
not
appear
to
have
any
influence
on
the
overall
conclusions
of
the
study.

Another
study
resulted
in
the
14­
day
LC
50
for
earthworms
exposed
to
purified
11098
Cry3Bb1
(
E.
coli
­
produced)
protein
in
an
artificial
soil
substrate
to
be
greater
than
166.6
mg
/
kg
dry
soil
(
the
highest
concentration
tested),
or
greater
than
20
times
the
worst
case
EEC
in
a
corn
field.
There
was
no
apparent
effect
of
the
phosphate
buffer
on
the
earthworms.
There
were
no
earthworm
mortalities
in
the
any
of
the
controls
or
Cry­
protein
treated
soils
during
the
14
day
study.
Changes
in
average
body
weights
were
not
statistically
different
(
p>
0.05)
among
the
controls
and
protein­
amended
soils.
There
were
no
other
remarkable
observations.
At
the
end
of
the
study,
mortality
in
the
10
and
20
mg
chloroacetamide/
kg
soil
was
2.5%
(
1
of
40)
and
85%
(
34
of
40),
respectively.
Percent
mortality
of
earthworms
in
the
reference
substance
(
chloroacetamide)
groups
was
consistent
with
historical
results,
and
further
confirmed
the
11
adequacy
and
consistency
of
the
protocol
used
in
the
definitive
test.

Pest
Susceptibility
Insect
Resistance
Management
°
Growers
may
choose
to
plant
a
common
refuge
for
European
corn
borers
and
corn
rootworms
or
separate
refuges
for
each
target
pest.
°
Common
Refuge
Option.
A
20%
non­
Bt
corn
refuge
should
be
planted
adjacent
to
or
within
YieldGard
®
Plus
Corn
fields.
If
row
strips
within
a
corn
field
are
implemented,
then
at
least
6,
and
preferably
12
consecutive
rows
should
be
planted
at
least
0.5
meters
apart
and
Bt
corn
strips
should
be
9
to
18
meters
from
refuge
rows.
Refuge
acres
may
be
treated
to
control
CRW
larvae
with
chemical
insecticides,
but
insecticides
should
not
be
used
on
refuges
to
control
CRW
adults.
Non­
Bt
insecticides
may
also
be
used
to
control
corn
borers
if
economic
injury
levels
occur.
Alternate
hosts
and
seed
mixes
should
not
be
utilized
as
refuges.
°
Separate
Refuge
Option.
A
20%
non­
corn
rootworm
protected
corn
refuge
must
be
planted
to
delay
corn
rootworm
resistance.
An
additional
20%
non­
Bt
corn
must
also
be
planted
to
delay
European
corn
borer
resistance.
The
corn
rootworm
refuge
must
be
planted
with
corn
that
does
not
contain
the
Cry3Bb
protein.
However,
corn
that
only
contains
the
Cry1Ab
protein
may
be
planted
if
a
separate
non­
Bt
corn
refuge
is
planted
to
delay
European
corn
borer
resistance.
The
corn
rootworm
refuge
should
be
planted
as
continuous
blocks
adjacent
to
or
within
fields,
perimeter
strips
or
strips
within
YieldGard
®
Plus
Corn
(
at
least
6
rows
and
preferably
12
rows
wide)
and
utilize
comparable
agronomic
practices
as
the
YieldGard
®
Plus
Corn
acres.
European
corn
borer
refuges
may
be
planted
within
fields
as
blocks
or
strips
(
at
least
4
rows
and
preferably
6
rows
wide),
adjacent
to
fields
or
up
to
½
mile
(
1/
4
mile
preferred)
from
YieldGard
®
Plus
Corn
acres.
Non­
Bt
insecticides
may
be
applied
to
refuge
acres
to
control
corn
rootworm
larvae,
but
may
only
be
applied
to
refuge
acres
when
corn
rootworm
adults
are
present
if
YieldGard
®
Plus
Corn
acres
are
also
treated.
Non­
Bt
insecticides
may
be
applied
to
refuges
to
control
the
European
corn
borer,
corn
earworm
or
southwestern
corn
borer
if
economic
injury
levels
occur.

°
If
growers
spray
their
corn
fields
with
insecticides
to
control
pests
other
than
CRW,
then
all
acres
(
Bt
and
non­
Bt)
should
be
treated
identically.
Bt
fields
and
the
non­
Bt
refuge
acres
should
be
treated
with
identical
agronomic
practices
such
as
irrigating
all
corn
(
Bt
and
non­
Bt)
at
the
same
time.
To
ensure
the
production
of
similar
numbers
of
CRW,
Bt
and
non­
Bt
corn
should
be
planted
in
fields
with
similar
backgrounds.

°
Expected
and
confirmed
resistance
should
be
determined
by
the
methods
outlined
in
the
Cry1Ab
and
Cry3Bb1
registrations.
A
remedial
action
plan
is
also
necessary.
If
resistance
is
confirmed,
all
acres
(
Bt
fields
and
non­
Bt
refuges)
should
be
treated
with
insecticides
1
The
data
collected
for
the
DCI
provide
a
deposition
curve
of
pollen
distribution
outside
a
corn
field.
A
conservative
estimate
of
about
300
pollen
grains
(
frequency
of
occurrence
0.017)
per
square
centimeter
is
found
at
the
edge
of
a
corn
field
and
the
levels
drop
off
rapidly
within
a
few
meters
of
the
corn
field
edge
(
Pleasants,
et
al,
2001).

12
targeted
at
CRW
adults
as
well
as
larvae.

°
Growers
should
be
required
to
sign
a
technology
use
agreement
that
outlines
IRM
requirements
and
acknowledges
the
growers
responsibility
to
comply
with
them
on
an
annual
basis.
The
agreement
should
also
state
that
growers
received
the
Product
Use
Guide.

°
Monsanto
must
develop
and
implement
a
monitoring
for
resistance
plan,
remedial
action
plan,
grower
education
and
compliance
assurance
program
that
is
applicable
for
the
European
corn
borer
and
corn
rootworm.
Results
of
the
IRM
grower
agreement,
grower
education
and
compliance
assurance
programs
should
be
submitted
to
EPA
annually
by
January
31.

Endangered
Species
Impact
Assessment
Since
Cry1Ab
is
active
against
lepidopteran
insects,
endangered
lepidopterans
were
evaluated
for
potential
risk.
The
majority
of
listed
lepidopteran
species
have
very
restricted
habitat
ranges.
Examination
of
an
overlay
map
showing
the
county
level
distribution
of
lepidopteran
species
relative
to
corn
production
counties
in
the
US
as
listed
by
the
U.
S.
Fish
and
Wildlife
Service
(
USFWS,
1997)
shows
that
as
a
rule,
listed
lepidopteran
species
do
not
occur
in
agricultural
areas
where
corn
is
grown
nor
is
corn
considered
a
host
plant
for
these
species.
The
map
clearly
indicates
that
any
potential
concern
regarding
range
overlap
with
corn
production
is
restricted
to
the
Karner
blue
butterfly
(
Lyceides
melissa
samuelis).
The
butterfly
is
found
along
the
northern
extent
of
the
range
of
wild
lupine
(
its
host
plant),
where
there
are
prolonged
periods
of
winter
snow
pack,
primarily
in
parts
of
Wisconsin,
Michigan,
Minnesota,
Indiana,
New
Hampshire
and
New
York.
Wild
lupine
grows
on
dry,
sandy
soils
in
pine
barrens,
oak
savannah,
forest
trails
and
previously
disturbed
habitats
such
as
utility
rights­
of­
way,
military
installations,
airports,
highway
corridors,
sand
roads
and
abandoned
sand
pits
(
US
Fish
and
Wildlife
Service,
2000a,
2000b).
No
corn
is
grown
in
the
area
in
New
Hampshire
where
Karner
blue
butterflies
are
found.

Toxicity
of
pollen
from
the
currently
registered
Cry1Ab
Bt
corn
products
to
Karner
blue
larvae
is
estimated
to
be
very
low.
At
least
12
lepidopteran
species
have
been
tested
to
determine
LC
50
levels
for
Cry1Ab.
The
most
sensitive
species
tested
is
the
monarch
butterfly.
Researchers
have
determined
that
the
concentration
producing
no
mortality
whatsoever
is
greater
than
(>)
4000
pollen
grains/
cm2
of
leaf
surface
(
Hellmich,
et
al,
2001).
Thus
the
actual
LD
50
for
monarchs
is
likely
to
be
substantially
higher.
Since
the
EEC
is
300
pollen
grains
/
cm2
or
less
at
the
field
edge,
and
200
at
one
meter
and
75
at
three
meters,
1
the
ratios
of
the
EEC/
LC
50
(
with
>
4000
pollen/
cm2
as
that
LC
50)
have
been
conservatively
calculated
to
be
1:>
13.3
for
Cry1Ab
at
the
field
edge,
13
1:>
20
at
one
meter
and
1:>
53
at
two
meters
from
the
field
edge
(
Vaituzis,
et
al,
2001).

On
the
basis
of
new
data
and
information
received
and
obtained
on
the
potential
impact
of
Bt
corn
on
Karner
blue,
EPA
has
conducted
an
ecological
risk
assessment
using
the
best
data
available,
and
determined
that
there
will
be
no
effect
on
the
Karner
blue
from
the
Bt
corn
registrations.
This
determination
is
based
on
a
number
of
factors
including
(
1)
if
wild
lupine
were
to
grow
adjacent
to
Bt
corn
fields,
the
amount
of
corn
pollen
shed
from
such
fields
onto
the
wild
lupine
would
be
insufficient
to
constitute
a
hazard
to
the
Karner
blue;
(
2)
relevant
data
and
information
indicate
that
there
will
be
relatively
little,
if
any,
wild
lupine
growing
in
the
areas
immediately
adjacent
to
corn
fields
that
are
reestablished
from
fallow
fields;
(
3)
the
amount
of
corn
pollen
shed
from
corn
fields
to
adjacent
areas
is
low;
(
4)
available
data
suggest
that
there
may
be
limited
overlap
between
the
period
of
pollen
shed
from
corn
fields
with
the
period
of
Karner
blue
larval
emergence.

Since
Cry3Bb1
is
active
against
coleopteran
insects,
endangered
coleopterans
were
evaluated
for
potential
risk.
Based
on
a
10­
fold
safety
factor
for
terrestrial
species
and
a
20­
fold
safety
factor
for
aquatic
species
and
a
lack
of
exposure,
no
unreasonable
adverse
effects
of
MON
863
to
endangered
Coleoptera
are
expected.
According
to
Monsanto's
submission,
the
LC
50
for
the
Colorado
potato
beetle,
the
most
sensitive
species
to
Cry3Bb1
protein,
is
2.4

g/
mL
diet.
A
maximum
exposure
of
0.23

g/
g
(
10­
fold
safety
margin)
is
expected
1
m
from
the
field
edge,
0.09

g/
g
(
28­
fold
safety
margin)
is
expected
2
m
from
the
field
edge
and
0.03

g/
g
(
82­
fold
safety
margin)
is
expected
4­
5
m
from
the
field
edge.
A
1­
ha
pond
with
a
2
m
depth
located
1
m
from
the
edge
of
the
corn
field
that
has
500
grains/
cm2
would
have
a
maximum
exposure
of
5.81250
×
10­
5
which
represents
a
41290
safety
margin.
These
data
indicate
that
pollen
drift
from
Cry3Bb1
corn
fields
will
not
pose
a
terrestrial
or
aquatic
exposure
risk.

Many
of
the
endangered
and
threatened
beetles
occur
in
cave
or
aquatic
habitats.
None
of
the
endangered
beetles
are
expected
to
occur
in
or
near
corn
fields.
The
American
burying
beetle
may
occur
in
old
fields
or
cropland
hedge
rows.
However,
based
upon
the
feeding
habits
of
the
American
burying
beetle,
it
is
not
expected
to
occur
within
corn
fields
nor
will
it
be
exposed
to
Cry3Bb1
protein.
Adult
American
burying
beetles
are
classified
as
opportunistic
scavengers
that
feed
on
anything
dead
and
bury
vertebrate
carcasses
which
larvae
feed
on.
Carrion
regurgitated
by
adults
is
fed
to
larvae
until
they
are
able
to
feed
directly
on
a
carcass.

Soil
Degradation
A
study
(
MRID
No.
436960­
01)
submitted
in
support
of
the
event
MON
810
registration
resulted
in
Cry1Ab
protein
bioactivity,
(
when
added
to
the
soil
as
a
component
of
corn
line
#
754­
10­
1
tissue)
decrease
with
an
estimated
DT
50
(
Degradation
Time)
of
1.6
days
and
an
estimated
DT
90
of
15
days.
Cry1Ab
protein
bioactivity
of
corn
line
#
754­
10­
1
tissue
incubated
without
soil
decreased
with
an
estimated
DT
50
of
25.6
days,
and
a
DT
90
of
40.7
days.
The
bioactivity
of
purified
Cry1Ab
protein
in
soil
decreased
with
an
estimated
DT
50
of
8.3
days
and
a
DT
90
of
32.5
days.
14
In
the
March
12,
2001
SAP
Report
No.
2000­
07
on
Bt
Plant­
Pesticides
Risk
and
Benefits
Assessment,
the
October
2000
SAP
concluded
that
published
data
at
that
time
did
not
adequately
address
the
persistence
of
Cry
proteins
from
Bt
crops
in
the
soil.
Since
it
is
difficult
to
correlate
the
relevance
of
published
laboratory
studies
to
field
situations,
the
SAP
recommended
field
studies
be
conducted
in
established
fields
in
a
variety
of
soil
types
and
climatic
conditions.
The
SAP
suggested
the
determination
of
the
amount,
concentration
and
persistence
of
biological
activity
of
Cry
proteins
in
the
soil
are
areas
that
should
be
investigated.
The
EPA
agrees
with
the
SAP
that
actual
field
data
on
Cry
protein
levels
in
the
soil
will
yield
relevant
data
on
persistence
and
natural
variation
of
plant­
produced
Bt
proteins
in
soil.
If
high
levels
of
Cry
proteins
are
found
in
field
soils,
reevaluation
of
the
risks
to
certain
non­
target
organisms
might
be
required.
Therefore,
EPA
is
requiring
supplementary
studies
regarding
Cry
protein
in
soil.

Soil
degradation
studies
were
also
submitted
for
the
Cry3Bb1
in
MON
863
registration.
Finely
ground
corn
leaf
tissue
in
sandy
loam
field
soil
degradation
data
at
worst­
case
field
concentrations
show
that
the
Cry3Bb1
protein
DT
50
based
on
insect
bioassays
and
ELISA
were
2.37
and
2.76
days
respectively.
The
DT
90
estimates
for
the
insect
bioassays
and
ELISA
were
7.87
and
9.16
days
respectively.
At

28
days
the
CryBb1
protein
was
below
the
detection
level.
These
results
verify
that
the
Cry3Bb1
protein
degrades
rapidly
and
does
not
accumulate
in
the
soil.
Additional
testing
in
different
soil
types
is
requested.

A
condition
of
the
MON
863
registration
is
that
Monsanto
must
submit
field
degradation
studies
evaluating
accumulation
and
persistence
of
Cry3Bb1
in
several
different
soils
in
various
strata.
Representative
fields
must
have
been
planted
with
MON
863
and
include
both
conventional
tillage
and
no­
till
samples
and
be
harvested
under
typical
agronomic
conditions.
Sampling
must
continue
until
the
limit
of
detection
is
reached.
Studies
should
include
soils
with
high
levels
of
a
variety
of
clays.
Both
ELISA
and
insect
bioassays
need
to
be
conducted
and
compared
to
determine
if
Cry3Bb1
is
accumulating
or
persisting
in
soil
samples.
The
same
studies
field
degradation
studies
requested
for
MON
863
corn
should
be
conducted
for
YieldGard
®
Plus
Corn.

Data
Evaluation
Report
(
DER)

Reviewed
by:
Robyn
Rose,
Entomologist,
BPPD
Secondary
Reviewer:
Zigfridas
Vaituzis,
Microbiologist,
BPPD
15
STUDY
TYPE:
Administrative
materials
including
the
product
label,
product
analysis,
residue,
non­
target
organism,
toxicology,
efficacy
and
insect
resistance
management
data
MRID
NO.:
457917­
00
CHEMICAL
NO.:
006498
TEST
MATERIAL:
Cry1Ab
and
Cry3Bb1
Proteins
STUDY
NO.:
02­
CR­
084E
SPONSOR:
Monsanto
Company,
600
13th
Street
N.
W.,
Suite
660,
Washington,
DC
20005
TITLE
OF
REPORT:
Administrative
Materials
for
Application
to
Register
Bacillus
thuringiensis
Cry1Ab
and
Cry3Bb1
Proteins
and
the
Genetic
Material
Necessary
for
their
Production
in
YieldGard
®
Plus
Corn
AUTHOR:
Dennis
P.
Ward,
Ph.
D.,
Regulatory
Affairs
Manager
STUDY
COMPLETED:
August
20,
2002
CLASSIFICATION:
Acceptable
Summary
of
Volume
1
of
4
in
Support
of
Registering
YieldGard
®
Plus
Corn:

Product
Label
The
YieldGard
®
Plus
Corn
label
lists
Bt
Cry1Ab
protein
and
the
genetic
material
necessary
for
its
production
in
corn
(
0.00007%
­
0.00013%)
and
Bt
Cry3Bb1
protein
and
the
genetic
material
necessary
for
its
production
in
corn
(
0.0043%
­
0.0092%)
as
the
active
ingredients.
Other
ingredients
listed
on
the
label
include
the
substance
produced
by
a
marker
gene
and
the
genetic
material
necessary
for
its
production
in
corn
(<.
00001%).
According
to
the
label,
the
Cry1Ab
and
Cry3Bb1
Bt
insecticidal
proteins
in
YieldGard
®
Plus
Corn
"
are
effective
in
controlling
leaf,
stalk
and
ear
damage
caused
by
European
corn
borer
(
Ostrinia
nubilalis),
southwestern
corn
borer
(
Diatrea
grandiosella),
southern
cornstalk
borer
(
D.
crambidoides),
corn
earworm
(
Helicoverpa
zea),
fall
armyworm
(
Spodoptera
frugiperda),
and
stalk
borer
(
Papaipema
nebris),
and
effective
in
controlling
root
feeding
damage
caused
by
western,
northern
and
Mexican
corn
rootworm
larvae
(
Diabrotica
spp.)."
The
label
also
states
that
an
insect
resistance
management
plan
consisting
of
a
20%
structured
refuge
within
or
adjacent
to
YieldGard
®
Plus
Corn
fields
is
required.
According
to
the
label,
a
Grower
Agreement
that
requires
growers
to
adhere
to
a
Technology
Use
Guide
that
specifies
refuge
requirements
is
necessary.

Reviewer
Comments
on
the
Product
Label
Based
on
information
provided
in
support
of
registration,
there
are
no
additional
comments
to
Monsanto's
proposed
label.

Product
Analysis
16
Volume
2
of
4
of
Monsanto's
submission
to
EPA
in
support
of
this
registration
request
includes
a
study
that
has
been
conducted
to
characterize
the
genetic
inserts
YieldGard
®
Plus
Corn
and
is
reviewed
in
a
separate
memorandum
and
DER.
Based
on
a
Southern
blot
analysis,
Monsanto
concluded
that
events
MON
810
and
MON
863
are
present
intact
in
YieldGard
®
Plus
Corn
(
Borovkov
et
al.
2001).
Volume
3
of
4
includes
the
summary
of
studies
conducted
to
determine
the
concentration
of
Cry1Ab
and
Cry3Bb1
proteins
in
plant
tissue
of
the
combined
trait
hybrids
(
Dudin
et
al.
2001).
Harvested
grain
and
forage
from
single
trait
hybrids
and
stack
hybrids
were
analyzed
using
enzyme­
linked
immunosorbent
assays
(
ELISA)
to
determine
Cry1Ab
and/
or
Cry3Bb1
levels.
According
to
this
submission,
results
of
these
data
are
highly
variable
with
a
lot
of
overlap
of
Bt
expression
levels
in
all
tissue.
Overall,
the
study
found
in
Volume
3
suggest
slightly
higher
Cry1Ab
and
Cry3Bb1
protein
levels
in
stacked
hybrids
than
single
trait
hybrids.
Additional
variability
was
evidenced
when
MON
863
single
trait
hybrids
from
the
previous
growing
season
were
compared
to
the
stacked
hybrids,
Cry3Bb1
expression
levels
were
higher
in
the
single
trait
hybrids.
According
to
Monsanto,
"[
t]
his
variability
in
protein
levels
is
most
likely
attributable
to
differences
in
germplasm
and
environmental
conditions."

Reviewer's
Comments
on
Product
Analysis
Reviews
of
the
product
analysis
data
found
in
Volumes
2
(
MRID
No.
457917­
01)
and
3
(
MRID
No.
457917­
02)
of
Monsanto's
submission
requesting
the
registration
of
YieldGard
®
Plus
Corn
will
be
reviewed
by
EPA
to
verify
that
adequate
data
has
been
submitted
to
support
this
registration.

Residue
Data
An
exemption
from
the
requirement
of
a
tolerance
was
granted
on
August
2,
1996
for
the
Bt
Cry1Ab
delta­
endotoxin
and
the
genetic
material
necessary
for
its
production
in
all
plants
including
corn.
This
tolerance
exemption
is
published
in
the
40
CFR
§
180.1173.
A
time­
limited
exemption
from
the
requirement
of
a
tolerance
was
granted
on
May
11,
2001
and
published
in
the
40
CFR
§
180.1214
for
the
Bt
Cry3Bb1
delta­
endotoxin
and
the
genetic
material
necessary
for
its
production
in
corn.
Monsanto
is
currently
requesting
that
a
permanent
exemption
from
the
requirement
of
a
tolerance
replace
the
current
time­
limited
tolerance
exemption
for
Cry3Bb1.
The
nptII
(
neomycin
phosphotransferase
II)
marker
gene
and
the
genetic
material
necessary
for
its
production
in
all
agricultural
commodities
including
YieldGard
®
Plus
Corn
has
also
been
granted
an
exemption
from
the
requirement
of
a
tolerance
in
the
40
CFR
§
180.1134
that
expires
in
May
2004.

Reviewer's
Comments
on
Residue
Data
Cry1Ab,
Cry3Bb1,
nptII
and
the
genetic
material
necessary
for
their
production
in
corn
have
each
been
granted
an
exemption
from
the
requirement
of
a
tolerance.
However,
the
Cry3Bb1
tolerance
17
exemption
is
currently
time­
limited.
EPA
is
currently
reviewing
Monsanto's
request
for
a
permanent
exemption
from
the
requirement
of
a
tolerance
for
Cry3Bb1.
The
Cry1Ab
and
Cry3Bb1
tolerance
exemptions
are
applicable
to
YieldGard
®
Plus
Corn.

Non­
target
Organism
Data
Monsanto
is
citing
previously
submitted
non­
target
organism
data
to
support
registration
of
YieldGard
®
Plus
Corn
(
Table
1).
In
addition,
Monsanto
has
reference
studies
verifying
that
the
Cry3Bb1
protein
is
only
toxic
to
chrysomelid
beetles
(
e.
g.,
corn
rootworm
species,
Colorado
potato
beetles).

Table
1.
Studies
cited
to
support
non­
target
organism
requirements
for
YieldGard
®
Plus
Corn
Title
MRID
No.
Status,
Classification
&
Comments
Evaluation
of
the
Dietary
Effects
of
Purified
B.
t.
k.
Endotoxin
Proteins
on
Honey
Bee
Larvae
434392­
02
Btk
HD­
1
Cry1Ab
protein
at
20
ppm
showed
no
toxicity
to
larval
honey
bees.
An
LC50
was
not
possible
to
calculate
since
this
was
a
single
dose
test.
Therefore,
the
NOEL
is
greater
than
20
ppm.

Evaluation
of
the
Dietary
Effects
of
Purified
B.
t.
k.
Endotoxin
Proteins
on
Honey
Bee
Adults
434392­
03
There
were
no
statistically
significant
differences
among
the
various
groups.
However,
sizable
mortality
occurred
in
all
treatments.
Btk
HD­
1
protein
at
20
ppm
resulted
in
a
mean
mortality
of
16.2%.
Because
mortality
was
observed
at
the
single
dose
tested,
a
NOEL
could
not
be
determined
from
this
study,
but
it
was
less
than
20
ppm.
20
ppm
was
determined
to
be
significantly
higher
than
exposure
conditions
in
the
environment.

Stability
of
the
CryIA(
b)
Insecticidal
Protein
of
Bacillus
thuringiensis
var.
kurstaki
(
B.
t.
k.
HD­
1)
in
Sucrose
and
Honey
Solutions
Under
Non­
refrigeration
Temperature
Conditions
434680­
02
Test
substance
was
stable
for
up
to
7
days
in
1:
1
honey:
sucrose
solution.
Test
material
was
bioactive.

Activated
Btk
HD­
1
Protein:
A
Dietary
Toxicity
Study
with
Green
Lacewing
Larvae
434680­
03
Btk
HD­
1
Cry1Ab
protein
at
16.7
ppm
showed
no
toxicity
to
green
lacewing
larvae
after
7
days.
The
NOEL
is
greater
than
16.7
ppm.

Activated
Btk
HD­
1
Protein:
A
Dietary
Toxicity
Study
with
Parasitic
Hymenoptera
(
Brachymeria
intermedia)
434680­
04
Btk
HD­
1Cry1Ab
protein
at
20
ppm
showed
no
toxicity
to
Brachymeria
intermedia.
Since
this
is
a
single
dose
study,
an
LC50
cannot
be
calculated.
The
NOEL
is
greater
than
20ppm.

Activated
Btk
HD­
1
Protein:
A
Dietary
Toxicity
Study
with
Ladybird
Beetles
434680­
05
Btk
HD­
1
protein
at
20
ppm
showed
no
toxicity
to
ladybird
beetles
(
Hippodamia
convergens).
The
NOEL
is
greater
than
20
ppm.
Title
MRID
No.
Status,
Classification
&
Comments
18
A
Dietary
Toxicity
Study
with
MON
80187
Meal
in
the
Northern
Bobwhite
435332­
05
No
treatment
related
mortality
or
differences
in
food
consumption,
body
weight
or
behavior
occurred
after
14
days
in
birds
fed
50,000
or
100,000
ppm
transgenic
corn
meal
derived
from
Monsanto's
MON
80187
corn
line
(
which
contains
Cry1Ab
protein)
relative
to
birds
fed
corn
meal
made
from
parental
corn
lines
which
did
not
express
Bt
protein.

Evaluation
of
the
European
Corn
Borer
Resistant
Corn
Line
MON
801
as
a
Feed
Ingredient
for
Catfish
438879­
01
Feed
per
fish,
feed
conversion
ratios,
final
weight,
percentage
weight
gain
and
survival
were
not
significantly
different
between
fish
fed
the
control
MON
800
diet
when
compared
to
those
fed
the
diet
containing
transgenic
corn
from
the
test
line
MON
801
expressing
the
Cry1Ab
protein.
Body
composition
data
exhibited
no
significant
differences
in
percentage
moisture,
fat,
or
ash,
with
a
higher
protein
content
in
the
test
fish
on
a
dry
weight
basis.
This
difference
in
protein
content
disappears
when
one
expresses
the
results
on
a
wet
weight
basis.
Data
in
this
study
are
consistent
with
historical
controls
for
catfish
grown
at
the
Delta
Research
and
Extension
Center.

Cry1Ab
Insecticidal
Protein:
An
Acute
Toxicity
Study
with
the
Earthworm
in
an
Artificial
Soil
Substrate
438879­
02
The
14­
Day
LC50
value
for
earthworms
exposed
to
Cry1Ab
insecticidal
protein
derived
from
E.
coli
in
an
artificial
soil
substrate
was
determined
to
be
greater
than
200
mg/
kg
(
ppm),
which
was
the
single
concentration
tested.
There
were
no
statistically
significant
effects
at
the
single
dose
tested,
therefore
the
NOEL
is
greater
than
200
ppm.
Although
this
study
was
graded
supplemental,
Bt
Cry1Ab
proteins
expressed
in
the
corn
plant
are
not
expected
to
generate
a
toxic
effect
in
the
earthworm,
therefore,
no
additional
follow­
up
of
this
study
is
required.

Effect
of
the
Bacillus
thuringiensis
Insecticidal
Proteins
CryIA(
b),
CryIA(
c),
CryIIA,
and
CryIIIA
on
Folsomia
candida
and
Xenylla
grisea
(
Insecta:
Collembola)
439416­
01
In
the
cited
study,
purified
Btk
insecticidal
proteins
derived
from
E.
coli
(
200
ppm),
including
Cry1Ab
protein,
had
no
observable
toxicological
effect
on
two
species
of
Collembola:
Folsomia
candida
and
Xenylla
grisea.
The
Agency
has
required
a
Collembola
study
using
leaf
material
rather
than
bacterially­
derived
Cry1Ab.
Title
MRID
No.
Status,
Classification
&
Comments
19
Chronic
Exposure
of
Folsomia
candida
to
Corn
Tissue
Expressing
CryIA(
b)
Protein
442715­
01
This
study
determined
that
the
LD50
of
lyophilized
MON
810
corn
leaf
tissue
containing
the
Cry1Ab
protein
to
Collembola
(
Folsomia
candida)
over
a
28­
day
exposure
period
is
greater
than
50%
(
by
weight)
of
the
diet.
The
noeffect
level
for
mortality
was
50%
of
the
diet.
This
same
concentration
in
the
diet
had
no
effect
on
the
reproduction
of
Collembola.
According
to
the
sponsor,
the
estimated
concentration
of
Cry1Ab
protein
was
50.6

g/
g
in
lyophilized
tissue
and
6.27

g/
g
in
fresh
tissue.
The
control
substance
was
lyophilized
leaf
tissue
from
the
nontransgenic
corn
line
MON
823
which
has
a
genetic
background
similar
to
the
MON
810
line
but
does
not
carry
the
gene
responsible
for
the
Cry1Ab
protein.
Thiodicarb
was
used
as
a
positive
control
or
reference
substance.

Corn
Pollen
Containing
the
CryIA(
b)
Protein:
48­
Hour
Static­
Renewal
Acute
Toxicity
Test
with
Cladoceran
(
Daphnia
mangna)
442715­
02
This
study
,
submitted
to
support
MON
810
corn,
is
scientifically
sound
and
no
treatment
mortality
or
behavior
change
were
observed
between
the
dosed
and
control
replicates.
These
results
indicate
that
Daphnia
magna,
a
sensitive
aquatic
invertebrate
species,
is
not
affected
by
a
48
hour
exposure
to
100
mg
of
Cry1Ab
protein
containing
MON810
corn
pollen/
L.
This
study
adequately
address
potential
aquatic
toxicity
concerns
for
MON
810
corn
pollen
expressing
Cry1Ab
protein.
The
data
suggest
that
at
the
expected
environmental
concentration
the
proposed
use
of
Cry1Ab
protein
in
corn
is
not
likely
to
have
any
measurable
effects
on
aquatic
invertebrates.

Evaluation
of
the
Dietary
Effects
of
Purified
Bacillus
thuringiensis
Protein11231
on
Honey
Bee
Larvae
449043­
10
The
LC50
for
honeybee
larvae
and
maturation
to
adult
bees
was
determined
to
be
>
1,790
ppm
Cry3Bb1
protein,
(
100X
the
concentration
in
pollen)
in
a
maximum
hazard
dose
study.
Therefore
no
hazard
to
honeybee
larvae
and
adult
bee
emergence
is
anticipated.
Classification:
Acceptable.

Evaluation
of
the
Dietary
Effects
of
Purified
Bacillus
thuringiensis
Protein11231
on
Adult
Honey
Bees
(
Apis
melliferaL.)
449043­
11
An
adult
honeybee
maximum
hazard
dose
feeding
study
showed
the
LC50
of
the
Cry3Bb1
protein
to
be
>
360

g/
mL.
(
20X
the
concentration
found
in
pollen).
Therefore,
no
hazard
from
the
Cry3Bb1
protein
to
honeybees
is
expected.
Classification:
Acceptable.

Bacillus
thuringiensis
Protein11231:
A
Dietary
Study
with
Green
Lacewing
Larvae
(
Chrysoperla
carnea)
449043­
12
The
LC50
for
green
lacewing
larvae
was
determined
to
be
>
8,000
ppm
Cry3Bb1
protein
(
20X
field
exposure).
Based
on
these
results
it
can
be
concluded
that
green
lacewing
will
not
be
adversely
affected
when
exposed
to
Cry3Bb1
in
the
field.
Because
of
questionable
ingestion
of
the
test
material
another
species
(
e.
g,
minute
pirate
bug,
predatory
carabid)
more
likely
to
be
exposed
to
Cry3Bb1
should
be
tested.
Classification:
Supplemental.
9­
29­
03
Title
MRID
No.
Status,
Classification
&
Comments
20
Bacillus
thuringiensis
Protein11231:
A
Dietary
Study
with
the
Parasitic
Hymenoptera
(
Nasonia
vitripennis)
449043­
13
The
LC50
for
parasitic
Hymenoptera
was
determined
to
be
>
400
ppm
Cry3Bb1
protein.
Although
400
ppm
Cry3Bb1
protein
is
only
1X
field
concentration
in
plants
rather
than
10X,
parasitic
Hymenoptera
are
not
expected
to
feed
directly
on
corn
plant
tissue.
Therefore,
minimal
exposure
and
no
hazard
to
parasitic
Hymenoptera
from
Cry3Bb1
protein
is
expected.
Testing
of
a
species
more
common
to
corn
fields
is
recommended.
Classification:
Acceptable.

Bacillus
thuringiensis
Protein11231:
A
Dietary
Study
with
the
Ladybird
Beetle
(
Hippodamia
convergens)
449043­
14
This
maximum
hazard
dose
study
showed
that
the
LC50
for
Cry3Bb1when
fed
to
adult
H.
convergens
is
>
8,000

g
purified
Bt
protein/
mL
diet.,
equivalent
to
20X
the
maximum
Bt
protein
concentration
in
plant
tissue.
A
follow­
up
pollen
feeding
study
was
requested.
Classification:
Acceptable.

Bacillus
thuringiensis
Protein11231:
A
Dietary
Study
with
the
Northern
Bobwhite
Quail
449043­
15
The
dietary
LC50
value
for
Cry3Bb1
corn
grain
to
juvenile
Northern
Bobwhite
was
greater
than
70,000
ppm
(
10%
of
the
diet)
in
a
8­
day
study
(
eight
day
observation).
No
adverse
effects
on
avian
wildlife
is
expected
from
incidental
field
exposure
to
Cry3Bb1
corn.
A
higher
corn
concentration
and
longer
duration
broiler
study
with
MON863
corn
is
recommended.
Classification:
Supplemental.

Bacillus
thuringiensis
Protein
11231:
A
Dietary
Study
with
Earthworm
in
an
Artificial
Soil
Substrate
449043­
16
A
maximum
hazard
dose
14­
day
LC50
for
earthworms
exposed
to
Cry3Bb1
protein
in
an
artificial
soil
substrate
was
determined
to
be
>
570
mg
Cry3Bb1
protein/
kg
dry
soil,
or
greater
than
10
times
the
maximum
EEC
of
the
protein.
The
data
show
that
no
adverse
effects
to
earthworms
are
expected
from
exposure
to
Cry3Bb1
protein
producing
corn
plants.
Classification:
Supplemental.

Assessment
of
Chronic
Toxicity
of
Corn
Tissue
Containing
the
Bacillus
thuringiensis
Protein
11098
to
Collembola
(
Folsomia
candida)
449043­
17
The
LC50
of
the
Cry3Bb1
protein
for
Collembola
was
found
to
be
>
872.5

g
(
50%
corn
leaf
tissue
in
the
diet).
No
adverse
reproductive
effects
were
noted.
It
can
be
concluded
from
this
test
that
Cry3Bb1protein
does
not
pose
a
hazard
to
Collembola,
a
representative
of
a
beneficial
decomposer
soil
inhabiting
species.
Classification:
Acceptable.

Bacillus
thuringiensis
Protein
11098
in
Corn
Pollen:
48­
Hour
Static
Renewal
Acute
Toxicity
Test
with
the
Cladoceran
(
Daphnia
mangna)
449043­
18
The
48­
hour
LC50
value
for
Cry3Bb1
corn
pollen
when
administered
to
neonate
daphnids
was
>
120
mg
pollen/
L,
a
maximum
hazard
dose.
No
other
adverse
effects
were
noted.
Therefore,
no
hazard
to
daphnia
are
expected
from
incidental
exposure
to
Cry3Bb1­
containing
corn
pollen.
Classification:
Acceptable.
Title
MRID
No.
Status,
Classification
&
Comments
21
Evaluation
of
Insect
Protected
Corn
Lines
MON
853
and
MON
859
as
a
Feed
Ingredient
for
Catfish
449043­
19
No
treatment
mortality
or
behavior
change
was
observed
among
channel
catfish
in
an
8
week
sub­
chronic
study
when
fed
diets
containing
35%
Cry3Bb1
corn
lines
MON
853
and
MON
859.
Classification:
Acceptable.

Research
on
the
Effects
of
Corn
Rootworm
Protected
Transgenic
Corn
Events
on
Nontarget
Organisms:
Preliminary
Results
453484­
02
457916­
01
455382­
06
Final
report
for
MRID
455382­
06
two
year
field
census
study.
MON863
showed
no
overall
differences
in
the
abundance
of
non­
target
invertebrates
and
had
less
impact
on
certain
beneficial
insects
compared
to
traditional
insecticides,
especially
soil
and
foliar
applications.
These
studies
are
supplemental
to
Tier
I
maximum
hazard
dose
testing
and
are
of
inadequate
statistical
power
for
long
term
effects
determination.
Classification:
Supplemental.

Dietary
Effects
of
Transgenic
Bacillus
thuringiensis
(
Bt)
Corn
Pollen
Expressing
a
Variant
of
Cry3Bb1
Protein
on
Adults
of
the
Ladybird
Beetle,
Coleomegilla
maculata
453613­
01
No
significant
adverse
effects
were
noted
in
a
30
day
50%
pollen
feeding
study.
Based
on
these
results,
no
hazard
to
Coleomegilla
maculata
is
expected
when
feeding
on
Cry3Bb1
corn
pollen
in
the
field.
Classification:
Acceptable.

Dietary
Effects
of
Transgenic
Bacillus
thuringiensis
(
Bt)
Corn
Pollen
Expressing
a
Variant
of
Cry3Bb1
Protein
on
the
Ladybird
Beetle,
Hippodamia
convergens
453613­
02
No
significant
adverse
effects
were
noted
in
a
15
day
50%
pollen
in
honey
water
feeding
study.
Based
on
these
results
,
no
hazard
to
Hippodamia
convergens
is
expected
if
feeding
on
Cry3Bb1corn
pollen
in
the
field.
Classification:
Acceptable.

Dietary
Effects
of
Transgenic
Bacillus
thuringiensis
(
Bt)
Corn
Pollen
Expressing
a
Variant
of
Cry3Bb1
Protein
on
Larvae
of
the
Ladybird
Beetle,
Coleomegilla
maculata
455382­
04
The
LC50
for
Cry3Bb1
expressed
in
pollen
is
>
93

g/
g
fresh
pollen
weight.
The
larvae
were
observed
through
pupation
to
adult
emergence.
It
can
be
concluded
from
this
study
that
Coleomegilla
maculata
larvae
will
not
be
adversely
affected
by
Cry3Bb1
field
corn
pollen.
Classification:
Acceptable.

Determination
of
the
Toxicity
of
Corn
Pollen
Expressing
a
Cry3Bb1
Variant
Protein
to
First
Instar
Monarch
Butterfly
Larvae
(
Danus
plexippus)
via
Laboratory
Bioassay
455382­
05
This
study
has
demonstrated
that
corn
pollen
expressing
the
Cry3Bb1
protein
will
not
result
in
acute
toxic
or
developmental
effects
to
monarch
larvae.
The
SAP
recommended
testing
Tetraopes
(
red
milkweed)
beetles
as
a
more
logical
choice
than
the
monarch
butterfly.
Classification:
Supplemental.

Field
Evaluation
of
the
Ecological
Impact
of
Corn
Rootworm
Insect­
Protected
Corn
on
Non­
target
Organisms
455382­
06
Preliminary
results
from
two
year
Tier
IV
field
census
studies.
These
studies
are
supplemental
to
Tier
I
maximum
hazard
dose
testing.
The
data
do
not
show
any
MON
863
corn
related
adverse
effect
on
non­
target
and
beneficial
invertebrate
abundance
in
the
field.
Classification:
Supplemental.
Title
MRID
No.
Status,
Classification
&
Comments
22
Insecticidal
Spectrum
of
Activity
for
Cry3Bb
Protein
in
vitro
455382­
07
Bioassays
of
six
Families
of
the
Order
Coleoptera
and
two
Lepidoptera
species
detected
activity
only
against
beetle
species
of
the
family
Chrysomelidae
(
corn
rootworm
and
Colorado
potato
beetle).
Classification:
Supplemental.

Endangered
Species
Impact
Assessment
for
Cry3Bb1
Protein
in
Transgenic
Corn
Event
MON
863
455770­
03
Monsanto
conducted
a
hazard
assessment,
exposure
assessment
and
risk
characterization
to
demonstrate
that
Cry3Bb1
does
not
pose
a
risk
to
endangered
Coleoptera.
The
Agency
performed
an
independent
assessment
and
determined
that
no
adverse
affects
from
Cry3Bb1event
MON
863
are
expected
to
endangered/
threatened
Coleoptera
species
listed
by
the
USFWS.
Non­
coleopteran
endangered
species
are
not
expected
to
be
susceptible
to
MON
863.

Research
on
the
Effects
of
Corn
Rootworm
Protected
Transgenic
Corn
Events
on
Nontarget
Organisms:
Preliminary
Results
456530­
03
Summary
(
without
data)
of
preliminary
findings
from
several
one
year
supplemental
higher
Tier
field
and
laboratory
studies
not
triggered
by
Tier
I
maximum
hazard
dose
testing
data.
Final
report
of
studies
to
be
submitted.
Classification:
Supplemental.

In
Monsanto's
Volume
1
submission
to
EPA,
there
is
a
discussion
of
the
general
lack
of
potential
non­
target
organism
effects
from
Bt
proteins
due
to
their
natural
occurrence
in
the
soil
and
specific
toxic
mode
of
action
on
susceptible
species.
Published
research
has
shown
that
an
insect
must
ingest
a
Bt
Cry
protein
which
is
solubilized
in
the
midgut.
The
toxin
is
activated
in
the
insects
midgut
by
digestive
enzymes
which
leads
to
the
toxin
binding
to
receptors
in
the
insect's
midgut
epithelial
cells.
Ultimately,
pores
are
formed
in
the
cellular
membranes
and
homeostasis
of
cells
occurs
(
Schnepf
et
al.
1998).
The
binding
of
the
toxin
to
the
midgut
receptor
is
critical
in
Bt
mode
of
action
and
only
occurs
in
susceptible
insects.
Lepidopterans
have
an
alkaline
midgut
(
pH

10.5­
11.0)
which
is
necessary
to
solubilize
the
Cry1Ab
crystal
proteins.
Whereas,
coleopterans
have
a
midgut
pH

6.5
­
7.0
which
suggest
that
the
alkaline­
solubilization
step
is
not
necessary.

The
host
specificity
of
Cry1Ab
and
CryBb1
proteins
may
be
due
to
the
difference
in
amino
acid
sequence.
Of
the
1155
amino
acid
residues
in
Cry1Ab
proteins,
633
make
up
the
active
toxin
and
522
are
the
protoxin
segment.
The
protoxin
is
needed
to
form
the
Bt
crystals
(
Cry)
and
proteolytic
removal
of
the
carboxyl­
terminal
protoxin
fragment
is
needed
to
activate
the
insecticidal
activity
of
Cry1Ab.
There
are
652
amino
acids
and
no
protoxin
segment
in
Cry3Bb1
so
activation
is
not
needed
for
insecticidal
activity
and
a
protoxin
is
not
needed
to
form
the
Bt
crystal.

An
unpublished
study
was
conducted
by
Monsanto
comparing
the
crystallographic
structure
of
Cry3Bb
to
the
active
portion
of
Cry1Aa.
Cry1Aa
is
92%
identical
to
Cry1Ab
so
results
of
this
study
may
be
correlated
to
Cry1Ab.
There
are
three
Domains
in
Cry1Aa
and
Cry3Bb
proteins
with
Domains
2
and
3
serving
as
binding
sites
to
midgut
cells.
According
to
Monsanto,
"[
s]
urface
exposed
amino
acids,
particularly
in
domains
2
and
3,
that
would
likely
serve
as
contact
points
23
with
midgut
cell
binding
proteins,
are
not
well
conserved
between
the
two
proteins.
This
would
suggest
that
these
proteins
act
by
binding
to
different
receptors,
presumably
lepidopteran­
specific
proteins
for
Cry1Ab
and
coleopteran­
specific
proteins
for
Cry3Bb.

At
EPA's
request,
Monsanto
has
addressed
the
potential
of
additive
or
synergistic
effects
of
Cry1Ab
and/
or
Cry3Bb1
in
YieldGard
®
Plus
Corn
on
non­
target
organisms
particularly
invertebrates.
According
to
Monsanto,
"
there
is
no
scientific
basis
for
concluding
that
there
will
be
any
interactions
between
these
two
proteins
that
will
result
in
an
unreasonable
risk
of
harm
for
nontarget
organisms."
Cry1A
proteins
are
specifically
toxic
to
lepidopteran
species,
whereas
Cry3
proteins
are
specifically
toxic
to
coleopterans.
Cry3Bb1
is
only
known
to
be
toxic
to
chrysomelid
beetles
and
Cry1
proteins
have
not
shown
any
activity
against
coleopteran
species.
Therefore,
Monsanto
has
concluded
that
no
interaction
between
Cry1Ab
and
Cry3Bb1
proteins
is
expected
against
target
or
non­
target
organisms.

An
additive
effect
would
occur
when
the
sum
of
the
effects
of
two
toxins
equals
each
of
the
toxins
effects
alone.
Whereas,
a
synergistic
effect
results
when
the
effects
of
two
toxins
is
greater
than
the
sum
of
each
toxin
alone.
Another
possible
effect
of
combining
toxins
is
potentiation
which
occurs
when
a
substance
is
not
toxic
until
it
is
combined
with
another
toxin.
An
antagonistic
effect
occurs
when
combining
two
toxins
interferes
with
each
other
resulting
in
reduced
toxicity.
Synergistic
effects
have
occurred
with
CytA
and
Cry4
toxins
against
mosquitoes
(
Aedes
aegypti)
and
there
have
been
other
reports
of
interactions
between
various
Cry
proteins.

Currently
there
is
no
evidence
of
synergistic
effects
of
combining
Cry1A
proteins
against
lepidopteran
insects.
Effects
of
combining
Cry1A
proteins
has
been
found
to
be
additive
and
there
has
been
no
evidence
of
Cry3
proteins
interacting
with
other
Cry
proteins.
In
addition,
Cry1
proteins
are
not
known
to
be
toxic
to
coleopteran
insects
and
Cry3
proteins
are
not
known
to
be
toxic
to
lepidopterans.
A
study
conducted
by
MacIntosh
et
al.
(
1990)
showed
no
synergism
between
Cry1Ac
and
Cry3Aa
against
16
agronomically
important
insect
species.
Monsanto
also
refers
to
additional
in
vitro
bioassay
and
growth
chamber
studies
that
do
not
show
any
interaction
of
Cry1
and
Cry3
proteins
on
target
insects
tested.
One
study
that
Monsanto
uses
as
an
example
looked
at
microbial
pest
control
agents
containing
Cry1Ac,
Cry3Aa
and
Cry3Bb.
There
was
no
interaction
found
between
the
Cry1A
and
Cry3
proteins.
However,
there
was
an
additive
effect
of
Cry3Aa
and
Cry3Bb
against
the
Colorado
potato
beetle
(
Leptinotarsa
decemlineata)
because
both
toxins
are
acting
on
the
same
binding
site.

Monsanto
has
conducted
a
bioassay
by
feeding
corn
leaf
tissue
containing
single
and
stacked
Cry1Ab
and
Cry3Bb1
protein
and
nontransgenic
corn
leaf
tissue
to
fall
armyworm
(
Spodoptera
frugiperda)
for
three
days.
An
ELISA
verified
that
Cry1Ab
and
Cry3Bb
protein
levels
were
comparable
among
the
single
and
stacked
hybrids
(
Table
2).
Fall
armyworm
feeding
damage
and
development
was
comparable
among
MON
810
and
YieldGard
®
Plus
Corn
and
MON
863
had
no
effect.
Therefore,
Monsanto
concluded
that
there
are
no
additive
or
synergistic
effects
of
YieldGard
®
Plus
Corn
against
target
species.
24
Table
2.
Cry
protein
levels
in
corn
leaf
tissue
(

g
protein/
g
fresh
weight
tissue)

Protein
MON
810
MON
863
YieldGard
®
Plus
Corn
Cry1Ab
11.8
±
1.4
not
detectable
12.4
±
2.2
Cry3Bb
not
detectable
4.9
±
1.81
6.4
±
3.4
A
growth
chamber
study
discussed
by
Monsanto
provides
additional
support
of
a
lack
of
interactive
effects
of
Cry1Ab
and
Cry3Bb1
proteins.
This
study
compared
the
efficacy
of
MON
810,
MON
863
and
YieldGard
®
Plus
Corn
against
the
European
corn
borer
feeding
on
leaf
tissue
and
western
corn
rootworm
(
Diabrotica
virgifera
virgifera)
feeding
on
roots.
Leaf
feeding
damage
by
the
European
corn
borer
was
comparable
among
MON
810
and
root
damage
from
the
corn
rootworm
was
comparable
among
MON
863
and
YieldGard
®
Plus
Corn.

Reviewer's
Comments
on
Non­
target
Organism
Data
Monsanto
has
cited
non­
target
organism
data
submitted
and
reviewed
by
EPA
for
Cry1Ab
and
Cry3Bb1
single
trait
corn
registrations
in
support
of
registering
YieldGard
®
Plus
Corn.
Acceptable
studies
were
submitted
to
EPA
or
waived
for
mammals,
avian
species,
aquatic
species,
aquatic
invertebrate,
fish,
plants,
honey
bees,
lady
beetles,
earthworms
and
Collembola.
Acceptable
green
lacewing
and
parasitic
Hymenoptera
studies
were
also
submitted
for
Cry1Ab
but
these
studies
were
not
acceptable
for
Cry3Bb1.
As
a
condition
of
the
Cry3Bb1
registration,
EPA
has
required
two
additional
beneficial
insect
studies,
preferably
the
minute
pirate
bug,
Orius
insidiosus
and
a
carabid
(
e.
g.,
Pterostichus
spp.).
These
studies
will
also
be
relevant
for
YieldGard
®
Plus
Corn.

During
a
conference
call
held
on
May
15,
2002,
EPA
requested
Monsanto
address
the
potential
effects
of
interaction
(
e.
g.,
synergistic
or
additive
effects)
of
the
Cry
proteins
in
YieldGard
®
Plus
Corn
on
non­
target
invertebrates.
Monsanto
notes
that
no
synergistic,
additive
or
antagonistic
effects
of
Bt
toxins
have
been
found
against
lepidopteran
insects.
However,
studies
conducted
thus
far
include
microbial
Bt
formulations
and
not
plant
incorporated
protectants
(
PIPs).
Pleiotropic
or
other
effects
of
inserting
Bt
genes
into
corn
plants
may
effect
the
concentration
level
in
the
plant
(
and
toxicity
to
target
or
non­
target
organisms.
Expression
levels
of
the
Cry1Ab
and
Cry3Bb1
proteins
are
generally
slightly
higher
in
the
stacked
trait
hybrids
(
Table
3).

Table
3.
Comparison
of
Cry1Ab
and
Cry3Bb1
protein
levels
in
single
trait
hybrids
and
YieldGard
®
Plus
Corn
25
Average
Cry1Ab
Protein
Levels
(

g/
g
fwt)
(
Range)
Average
Cry3Bb1
Protein
Levels
(

g/
g
fwt)
(
Range)

Tissue
Type
and
Collection
Time
(
Days
Post­
Planting)
MON
863
x
MON
810
MON
810
MON
863
x
MON
810
MON
863
Young
Leaf
(

18)
17.9
(
14.1
­
27.5)
13.0
(
9.8
­
15.4)
46.7
(
35.5
­
53.2)
30.0
(
21.3
­
47.2)

Forage
(

90)
7.9
(
3.9
­
11.9)
5.6
(
3.0
­
8.2)
23.6
(
6.7
­
39.7)
12.8
(<
0.22
­
28.8)

Grain
(

117)
0.84
(
0.63
­
1.2)
0.46
(
0.24
­
0.77)
61.1
(
38.5
­
83.1)
43.7
(<
0.096
­
84.1)

Pollen
(

60)
<
0.08
(<
0.08
­
0.18)
<
0.08
(<
0.08)
79.6
(
65.1
­
96.5)
60.4
(
29.7
­
90.7)

Mature
Root
(

90)
N/
A
N/
A
19.7
(
6.0
­
41.7)
16.2
(<
0.76
­
49.8)

Over­
season
Root
(

46)
N/
A
N/
A
22.0
(
N/
A)
20.0
(
N/
A)

Non­
target
beneficial
insect
data
may
be
waived
because
the
susceptibility
of
target
pests
to
YieldGard
®
Plus
Corn
is
comparable
to
their
susceptibility
to
the
single
trait
Cry1Ab
and
Cry3Bb1
corn.
Since
there
is
no
change
in
susceptibility
among
susceptible
insects,
then
it
is
unlikely
that
there
will
be
a
difference
in
effects
of
the
stacked
versus
single
trait
hybrids
on
nontarget
insects
Toxicology
Data
Monsanto
has
requested
a
waiver
from
the
requirement
to
submit
toxicity
and
irritation
data
in
Volume
4
of
the
data
submitted
to
support
registration
of
YieldGard
®
Plus
Corn.
Volume
4
is
reviewed
in
a
separate
DER
from
this
review.
Monsanto
references
previously
submitted
studies
to
support
a
lack
of
toxicity
and
allergenicity
of
Cry1Ab
and
Cry3Bb1
proteins
in
corn
(
Table
4).
According
to
Monsanto,
studies
referenced
in
Table
4
include
data
demonstrating
equivalent
amounts
of
Bt
Cry
protein
used
in
test
material
and
in
corn
plants.
Oral
toxicity
testing
previously
submitted
to
EPA
have
shown
that
there
are
no
adverse
affects
to
mammals
from
Cry1Ab
and
Cry3Bb1
proteins.
In
addition,
human
exposure
to
Cry1Ab
and
Cry3Bb1
in
single
trait
or
stacked
hybrid
corn
will
be
extremely
low.

Both
proteins
have
been
shown
to
rapidly
degrade
in
a
gastric
matrix
and
will
not
result
in
allergenicity
to
mammals.
Cry1Ab
and
Cry3Bb1
proteins
are
not
stable
to
heat
and
they
don't
have
any
amino
acid
sequence
homology
with
proteins
known
to
cause
toxicity
or
allergenicity
to
mammals.
Based
on
studies
submitted
to
support
registrations
of
MON
810
and
MON
863
corn
containing
the
Cry1Ab
and
Cry3Bb1
proteins
respectively,
EPA
concluded
that
these
proteins
are
degraded
rapidly
by
gastric
fluid
in
vitro
unlike
known
food
allergens
that
tend
to
be
resistant
to
degradation.
Furthermore,
Bacillus
thuringiensis
is
a
bacterium
and
bacteria
are
not
typically
26
allergens
and
there
is
no
evidence
of
allergenicity
of
Cry1Ab
or
Cry3Bb1
proteins
to
mammals.
There
is
also
no
evidence
that
combining
Cry1Ab
and
Cry3Bb1
proteins
will
change
their
potential
as
an
allergen.

Table
4.
Studies
cited
to
support
lack
of
toxicity
and
allergenicity
for
YieldGard
®
Plus
Corn
Title
MRID
No.
Status,
Classification
&
Comments
Assessment
of
the
In
vitro
Digestive
Fate
of
Bacillus
thuringiensis
subsp.
kurstaki
HD­
1
Protein
434392­
01
The
tryptic
core
Cry1Ab
protein
is
significantly
degraded
by
2
minutes
incubation
in
gastric
fluid
but
not
significantly
affected
by
19.5
hours
in
intestinal
fluid
as
monitored
by
western
blot.
The
decrease
in
bioactivity
of
these
digestions
against
tobacco
budworm
is
similar
to
its
loss
of
immunorecogniton
in
western
blots.
Classification:
Acceptable
Acute
Oral
Toxicity
Study
of
Btk
HD­
1
Tryptic
Core
Protein
in
Albino
Mice
434680­
01
No
test
substance
related
deaths
occurred.
One
female
died
within
a
day
of
BSA
dose
administration
due
to
a
perforated
trachea.
The
majority
of
the
animals
failed
to
gain
weight
or
showed
a
slight
weight
reduction.
No
treatment
related
trends
in
these
losses
was
apparent.
Classification:
Acceptable.
Test
substance
is
given
a
Toxicity
category
IV
rating
although
highest
dose
administered
is
4000
mg/
kg
due
to
lack
of
any
evidence
of
a
dose/
effect
relation.

Compositional
Comparison
of
Bacillus
thuringiensis
subsp.
kurstaki
HD­
1
Protein
Produced
in
ECB
Resistant
Corn
and
the
Commercial
Product,
DIPEL
435332­
03
The
antiserum
reactions
revealed
many
western
blot
bands
in
both
the
Dipel
®
and
the
ECB
resistant
corn
extracts
not
treated
with
trypsin.
No
bands
clearly
related
to
the
Cry1Ab
toxin
were
seen
in
the
non­
transformed
plant
extracts
whereas
a
band
comigrating
with
the
full
length
Cry1Ac
standard
(
similar
in
size
to
Cry1Ab)
was
seen
in
both
Dipel
®
and
ECB
resistant
corn.
The
tryptic
digests
of
Dipel
®
and
ECB
resistant
corn
extracts
revealed
intensified
bands
that
comigrated
with
the
Cry1Ab
tryptic
core
standard.
Together
these
data
infer
that
the
same
Cry1Ab
protein
is
being
produced
in
ECB
resistant
corn
plants
as
is
found
in
the
microbial
product.
Classification:
Acceptable
Title
MRID
No.
Status,
Classification
&
Comments
27
Assessment
of
the
Equivalence
of
the
Bacillus
thuringiensis
subsp.
kurstaki
HD­
1
Protein
Produced
in
Escherichia
coli
and
European
Corn
Borer
Resistant
Corn
435332­
04
The
Cry1Ab
protein
produced
in
E.
coli
was
shown
by
SDS­
PAGE,
western
blot,
N­
terminal
amino
acid
sequencing,
glycosylation
and
bioactivity
to
be
substantially
equivalent
to
the
plant
produced
Cry1Ab.
The
test
results
showed
the
tryptic
core
of
the
plant
and
microbial
protein
were
of
essentially
identical
SDS­
PAGE
mobility,
immunoreactivity
in
western
blot
analysis
and
N­
terminal
amino
acid
sequence
for
the
first
15
positions.
A
comparison
of
the
dose
response
relationship
of
plant
and
microbial
extracts
against
Heliothis
virescens
and
Helicoverpa
zea
indicates
that
the
tested
proteins
are
of
similar
bioactivity.
Classification:
Acceptable.
These
results
allow
the
substitution
of
the
microbially
produced
Cry1Ab
protein
for
the
plant
source
in
toxicology
testing.

Evaluations
of
Insect
Protected
Corn
Lines
in
1994
U.
S.
Field
Test
Locations
and
Supplemental
Submission
on
the
Tissue
Expression
and
Corn
Earworm
(
Helicoverpa
zea)
Efficacy
of
the
Cry1Ab
Protein
in
Insect­
Protected
Corn
436655­
02
and
441686­
01
All
the
corn
lines
tested
by
ELISA
for
Cry1Ab
HD­
1
protein
showed
a
decrease
in
expressed
levels
over
the
season
for
the
leaves
sampled
from
the
Jerseyville
site.
The
initial
leaf
values
obtained
for
the
Jerseyville
site
also
fell
wthin
the
range
expressed
for
leaf
HD­
1
protein
across
the
six
Midwestern
states.
The
values
for
HD­
1
protein
in
leaves
has
the
greatest
expression
level
and
ranges
from
nondetectable
to
13.72

g/
g
frest
weight.
The
whole
plant
HD­
1
values
range
from
nondetectable
to
4.65

g/
g
fresh
weight.

Assessment
of
the
Equivalence
of
B.
t.
k.
HD­
1
Protein
Produced
in
Several
Insect
Protected
Corn
Lines
and
Escherichia
coli
436655­
03
The
results
of
the
western
blot
showed
the
trypsinized
extracts
of
corn
lines
MON
802,
805,
809,
810,
813,
and
814
expressed
proteins
that
comigrated
with
the
Cry1Ab
protein
as
found
in
MON
801
and
the
same
Cry1Ab
protein
purified
from
E.
coli.
These
bands
also
reacted
with
antiserum
#
B6
specific
for
the
tryptic
core
protein
of
Cry1Ab.
These
results
indicate
the
trypsinized
proteins
found
in
all
these
plants
were
of
same
molecular
size
(
63
kD)
and
immunoreactivity
with
the
reference
standards
of
Cry1Ab
expressed
in
E.
coli
and
corn
line
MON801.
Classification:
Acceptable
Bacillus
thuringiensis
subsp.
kurstaki
HD­
1
Insecticidal
Protein
(
B.
t.
k.
HD­
1
Protein
Shares
No
Significant
Sequence
Similarity
with
Proteins
Associated
with
Allergy
or
Coeliac
Disease
453849­
01
An
amino
acid
database
was
constructed
containing
amino
acid
sequences
of
known
protein
allergens
and
gliadins.
The
B.
t.
k.
HD­
1
protein
was
compared
to
this
database
and
no
significant
sequence
similarity
was
identified.
Based
upon
this
data,
there
does
not
appear
to
be
significant
sequence
similarity
between
HD­
1
and
known
protein
allergens
and
gliadins.
Title
MRID
No.
Status,
Classification
&
Comments
28
Amended
Report
for
MSL­
16559:
B.
t.
Cry3Bb1.11098
and
NPTII
Protein
Levels
in
Sample
Tissues
Collected
from
Corn
Event
MON
863
Grown
in
1999
Field
Trials
454240­
01
The
protein
titer
data
provided
shows
the
ranges
of
Cry3Bb1
protein
in
various
parts
of
the
plant,
as
well
as
geographical
variation.
Overall,
based
upon
the
ranges
provided,
there
appears
to
be
significant
variation
between
the
samples
analyzed
on
different
days
post­
planting
and
at
different
sites.
Ranges
of
Cry3Bb1
protein
levels
in
MON863
in
microgram
Cry3bb1
protein
per
gram
of
fresh
weight
tissue
were
30­
93
(
leaf),
49­
86
(
grain),
30­
93
(
pollen),
3.2­
66
(
root),
and
13­
54
(
above
ground
whole
plant).
Classification:
Acceptable.

Amended
Report
for
MSL­
16597:
Immuno­
detectability
of
Cry3Bb1.11098
and
Cry3Bb1.11231
Proteins
in
the
Grain
of
Insect
Protected
Corn
Events
MON
863
and
MON
853
After
Heat
Treatment
454240­
07
Heating
the
corn
flour
samples
at
204

C
for
30
minutes
destroys
both
the
immunoreactivity
and
insect
bioactivity
of
the
Cry3Bb1.11098
found
in
MON
863
corn.
The
Cry3Bb1
immunoreactivity
was
not
detectable
in
both
an
immunoblot
and
ELISA
format
for
MON
863.
For
MON
853,
Cry3Bb1
was
not
recognizable
in
an
immunoblot
and
reduced
more
than
1000­
fold
in
an
ELISA
format.
Since
the
rabbit
anti­
Cry3Bb1
antibody
employed
was
polyclonal
IgG,
it
is
also
suggestive
that
epitopes
were
destroyed
and
not
just
rendered
unrecognized
by
alteration
of
the
three
dimensional
configuration.
Classification:
Acceptable.

Bioinformatics
Evaluation
of
the
Cry3Bb1
Protein
Produced
in
Corn
Event
MON
863
Utilizing
Allergen
Toxin
and
Public
Domain
Protein
Databases
454240­
08
Several
amino
acid
database
comparison
tools
were
employed
to
compare
the
amino
acid
sequence
of
Cry3Bb
to
known
protein
allergens
and
gliadins.
The
UPDATE2
database
was
compiled
to
allow
for
comparison
of
Cry3Bb1.11098
and
Cry3Bb1.11231
to
these
proteins.
The
level
of
similarity
identified
does
not
indicate
significant
similarity
to
any
of
the
proteins
or
gliadins
contained
in
the
database.
In
addition,
no
contiguous
stretch
of
8
identical
amino
acids
was
identified
in
either
the
FASTA
or
IDENTITYSEARCH
algorithms
suggesting
a
lack
of
immunological
similarity.
Based
upon
this
data,
it
does
not
appear
that
Cry3Bb1
shares
significant
structural,
biological
or
immunological
similarity
with
known
protein
allergens
or
gliadins.
Classification:
Acceptable
Title
MRID
No.
Status,
Classification
&
Comments
29
Characterization
and
Equivalence
of
the
Cry3Bb1
Protein
Produced
by
Fermentation
and
Corn
Event
MON
863
455382­
01
MALDI­
TOF
analysis
of
the
microbial
and
corn
Cry3B1.11098(
Q349R)
proteins
yielded
an
agreement
of
from
42
to
50
amino
acid
fragments
predicted
from
the
theoretical
sequence.
The
N­
terminus
of
the
microbial
form
lacked
the
terminal
methionine
which
is
commonly
cleaved
in
expressed
proteins.
The
corn
form
was
apparently
not
only
lacking
the
terminal
methionine
but
the
N­
terminal
alanine
residue
was
acetylated
as
indicated
by
a
42
Dalton
greater
weight.
The
N­
terminal
amino
acid
sequence
analyses
were
flawed
in
that
unequivocal
determinations
were
not
possible
due
to
the
presence
of
multiple
residues
in
most
cycles.
However,
by
comparison
to
the
expected
sequences,
several
different
start
sites
for
N­
terminal
sequencing
could
be
detected.
In
the
E.
coli
Cry3Bb1,
the
sequence
started
at
both
position
2
and
32.
In
the
corn
Cry3Bb1,
three
different
starts
were
detected
at
position
19,
25
and
36.
The
immunoblot
analysis
gave
similar
positive
band
patterns
that
indicated
the
Cry3Bb1
protein
produced
in
both
corn
and
E.
coli
had
essentially
the
same
electrophoretic
mobility
and
immunoreactivity.
The
positive
bands
themselves
were
sometimes
rather
broad
(
74­
66kDa)
but
no
series
of
distinct
bands
could
be
discerned
from
the
photographs
provided.
The
molecular
weight
and
purity
analyses
for
the
corn
and
microbial
extracts
indicate
that
the
microbially
produced
samples
were
nearly
twofold
higher
purity
in
Cry3Bb1
proteins
compared
to
the
corn
extracts.
The
purity
for
Cry3Bb1
was
92.6%
and
53.9%
for
microbial
and
corn
extracts,
respectively.
Total
protein
concentrations
for
the
two
extracts
were
determined
as
0.58
mg/
ml
and
0.46
mg/
ml
for
microbial
and
corn
extracts,
respectively,
by
colormetric
assays.
The
glycosylation
analysis
for
the
Cry3Bb1
extracts
gave
no
positive
carbohydrate
staining
regions
for
either
the
microbial
or
corn
samples
in
the
expected
regions
for
Cry3Bb1
protein.
The
results
of
the
bioassays
for
the
two
Cry3Bb1extracts
against
Colorado
potato
beetle
larvae
(
table
5
attached)
indicate
that
there
was
a
dose/
response
in
all
tests
and
the
LC50
values
were
similar
and
had
overlapping
95%
confidence
intervals.
Classification:
Acceptable.
Title
MRID
No.
Status,
Classification
&
Comments
30
An
Acute
Oral
Toxicity
Study
in
Mice
with
E.
coli
Produced
Cry3Bb1.11098
(
Q349R)
Protein
455382­
02
There
did
not
appear
to
be
significant
adverse
affects
to
animals
resulting
from
exposure
to
Cry3Bb1.11098(
Q349R)
at
dose
amounts
of
300,
900
&
2700
mg/
kg
body
weight.
Observations
included
some
minor
clinical
affects
and
a
relatively
insignificant
lack
of
weight
gain
in
two
animals,
however,
these
do
not
appear
to
be
related
to
exposure
to
the
test
substance,
because
these
occurred
in
the
various
test
groups.
Based
upon
the
data
contained
in
this
submission,
the
LD50
for
Cry3Bb1.11098(
Q349R)
is
greater
than
3200
mg/
kg
body
weight
in
mice.
Classification:
Acceptable.

Assessment
of
the
in
vitro
Digestibility
of
Cry3Bb1
Protein
Purified
from
Corn
Event
MON
863
and
Cry3Bb1
Protein
Purified
from
E.
coli
455382­
03
The
tests
performed
in
this
study
show
that
the
Cry3Bb1
proteins
are
degraded
in
simulated
gastric
fluid.
Incubation
of
corn­
produced
and
E.
coli­
produced
Cry3Bb1
protein
in
SGF
results
in
the
loss
of
detectable
protein
by
the
15
second
observation
point,
as
detected
by
SDS­
PAGE.
Classification:
Acceptable.

Immuno­
detectability
of
NPTII
Protein
in
the
Grain
of
Insect
Protected
Corn
Event
MON
863
After
Heat
Treatment
455382­
09
The
immunoblot
shows
that
extraction
of
the
MON863
corn
grain
spiked
with
nptII
yielded
an
immunoreactive
band
that
comigrated
with
the
E.
coli
produced
nptII.
The
blot
also
showed
that,
regardless
of
the
extraction
buffer
used,
the
heat
treatment
effectively
removed
any
immunoreactive
bands
from
the
samples.
The
results
suggest
that,
even
if
detectable
levels
of
nptII
were
present
in
MON863
corn
grain,
the
heat
treatment
would
remove
them.
Unfortunately,
the
use
of
a
mouse
monoclonal
antibody
limits
the
ability
of
this
data
to
be
extrapolated.
A
heat
treatment
significantly
above
the
95.8

C
used
for
sample
preparation
for
SDS­
PAGE
destroyed
the
epitope(
s)
recognized
by
the
anti­
nptII
antibody
used.
Classification:
Acceptable
Assessment
of
the
in
vitro
Digestability
of
Cry3Bb1.11098
(
Q349R)
Protein
in
Simulated
Intestinal
Fluid
455770­
02
Simulated
intestinal
fluid
activity
was
verified
to
be
present
and
at
a
level
deemed
acceptable
by
SOP
GEN­
PRO­
058­
01.
The
gels
provided
indicate
that
the
Cry3Bb1.11098
(
Q349R)
protein
is
present
as
a
single
band
at
74
kDa
which
rapidly
degraded
to
two
bands
of
68
and
57kDa
at
the
first
assay
time
point
of
1
minute.
The
subsequent
samples
(
from
5
minute
to
24
hours)
all
gave
a
single
57
kDa
band
which
did
not
appear
to
decrease
in
intensity.
This
lack
of
degration
by
intestinal
fluids
is
similar
to
the
majority
of
Cry
proteins
which
are
resistant
to
the
action
of
trypsin.
Classification:
Acceptable
Efficacy
Data
Data
referenced
by
Monsanto
on
the
efficacy
of
Cry1Ab
proteins
against
lepidopteran
species
and
31
Cry3Bb1
against
corn
rootworm
are
listed
in
Table
5.
There
was
no
data
on
the
efficacy
of
YieldGard
®
Plus
Corn
against
lepidopteran
or
corn
rootworm
species
included
in
Volume
1
of
Monsanto's
submission.

Table
5.
Efficacy
studies
cited
to
support
registration
of
YieldGard
®
Plus
Corn
Title
MRID
No.
Classification
Public
Interest
Document
for
the
Plant
Pesticide,
Bacillus
thuringiensis
subsp.
kurstakiInsect
Control
Protein
as
Expressed
in
Corn
(
Zea
maysL.)
442889­
01
No
Data.
Benefits
document.

Application
for
Registration
of
Bacillus
thuringiensis
Cry3Bb
Protein
and
the
Genetic
Material
(
Vector
ZMIR13L)
Necessary
for
it
Production
in
Corn;
and
Amendment
of
the
Previous
Request
for
Exemption
from
the
Requirement
of
a
Tolerance
451568­
00
No
Data.
Application
for
Registration.

Efficacy
of
MON
863
Against
Corn
Rootworm
and
Comparison
to
Insecticide
Treatments
­
Results
of
Year
2000
Field
Trials
453613­
03
Based
on
the
review
of
the
submitted
field
efficacy
studies,
MON
863
corn
is
as
effective
or
more
effective
than
chemical
insecticides
in
protecting
corn
roots
from
CRW
larval
feeding
damage.
Chemical
pesticides
for
CRW
are
usually
applied
to
the
soil
at
the
time
of
planting.
However,
the
pesticide
may
dissipate
and
no
longer
be
effective
by
the
time
the
larvae
hatch.
Timing
is
not
a
problem
with
MON
863
corn
because
the
pesticide
is
incorporated
within
the
corn
roots
and
is
produced
at
a
relatively
constant
rate
in
growing
corn.

Public
Interest
Assessment
Supporting
Registration
of
Bacillus
thuringiensis
Cry3Bb1
Protein
and
the
Genetic
Material
(
Vector
ZMIR13L)
Necessary
for
its
Production
in
Corn
Event
MON
863
456530­
01
Based
upon
this
review,
the
use
of
MON
863
CRWprotected
corn
is
presumed
to
be
in
the
public
interest
under
section
IV.
A.
of
52
FR
7628
because
it
will
replace
or
reduce
the
use
of
a
number
of
higher
risk
pesticides
for
CRW
control
that
are
of
Agency
concern
as
discussed
above
(
e.
g.,
terbufos,
chlorpyrifos,
phorate).
It
also
has
clearly
identified
benefits
and
meets
the
criteria
in
part
IV.
B
of
the
FR
notice.
Therefore,
EPA
concludes
that
the
use
of
MON
863
CRW­
protected
corn
is
in
the
public
interest
and
supports
the
conditional
registration
under
FIFRA
section
3(
c)(
7)(
C).

Insect
Resistance
Management
Monsanto
has
developed
an
insect
resistance
management
(
IRM)
plan
for
YieldGard
®
Plus
Corn.
An
amendment
to
Monsanto's
IRM
plan
in
Volume
1
was
submitted
to
EPA
by
Monsanto
on
April
1,
2003.
Since
YieldGard
®
Plus
Corn
contains
the
Cry1Ab
and
Cry3Bb1
proteins,
an
IRM
32
plan
needs
to
consider
European
corn
borers
and
corn
rootworms.
Monsanto's
IRM
plan
for
YieldGard
®
Plus
Corn
considers
European
corn
borer
and
corn
rootworm
resistance
management
and
takes
the
conservative
approach
when
strategies
differ
between
the
target
pests.

Refuge
Requirements.
Based
upon
Growers
agronomic
practices
and
pesticide
use,
one
refuge
for
European
corn
borer
and
corn
rootworms
or
separate
refuges
may
be
planted
for
each
pest.
A
grower
that
adopts
the
Common
Refuge
option
would
be
required
to
plant
a
minimum
of
a
20%
non­
Bt
structured
refuge
adjacent
to
or
within
YieldGard
®
Plus
Corn
fields.
Refuges
acres
should
be
planted
as
continuous
blocks
adjacent
to
or
within
fields,
perimeter
strips
or
strips
within
YieldGard
®
Plus
Corn.
Monsanto
is
recommending
that
in­
field
strips
should
be
at
least
four
row
and
preferably
six
rows
wide.
Agronomic
practices
should
be
comparable
for
YieldGard
®
Plus
Corn
and
refuge
acres.
For
example,
if
YieldGard
®
Plus
Corn
acres
are
planted
continuously
or
as
first
year
corn,
then
the
non­
Bt
refuge
acres
should
also
be
planted
continuously
or
as
first
year
corn
respectively.
Non­
Bt
insecticides
may
be
applied
to
refuge
acres
to
control
corn
root
larvae,
but
may
only
be
applied
to
refuge
acres
when
corn
rootworm
adults
are
present
if
YieldGard
®
Plus
Corn
acres
are
also
treated.

Growers
that
choose
the
Separate
Refuge
option
must
plant
a
distinct
refuge
for
corn
rootworm
and
European
corn
borer.
A
20%
non­
corn
rootworm
protected
corn
refuge
must
be
planted
to
delay
corn
rootworm
resistance
to
YieldGard
®
Plus
Corn.
An
additional
20%
non­
Bt
corn
must
also
be
planted
to
delay
European
corn
borer
resistance.
The
corn
rootworm
refuge
must
be
planted
with
corn
that
does
not
contain
the
Cry3Bb
protein.
However,
corn
that
only
contains
the
Cry1Ab
protein
may
be
planted
if
a
separate
non­
Bt
corn
refuge
is
planted
to
delay
European
corn
borer
resistance.
The
corn
rootworm
refuge
should
be
planted
as
continuous
blocks
adjacent
to
or
within
fields,
perimeter
strips
or
strips
within
YieldGard
®
Plus
Corn
(
at
least
6
rows
and
preferably
12
rows
wide)
and
utilize
comparable
agronomic
practices
as
the
YieldGard
®
Plus
Corn
acres.
European
corn
borer
refuges
may
be
planted
within
fields
as
blocks
or
strips,
adjacent
to
fields
or
up
to
½
mile
(
1/
4
mile
preferred)
from
YieldGard
®
Plus
Corn
acres.
Non­
Bt
insecticides
may
be
applied
to
refuge
acres
to
control
corn
rootworm
larvae,
but
may
only
be
applied
to
refuge
acres
when
corn
rootworm
adults
are
present
if
YieldGard
®
Plus
Corn
acres
are
also
treated.
Non­
Bt
insecticides
may
be
applied
to
refuges
to
control
the
European
corn
borer,
corn
earworm
or
southwestern
corn
borer
if
economic
injury
levels
occur.

Table
6.
Comparison
of
event
MON
810
and
MON
863
IRM
requirements
with
Monsanto's
proposed
IRM
strategy
for
YieldGard
®
Plus
Corn
Requirements
MON
810
MON
863
YieldGard
®
Plus
Corn
Refuge
Size
 
20%
 
20%
 
20%

Refuge
Placement
 
½
mile
( 
¼
mile
preferred)
Adjacent
or
within
field
Adjacent
or
within
field
Requirements
MON
810
MON
863
YieldGard
®
Plus
Corn
33
Refuge
Configuration
Block,
in­
field
strips
( 
4
rows,
where
 
6
rows
preferred),
or
edges
Block
or
strips
( 
6
rows,
preferably
 
12
rows
wide)
Block
or
strips
( 
6
rows,
preferably
 
12
rows
wide)

Refuge
Management
Any
corn
rotation
meeting
placement
&
configuration
requirements.

Insecticides
can
be
used
in
refuge
to
control
ECB/
SWCB
when
above
economic
thresholds.

Microbial
Bt
insecticides
are
not
allowed.
Same
corn
rotation
as
YGRW
(
e.
g.,
first
year
corn
or
corn
followed
by
corn).

Conventional
insecticides
or
seed
treatments
can
be
used
in
refuge
to
control
CRW
larvae
&
other
soil
pests.
If
the
refuge
is
treated
with
a
foliar
insecticide
labeled
for
CRW
control
when
CRW
adults
are
present,
then
YGRW
also
must
be
treated.

(
Not
applicable)
Same
corn
rotation
as
YG
Plus
(
e.
g.,
first
year
corn
or
corn
followed
by
corn).

Conventional
insecticides
or
seed
treatments
can
be
used
in
refuge
to
control
CRW
larvae
&
other
soil
pests.
If
the
refuge
is
treated
with
a
foliar
insecticide
labeled
for
CRW
control
when
CRW
adults
are
present,
then
YG
Plus
also
must
be
treated.

Microbial
Bt
insecticides
are
not
allowed.

Refuge
Corn
Types
Conventional
Conventional
YGCB
(
a
CB
refuge
planted
 
½
mile
also
will
be
required)

Roundup
Ready
corn
Conventional
YGCB
(
an
additional
refuge
for
CB
will
be
required)

Roundup
Ready
corn
This
table
was
reproduced
from
pages
4
and
5
of
10
of
Monsanto's
April1,
2003
amendment
request
Grower
Agreements.
Growers
will
be
required
to
sign
an
agreement
similar
to
the
agreements
growers
currently
sign
to
plant
MON
810
or
MON
863
corn.
This
signed
agreement
contractually
obligates
growers
to
comply
with
refuge
requirements.

Grower
Education.
Grower
education
programs
are
required
in
an
IRM
strategy.
Monsanto
will
develop
and
implement
grower
education
programs
to
inform
growers
of
YieldGard
®
Plus
Corn
IRM
requirements
and
report
on
these
programs
to
EPA.

Compliance
Assurance
Plan.
Monsanto
is
currently
developing
a
program
that
will
evaluate
and
promote
grower
compliance.
A
grower
compliance
plan
is
currently
under
development
for
Cry1Ab
corn.
Monsanto
anticipates
that
the
compliance
requirements
for
Cry1Ab
corn
will
be
the
same
for
YieldGard
®
Plus
Corn.

Monitoring.
Monsanto
will
be
required
to
monitor
for
pest
resistance
to
the
Cry1Ab
and
Cry3Bb1
proteins
in
YieldGard
®
Plus
Corn.
Monsanto
will
develop
a
plan
for
YieldGard
®
Plus
34
Corn
to
monitor
for
changes
susceptibility
of
the
European
corn
borer,
southwestern
corn
borer,
corn
earworm
and
western
corn
rootworm.
According
to
Monsanto,
this
plan
is
"
contingent
on
the
availability
of
a
diagnostic
dose
assay.
Monsanto
suggests
the
need
for
a
panel
to
consider
corn
rootworm
monitoring
programs
similar
to
the
panel
of
experts
required
to
be
convened
by
the
Agricultural
Biotechnology
Stewardship
Technical
Committee
(
ABSTC)
to
evaluate
corn
borer
monitoring
in
Cry1Ab
corn.

Mitigation.
A
Remedial
Action
Plan
must
be
developed
and
implemented
if
resistance
is
detected.
European
corn
borer
and
corn
rootworm
Remedial
Action
Plan
will
be
the
same
for
YieldGard
®
Plus
Corn
as
single
trait
Cry1Ab
and
Cry3Bb1
corn
respectively.

Reporting.
Reporting
requirements
for
YieldGard
®
Plus
Corn
will
be
the
same
as
for
MON
810
and
MON
863
corn.
Sales
reports,
IRM
grower
agreement
results,
compliance
and
educational
programs
will
be
submitted
to
EPA
annually
by
January
31.
Monitoring
for
European
corn
borer
resistance
reports
will
be
submitted
to
EPA
annually.
However,
corn
rootworm
resistance
monitoring
reports
will
be
submitted
biannually
because
there
is
only
one
generation
per
year
of
corn
rootworms
compared
to
two
to
three
generations
of
European
corn
borers
per
year.

Reviewer's
Comment
to
the
Insect
Resistance
Management
Plan
Monsanto's
proposed
IRM
plan
for
YieldGard
®
Plus
Corn
is
acceptable.

References:

Borokov,
I.
G.,
T.
A.
Cavato
and
R.
P.
Lirette.
2001.
Confirmation
of
the
molecular
identity
of
YieldGard
®
and
corn
rootworm­
protected
combined
trait
corn
hybrid
MON
810
x
MON
863
by
Southern
Blot.
Report
MSL­
17466,
an
unpublished
study
conducted
by
Monsanto
Company,
included
as
Volume
2
of
this
submission.

Dudin,
Y.
A.,
B.
P.
Tonnu
and
R.
P.
Lirette.
2001.
Cry3Bb1,
Cry1Ab
and
nptII
protein
levels
in
the
dual­
trait
maize
hybrid
MON
863
x
MON
810
produced
in
Argentinian
field
trials
conducted
during
the
1999­
2000
growing
season.
Report
MSL­
17266,
an
unpublished
study
conducted
by
Monsanto
Company,
included
as
Volume
3
of
this
transmittal.

Hellmich,
R.
L.,
B.
D.
Siegfried,
M.
K.
Sears,
D.
E.
Stanley­
Horne,
H.
R.
Mattila,
T.
Spencer,
and
K.
Bidne
and
L.
C.
Lewis.
2001.
Monarch
caterpillar
sensitivity
to
Bacillus
thuringiensispurified
proteins
and
pollen.
Proc.
Natl.
Acad.
Sci.
USA,
10.1073/
pnas.
211297698.
98:
11925­
11930.

MacIntosh,
S.
C.,
T.
B.
Stone,
S.
R.
Sim,
P.
L.
Hunst,
J.
T.
Greenplate,
P.
G.
Marrone,
F.
J.
Perlak,
D.
A.
Fischoff
and
R.
L.
Fuchs.
1990.
Specificity
and
efficacy
of
purified
Bacillus
thuringiensis
proteins
against
agronomically
important
insects.
J.
Invert.
Pathol.
56:
258­
266.
35
Pleasants,
J.
M.,
R.
L.
Hellmich,
G.
Dively,
M.
K.
Sears,
D.
E.
Stanley­
Horne,
H.
R.
Matilla,
J.
E.
Foster,
T.
L.
Clark,
and
G.
D.
Jones.
2001.
Corn
pollen
deposition
on
milkweeds
in
and
near
corn
fields.
Proc.
Natl.
Acad.
Sci.
USA,
10.1073/
pnas.
211287498.
98:
11919­
11924
Schnepf,
E.,
N.
Crickmore,
J.
Van
Rie,
D.
Lereclus,
J.
Baum,
J.
Feitelson,
D.
R.
Zeigler
and
D.
H.
Dean.
1998.
Bacillus
thuringiensis
and
its
pesticidal
crystal
proteins.
Microbiol.
Molec.
Biol.
Rev.
62:
775­
806.

Vaituzis,
Z.
and
Gail
Tomimatsu.
2001.
Risk
Analysis
to
the
Karner
Blue
Butterfly
and
Other
Endangered
Lepidoptera
from
Bt
Cry1Ab
event
MON810
and
Bt11,
and
Bt
Cry1F
Corn
TC1507.
Biopesticides
and
Pollution
Prevention
Division.
U.
S.
Environmental
Protection
Agency.
Washington,
D.
C.

Wolt,
J.
D.
2000.
Non­
target
exposure
and
risk
assessment
for
environmental
dispersal
of
Cry1F
maize
protein.
Unpublished
report
of
Dow
AgroSciences.
MRID
450415­
02.
36
Data
Evaluation
Report
(
DER)

Reviewed
by:
Robyn
Rose,
Entomologist,
BPPD
Secondary
Reviewer:
Zigfridas
Vaituzis,
Microbiologist,
BPPD
STUDY
TYPE:
Evaluation
of
potential
interactions
between
the
Cry1Ab
and
Cry3Bb1
proteins
expressed
in
YieldGard
®
Plus
Corn
MRID
NO.:
460697­
01
CHEMICAL
NO.:
006498
TEST
MATERIAL:
Cry1Ab
and
Cry3Bb1
Proteins
STUDY
NO.:
MSL­
18726
SPONSOR:
Monsanto
Company,
600
13th
Street
N.
W.,
Suite
660,
Washington,
DC
20005
TITLE
OF
REPORT:
An
Investigation
into
the
Potential
for
Interactions
Between
the
Cry1Ab
and
Cry3Bb1
Proteins
Produced
in
YieldGard
®
Plus
Corn
AUTHORS:
Ty
T.
Vaughn,
Ph.
D.,
Christopher
R.
Brown,
Michael
J.
McKee,
Ph.
D.,
Robin
D.
Camp,
Michael
J.
Pleau,
Jian
J.
Duan,
Ph.
D.,
Dennis
P.
Ward,
Ph.
D.
STUDY
COMPLETED:
September
4,
2003
CLASSIFICATION:
Acceptable
Study
Summary
Objective
Studies
were
conducted
to
test
the
hypothesis
that
the
Cry1Ab
and
Cry3Bb1
proteins
do
not
interact
when
combined
in
YieldGard
®
Plus
Corn
Methods:

Seven
insect
species
were
tested
from
eggs
reared
at
Monsanto's
Insectary
for
their
susceptibility
to
the
Cry1Ab
or
Cry3Bb1
Bt
proteins
in
the
single
trait
YieldGard
®
Corn
compared
to
the
dual
trait
YieldGard
®
Plus
Corn.
The
four
insects
evaluated
for
their
susceptibility
to
the
Cry1Ab
protein
included
the
corn
earworm
(
Helicoverpa
zea;
CEW),
southwestern
corn
borer
(
Diatraea
grandiosella,
SWCB),
European
corn
borer
(
Ostrinia
nubilalis,
ECB)
and
fall
armyworm
(
Spodoptera
frugiperda,
FAW).
Three
insects
including
the
Colorado
potato
beetle
(
Leptinotarsa
decemlineata,
CPB),
southern
corn
rootworm
(
Diabrotica
undecimpunctata,
SCRW)
and
western
corn
rootworm
(
Diabrotica
virgifera,
WCRW)
susceptibility
to
the
37
Cry3Bb1
protein
were
also
evaluated.

Leaf
disk
and
whole
plant
insect
feeding
assays
were
conducted
with
single
trait
MON
810
(
Cry1Ab)
and
MON
863
(
Cry3Bb1)
hybrids,
a
combined
trait
YieldGard
®
Plus
Corn
(
Cry1Ab
&
Cry3Bb1)
and
nontransgenic
control
hybrid.
FAW,
CEW
and
SWCB
susceptibility
to
Cry1Ab
was
evaluated
from
leaf
disk
assays.
One
first
instar
FAW
or
CEW
larvae
or
three
SWCB
neonate
larvae
were
exposed
to
three
to
four
1
cm
leaf
disks
cut
from
ten
plants
at
the
V4
plant
growth
stage
from
MON
810,
YieldGard
®
Plus
and
nontransgenic
corn
plants.
Leaf
damage
was
rated
on
a
scale
of
0
to
11
after
FAW
fed
for
two
days
and
after
three
days
of
CEW
and
SWCB
feeding.
Feeding
damage
was
rated
from
no
damage
(
0
on
rating
scale)
and
increased
by
5%
for
each
rating
point
up
to
>
50%
damage
(
11
on
rating
scale).
FAW
larval
development
was
evaluated
and
instar
survival
was
determined
for
all
three
lepidopteran
species
at
test
termination.
FAW
larvae
were
expected
to
develop
to
third
instars
during
a
two
day
feeding
assay.
An
analysis
of
variance
(
ANOVA)
including
a
Tukey­
Kramer
means
separation
test
for
significance
(
p=
0.05)
was
conducted
with
data
from
leaf
disk
assays.

Lepidopteran
leaf
feeding
damage
and
coleopteran
root
feeding
damage
was
evaluated
from
whole
plant
growth
chamber
assays
to
evaluate
potential
differences
in
insecticidal
activity
of
single
trait
compared
to
dual
trait
Cry1Ab
and
Cry3Bb1
corn
hybrids.
ECB
and
SWCB
were
evaluated
for
their
susceptibility
to
the
Cry1Ab
protein
and
SCRW
and
WCRW
susceptibility
to
Cry3Bb1
was
evaluated.
Each
hybrid
was
planted
in
15
to
25
25.5
cm
diameter
and
30
cm
deep
pots.
V2
plants
infested
with
900
eggs
were
used
for
the
WCRW
assays
and
plants
at
the
V4
growth
stage
were
infected
with
1,000
eggs
for
the
SCRW
assays.
Lepidopteran
assays
included
40
to
50
ECB
or
35
SWCB
placed
in
the
whorl
of
each
plant
at
the
V6
growth
stage.
Plants
were
assayed
with
ECB
and
WCR
simultaneously
or
with
SWCB
and
SCRW
together
and
evaluated
for
leaf
or
root
feeding
damage
after
21
days.
The
modified
Guthrie
scale
was
used
to
rate
ECB
and
SWCB
damage
from
0
to
9
with
0
equally
no
damage
and
9
indicating
long
feeding
lesions.
Root
damage
was
rated
according
to
the
Iowa
1
to
6
scale
with
1
equaling
no
damage
and
a
6
correlating
to
at
least
six
damaged
nodes.
An
analysis
of
variance
(
ANOVA)
including
a
Tukey­
Kramer
HSD
means
separation
test
for
significance
(
p=
0.05)
was
conducted
with
data
from
the
whole
plant
assays.

ECB
and
CPB
larvae
were
evaluated
for
levels
of
mortality
in
in
vitro
artificial
diet
bioassays
with
Cry1Ab
and
Cry3Bb1
proteins
respectively
alone
and
combined
(
Table
1).
Both
Cry
proteins
were
purified
from
heterologous
E.
coli
fermentation
production
systems.
According
to
Monsanto,
the
"
Cry1Ab
protein
was
suspended
in
a
50
mM
sodium
bicarbonate
buffer
/
5
mM
NaCl,
pH
10.25
(
hereafter:
Buffer­
1),
and
Cry3Bb1
protein
was
suspended
in
a
10
mM
potassium
phosphate
buffer,
pH
7.0
(
hereafter:
Buffer­
2)."
Concentrations
of
Cry1Ab
ranging
from
0.0195
to
5

g
protein/
mL
diet
were
used
in
assays
with
and
without
the
incorporation
of
100

g/
mL
Cry3Bb1
protein
to
determine
the
Cry1Ab
LC
50
for
ECB.
The
Cry3Bb1
LC
50
for
CPB
was
determined
from
assays
conducted
with
Cry3Bb1
concentrations
ranging
from
0.375
to
6

g
protein/
mL
diet
alone
and
in
combination
with
30

g/
mL
of
Cry1Ab.
The
concentration
levels
of
100

g/
mL
Cry3Bb1
protein
and
30

g/
mL
of
Cry1Ab
were
used
because
they
are
slightly
higher
than
the
maximum
levels
found
in
YieldGard
®
Plus
plant
tissue.
A
buffer
and
a
38
water
control
were
included
for
each
dose
of
each
bioassay.
Each
bioassay
included
approximately
24
larvae
and
was
replicated
three
times
for
a
total
of
72
larvae
per
dose.
An
additional
study
was
conducted
to
ensure
that
Cry1Ab
was
not
affecting
CPB
and
Cry3Bb1
was
not
affecting
ECB.

Table
1.
Key
experimental
design
components
for
each
replicate
in
the
ECB
and
CPB
diet
incorporation
bioassays
with
the
Cry1Ab
and
Cry3Bb1
proteins.

Species
Treatment
Cry1Ab
(
µ
g/
ml)
Cry3Bb1
(
µ
g/
ml)

ECB
Cry1Ab
0.0195
 
5
0
ECB
Cry1Ab
+
Cry3Bb1
0.0195
 
5
100
ECB
Water
control
0
0
ECB
Buffer­
1
control
0
0
ECB
Buffer­
1
+
Buffer­
2
control
0
0
CPB
Cry3Bb1
0
0.375
­
6
CPB
Cry3Bb1
+
Cry1Ab
30
0.375
­
6
CPB
Water
control
0
0
CPB
Buffer­
2
control
0
0
CPB
Buffer­
1
+
Buffer­
2
control
0
0
Table
copied
from
page
9
of
17
of
MRID
No
460697­
01
Larval
survival
was
recorded
six
to
seven
days
after
test
initiation
and
weights
were
recorded
although
the
weight
data
wasn't
analyzed.
LC
50
values
were
calculated
from
a
Probit
analysis
for
ECB
for
Cry1Ab
alone
and
combined
with
Cry3Bb1
as
well
as
for
Cry3Bb1
alone
and
combined
with
Cry1Ab
for
CPB.
A
dose
response
was
also
calculated
from
an
Integrated
Probit
analysis.
Rates
of
mortality
were
compared
between
the
water,
buffer
and
protein
background
control
groups
by
Fisher's
Exact
Test
(
p=
0.05).

Results
Single
and
dual
trait
hybrids
containing
Cry1Ab
significantly
reduced
FAW,
CEW
and
SWCB
larvae
feeding
compared
to
hybrids
without
Cry1Ab
in
leaf
disk
assays
(
Table
2.).
There
was
more
damage
caused
by
FAW
than
CEW
or
SWCB
suggesting
FAW
is
less
susceptible
to
the
Cry1Ab
protein
than
CEW
or
SWCB.
MON
810
and
YieldGard
®
Plus
Corn
provided
comparable
protection
from
FAW,
CEW
and
SWCB
feeding
(
Table
2).
Hybrids
containing
the
Cry1Ab
protein
significantly
reduced
CEW
and
SWCB
survival
compared
to
hybrids
without
Cry1Ab
(
Table
3).
.
Survival
of
FAW
did
not
differ
between
any
of
the
single
trait,
dual
trait
and
control
hybrids.
However,
FAW
feeding
on
hybrids
containing
the
Cry1Ab
protein
resulted
in
reduced
growth
rate
compared
to
MON
863
or
control
corn
hybrids
(
p<
0.0001).
FAW
larvae
feeding
on
MON
810
and
YieldGard
®
Plus
Corn
developed
to
developmental
ratings
of
1.30
±
0.09
and
1.68
±
0.12
respectively
and
to
2.30
±
0.11
and
2.26
±
0.13
for
nontransgenic
and
MON
863
hybrids
respectively.
In
general,
the
presence
of
MON
863
did
not
affect
survival
or
development
of
CEW,
SWCB
or
FAW
in
the
leaf
disk
assays.
39
Table
2.
Leaf
disk
damage
rating
results
for
FAW,
CEW,
and
SWCB.
The
extent
of
leaf
disk
feeding
damage
was
rated
on
a
scale
of
0
 
11;
each
increase
in
rating
value
represents
an
additional
5%
loss
of
tissue
mass.
The
values
presented
are
the
mean
damage
ratings
(
±
SEM)
for
each
treatment
and
species
tested.
The
value
in
parenthesis
is
the
number
of
leaf
disks
rated
for
each
treatment.
An
ANOVA
p
value
of
<
0.05
indicates
an
overall
treatment
effect.
Significant
differences
between
treatment
means
within
a
row
are
denoted
by
letter
code:
`
a'
is
different
from
`
b'
(
p<
0.05,
Tukey­
Kramer
HSD).

Leaf
Damage
Ratings
Species
MON
810
MON
863
YGPlus
Control
ANOVA
FAW
6.00
±
0.74
a
9.30
±
0.63
b
7.03
±
0.67
a
10.67
±
0.24
b
p<
0.0001
(
30)
(
30)
(
30)
(
30)

CEW
1.22
±
0.11
a
9.39
±
0.68
b
1.43
±
0.16
a
10.31
±
0.48
b
p<
0.0001
(
27)
(
28)
(
30)
(
29)

SWCB
1.98
±
0.10
a
11
±
0
b
1.68
±
0.11
a
11
±
0
b
p<
0.0001
(
40)
(
40)
(
40)
(
40)

Table
2
copied
from
page
10
of
17
of
MRID
No
460697­
01
Table
3.
Instar
survival
results
for
FAW,
CEW,
and
SWCB.
The
values
presented
are
the
mean
three­
day
percent
survival
rates
(
±
SEM)
for
each
treatment
and
species
tested
in
leaf
disk
feeding
assays.
The
value
in
parenthesis
is
the
number
of
plants
used
for
each
treatment.
An
ANOVA
p
value
of
<
0.05
indicates
an
overall
treatment
effect.
Significant
differences
between
treatment
means
within
a
row
are
denoted
by
letter
code:
`
a'
is
different
from
`
b'
(
p<
0.05,
Tukey­
Kramer
HSD).

Instar
Survival
(%)
Species
MON
810
MON
863
YGPlus
Control
ANOVA
FAW
90
±
6
a
90
±
6
a
93
±
5
a
100
±
0
a
p=
0.31
(
10)
(
10)
(
10)
(
10)

CEW
4
±
5
a
86
±
5
b
7
±
5
a
93
±
5
b
p<
0.0001
(
9)
(
10)
(
10)
(
10)

SWCB
5
±
2
a
100
±
0
b
0
±
0
a
100
±
0
b
p<
0.0001
(
10)
(
10)
(
10)
(
10)

Table
copied
from
page
10
of
17
of
MRID
No
460697­
01
Cry1Ab
in
MON
810
and
YieldGard
®
Plus
Corn
had
significantly
less
leaf
damage
from
ECB
and
SWCB
feeding
in
whole
plant
assays
than
hybrids
without
Cry1Ab
(
Table
4).
There
was
no
statistical
difference
between
MON
810
and
YieldGard
®
Plus
damage
but
MON
863
and
control
plants
had
high
damage
ratings.
Results
of
the
whole
plant
assays
also
showed
that
the
presence
of
Cry3Bb1
does
not
affect
the
insecticidal
potential
of
Cry1Ab
in
corn.
40
Table
5.
Leaf
damage
rating
results
for
SWCB
and
ECB.
The
extent
of
leaf
disk
feeding
damage
was
rated
on
a
scale
of
0
 
9
(
modified
Guthrie
scale).
The
values
presented
are
mean
damage
ratings
(
±
SEM)
for
each
treatment
and
species
tested.
The
value
in
parenthesis
is
the
number
of
plants
rated
for
each
treatment.
An
ANOVA
p
value
of
<
0.05
indicates
an
overall
treatment
effect.
Significant
differences
between
treatment
means
are
denoted
by
letter
code:
`
a'
is
different
from
`
b'
(
p<
0.05,
Tukey­
Kramer
HSD).

Leaf
Damage
Ratings
Species
MON
810
MON
863
YGPlus
Control
ANOVA
SWCB
1.6
±
0.25
a
8.9
±
0.07
b
1.4
±
0.19
a
9
±
0
b
p<
0.0001
(
15)
(
15)
(
15)
(
15)

ECB
0.21
±
0.08
a
7.52
±
0.54
b
0.38
±
0.13
a
8.35
±
0.15
b
p<
0.0001
(
24)
(
21)
(
16)
(
20)

Table
copied
from
page
12
of
17
of
MRID
No
460697­
01
Whole
plant
assays
with
the
SCRW
and
WCRW
resulted
in
reduced
root
damage
ratings
for
MON
863
and
YieldGard
®
Plus
Corn
compared
to
MON
810
and
control
hybrids
which
had
high
damage
ratings
(
Table
5).
There
was
no
statistical
difference
in
damage
ratings
of
MON
863
and
YieldGard
®
Plus
Corn
which
contain
the
Cry3Bb1
protein.
These
data
demonstrate
that
Cry1Ab
does
not
have
interactive
effects,
meaning
it
does
not
enhance
or
diminish
effects,
on
target
Chrysomelids
when
combined
with
Cry3Bb1.

Table
5.
Root
damage
rating
results
for
SCRW
and
WCRW.
The
extent
of
larval
feeding
damage
was
rated
on
a
scale
of
1
 
6.
The
values
presented
are
the
mean
root
damage
ratings
(
±
SEM)
for
each
treatment
and
species
tested.
The
value
in
parenthesis
is
the
number
of
plants
rated
for
each
treatment.
An
ANOVA
p
value
of
<
0.05
indicates
a
significant
difference
between
the
treatment
means.
Significant
differences
between
treatment
means
within
a
row
are
denoted
by
letter
code:
`
a'
is
different
from
`
b'
(
p<
0.05,
Tukey­
Kramer
HSD).

Root
Damage
Ratings
Species
MON
810
MON
863
YGPlus
Control
ANOVA
SCRW
3.6
±
0.16
a
2.3
±
0.12
b
2.1
±
0.07
b
3.3
±
0.16
a
p<
0.001
(
15)
(
15)
(
15)
(
15)

WCRW
4.8
±
0.16
a
2.1
±
0.08
b
2.1
±
0.08
b
4.6
±
0.11
a
p<
0.001
(
20)
(
17)
(
21)
(
19)

Table
copied
from
page
13
of
17
of
MRID
No
460697­
01
The
Cry1Ab
LC
50
values
for
ECB
were
comparable
for
the
single
and
dual
trait
hybrids
according
to
the
Integrated
Probit
analysis
(
p=
0.82)
(
Table
6).
The
Fisher's
Exact
Test
resulted
in
significant
differences
between
results
of
the
different
control
groups
and
replicates
(
p<
0.0001).
There
was
no
ECB
mortality
in
the
second
two
control
replicates,
however
there
was
29%
mortality
in
the
first
control
replicate,
13%
mortality
in
Buffer­
1
and
20%
mortality
in
the
Buffer­
41
1

Buffer­
2
control
groups.
Although
there
was
mortality
in
the
first
control
replicate
for
unknown
reasons,
analysis
of
this
data
alone
showed
no
significant
differences
in
mortality
in
this
group
(
p=
0.37).

Table
6.
Results
of
ECB
LC
50
and
95%
confidence
interval
(
CI)
determinations
for
Cry1Ab
tested
alone
and
in
combination
with
a
background
dose
of
100
µ
g/
ml
Cry3Bb1.

Replicate
Treatment
LC50
(
µ
g/
ml)
95%
CI
Treatment
LC50
(
µ
g/
ml)
95%
CI
1
Cry1Ab
0.165
0.109
­
0.247
Cry1Ab+
Cry3Bb1
0.204
0.122
­
0.334
2
Cry1Ab
0.279
na
Cry1Ab+
Cry3Bb1
0.251
0.178
­
0.345
3
Cry1Ab
0.333
0.197
­
0.562
Cry1Ab+
Cry3Bb1
0.294
na
Mean
0.259
Mean
0.250
na
=
not
able
to
compute
due
to
variability
in
dose
response
Table
copied
from
page
14
of
17
of
MRID
No
460697­
01
The
Cry3Bb1
LC
50
values
for
CPB
were
comparable
for
the
single
and
dual
trait
hybrids
according
to
the
Integrated
Probit
analysis
(
p=
0.06)
(
Table
7).
The
Fisher's
Exact
Test
did
not
result
in
significant
differences
between
the
different
control
groups
and
replicates
(
p=
1.0).

Table
7.
Results
of
CPB
LC
50
and
95%
confidence
interval
(
CI)
determinations
for
Cry3Bb1
tested
alone
and
in
combination
with
a
background
dose
of
30
µ
g/
ml
Cry1Ab.

Replicate
Treatment
LC50
(
µ
g/
ml)
95%
CI
Treatment
LC50
(
µ
g/
m
l)
95%
CI
1
Cry3Bb1
2.02
1.60
 
2.59
Cry3Bb1+
Cry1Ab
1.59
1.26
 
2.02
2
Cry3Bb1
2.33
1.88
 
2.92
Cry3Bb1+
Cry1Ab
1.87
1.98
 
123
3
Cry3Bb1
3.04
2.32
 
4.26
Cry3Bb1+
Cry1Ab
2.11
1.63
 
2.81
Mean
2.47
Mean
1.86
Table
copied
from
page
15
of
17
of
MRID
No
460697­
01
CPB
larval
survival
was
not
affected
by
the
presence
of
30

g
Cry1Ab
protein/
mL
diet
(
p=
1.0).
ECB
larval
survival
was
not
affected
by
the
presence
of
100

g
Cry3Bb1
protein/
mL
diet
(
p=
0.97).
According
to
Monsanto,
"[
t]
hese
data
provide
further
evidence
that
the
lepidopteranactive
Cry1Ab
protein
does
not
affect
Coleoptera
species
(
e.
g.,
CPB)
and
the
coleopteran­
active
Cry3Bb1
protein
does
not
affect
Lepidoptera
species
(
e.
g.,
ECB).

Study
Author's
Conclusions
YieldGard
®
Plus
Corn
did
not
enhance
or
diminish
ECB,
CEW,
FAW
or
SWCB
leaf
feeding
damage
compared
to
single
trait
MON
810
corn
containing
the
Cry1Ab
protein
in
five
in
planta
assays.
YieldGard
®
Plus
Corn
also
did
not
enhance
or
diminish
WCRW
and
SCRW
larval
feeding
on
roots
compared
to
single
trait
MON
863
corn
containing
the
Cry3Bb1
protein.
Leaf
disk
assays
resulted
in
no
difference
in
insecticidal
activity
against
FAW
between
YieldGard
®
Plus
and
single
trait
MON
810
corn.
The
presence
of
Cry3Bb1
in
YieldGard
®
Plus
Corn
did
not
affect
FAW
nor
did
the
presence
of
Cry1Ab
affect
CPB
in
leaf
disk
assays.
Insect
bioassays
42
conducted
with
purified
protein
verified
that
Cry3Bb1
will
not
effect
ECB
survival
and
Cry1Ab
will
not
effect
CPB
survival.
LC
50
values
for
ECB
and
CPB
were
similar
for
the
single
trait
hybrids
(
MON
810
and
MON
863)
and
dual
trait
hybrids
()
and
dose
response
curves
did
not
differ.

According
to
Monsanto,
"[
c]
ollectively
these
data
provide
evidence
that
the
Cry1Ab
and
Cry3Bb1
proteins
produced
in
YieldGard
®
Plus
Corn
do
not
interact
in
an
antagonistic,
additive,
or
synergistic
manner."
Results
of
these
assays
verify
that,
no
interactive
affects
occur
which
was
expected
since
different
physiological
conditions
are
needed
for
the
Cry1Ab
and
Cry3Bb1
proteins
to
function.
Protection
against
lepidopteran
and
coleopteran
target
pests
were
equivalent
for
the
single
trait
and
stacked
hybrids.
Based
on
the
lack
of
interactive
effects
on
susceptible
pests,
Monsanto
concluded
that
"
it
is
extremely
unlikely
that
the
Cry1Ab
and
Cry3Bb1
proteins
contained
in
YieldGard
®
Plus
Corn
will
impart
any
safety
concerns
for
nontarget
organisms
exposed
to
these
hybrids
in
the
environment."

Reviewer's
Comments
It
can
be
concluded
from
the
leaf
disk,
whole
plant
and
in
vitro
studies
with
purified
Bt
protein
that
there
are
no
interactive
effects
on
susceptible
insect
pests
when
the
Cry1Ab
and
Cry3Bb1
proteins
are
combined
in
YieldGard
®
Plus
Corn.
Since
combining
these
proteins
in
YieldGard
®
Plus
Corn
does
not
change
the
level
of
susceptibility
of
susceptible
pests
compared
to
single
trait
MON
810
and
MON
863
corn,
it
can
be
concluded
that
there
will
not
be
a
difference
for
nontarget
insects
not
susceptible
to
the
Cry1Ab
or
Cry3Bb1
proteins.
Therefore,
non­
target
beneficial
insect
data
may
be
waived
for
YieldGard
®
Plus
Corn.
