Document ID: EPA-HQ-OPP-2004-0182-0024
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2005-10-11T04:00Z

OCT
17
2003
MEMORANDUM
SUBJECT:
Assessment
of
the
product
characterization
of
the
Cry1Ab
/
Cry3Bb1
combined
trait,
insect
control
proteins
as
expressed
in
maize,
a
Bacillus
thuringiensis
­
based
plant­
incorporated
protectant
for
control
of
the
corn
borer
and
rootworm
complexes,
and
consideration
of
a
data
waiver
request
for
further
acute
oral
toxicity
evaluation.

TO:
Mike
Mendelsohn,
Product
Manager
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
FROM:
Chris
A.
Wozniak,
Ph.
D.,
Biologist
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
THROUGH:
John
L.
Kough,
Ph.
D.,
Senior
Scientist
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
ACTION
REQUESTED
To
review
the
product
characterization
considerations
associated
with
the
approval
of
a
Section
3
registration
for
the
insect
control
proteins
Cry3Bb1
and
Cry1Ab
from
Bacillus
thuringiensis
as
expressed
in
maize
as
a
dual
trait
product.

BACKGROUND
Monsanto
corporation
has
submitted
a
registration
request
for
the
Cry1Ab
/
Cry3Bb1
 ­
endotoxins
as
a
combined
trait
product
in
maize
as
a
means
of
controlling
the
European
corn
borer
(
Ostrinia
nubilalis)
and
other
lepidopteran
pests
(
i.
e.,
Diatraea
grandosiella,
D.
crambidoides,,
Helicoverpa
zea,
Spodoptera
frugiperda,
Papaipema
nebris)
and
the
corn
rootworm
complex,
Diabrotica
spp.
These
products
are
based
on
the
insecticidal
protein
of
B.
thuringiensis
strain
HD­
1
/
HD­
73
for
Cry1Ab
protein
and
strain
EG11098,
a
modified
Cry3Bb1
2
protein,
which
represents
the
active
ingredient
of
this
plant­
incorporated
protectant.
The
combined
trait
product
will
be
known
as
YieldGard
®
Plus
Corn
and
was
developed
through
traditional
breeding
of
maize
plants
containing
cry1Ab
(
MON
810)
and
cry3Bb1
(
MON
863).
Maize
plants
also
express
neomycin
phosphotransferase
(
nptII)
which
was
used
for
tolerance
to
the
antibiotic
kanamycin
during
in
vitro
culture
and
selection.

DATA
REVIEW
RECORD
Active
Ingredient:
Bacillus
thuringiensis
Cry1Ab
/
Cry3Bb1
protein
as
expressed
in
maize
Product
Name:
Event
MON
810
x
MON
863,
Cry1Ab
/
Cry3Bb1
proteins
as
expressed
in
maize
ID
No.:
000524­
LUL
Submission
No.:
S627345
Chemical
No.:
006484
Bacillus
thuringiensis
Cry3Bb1
protein
and
genetic
material
for
its
production.
DP
Barcode:
D287601
Sponsor:
Monsanto
Corporation,
700
Chesterfield
Parkway,
St.
Louis,
MO
63198
MRID/
Study
Titles:
457917­
01
­
Confirmation
of
the
molecular
identity
of
YieldGard
®
and
corn
rootworm
protected
combines
trait
corn
hybrid
MON
810
x
MON
863
by
Southern
blot
analysis.
457917­
02
­
Cry3Bb1,
Cry1Ab
and
NPTII
protein
levels
in
the
dual
trait
maize
hybrid
MON
863
x
MON
810
produced
in
Argentina
field
trials
conducted
during
the
1999­
2000
growing
season.
Data
Waiver
Request:
Acute
toxicity
and
irritation
data
requirements
for
the
end­
use
product,
YieldGard
®
Plus
Corn
seed
containing
Cry1Ab
and
Cry3Bb1
proteins
from
Bacillus
thuringiensis.

SUMMARIES
OF
REVIEW
MRID#
457917­
01
­
Confirmation
of
the
molecular
identity
of
YieldGard
®
and
corn
rootworm
protected
combines
trait
corn
hybrid
MON
810
x
MON
863
by
Southern
blot
analysis.
The
corn
hybrid
MON
810
x
MON
863
containing
the
two
transformation
events,
MON
810
(
cry1Ab)
and
MON
863
(
cry3Bb1)
was
examined
for
the
presence
of
these
two
genes
encoding
 ­
endotoxins
in
the
resulting
hybrid.
Probes
for
the
cry1Ab
and
cry3Bb1
genes
were
obtained
from
previous
studies
and
corresponded
to
the
first
900
bp
(
cry1Ab)
or
the
entire
length
of
the
gene
(
cry3Bb1).
The
radiolabelled
(
32P)
probe
for
cry1Ab
hybridized
to
restricted
DNA
samples
on
nylon
membranes
and
resulted
in
a
lack
of
any
signal
detection
for
the
samples
from
MON
846
(
non­
transgenic)
and
MON
863
samples,
but
did
detect
the
presence
of
cry1Ab
in
the
MON
810
and
MON
810
x
MON
863
hybrid
plant
samples.
When
DNA
samples
were
probed
3
with
the
cry3Bb1
sequence,
hybridization
confirmed
the
presence
of
this
gene
in
MON
863
plants
and
the
MON
810
x
MON
863
hybrid,
but
again
failed
to
detect
the
presence
of
this
gene
in
the
negative
control
MON
846.
Plasmid
DNA
from
plasmids
containing
either
gene
separately
did
react
positively
with
the
appropriate
probes
when
the
restricted
plasmid
DNA
was
coelectrophoresed
with
MON
846
DNA
and
hybridized
with
the
respective
probe.
From
these
results
it
is
evident
that
the
hybrid
MON
810
x
MON
863
contains
the
cry1Ab
and
cry3Bb1
genes.
Additionally,
the
restriction
patterns
noted
on
the
Southern
blot
provided
suggest
that
there
have
been
no
major
alterations
or
rearrangements
in
the
conventional
cross
of
these
two
events
(
hybrids)
for
these
two
gene
inserts.

457917­
02
­
Cry3Bb1,
Cry1Ab
and
NPTII
protein
levels
in
the
dual
trait
maize
hybrid
MON
863
x
MON
810
produced
in
Argentina
field
trials
conducted
during
the
1999­
2000
growing
season.
The
hybrid
maize
resulting
from
the
MON
863
x
MON
810
cross
represents
the
test
substance
for
this
study.
This
dual
trait
or
stacked
trait
hybrid
(
also
known
as
MT­
02)
results
from
the
contribution
of
the
cry3Bb1
and
nptII
genes
from
MON
863
and
the
cry1Ab
gene
from
MON
810.
The
Cry3Bb1
protein
from
MON
863
is
also
known
as
Cry3Bb1.11098,
but
will
be
referred
to
in
this
review
as
simply
Cry3Bb1.
Reference
protein
Cry3Bb1standard
for
the
ELISA
was
produced
by
fermentation
of
Bacillus
thuringiensis
encoding
Cry3Bb1.11231
and
stored
as
a
lyophilized
powder.
Cry1Ab
protein,
produced
in
E.
coli,
was
trypsinized
and
established
as
the
reference
standard.
The
coding
sequence
for
nptII
was
cloned
and
transferred
to
E.
coli
for
fermentation.
Four
field
sites
within
the
province
of
Buenos
Aires
(
Argentina)
were
used
to
culture
plants
for
sample
generation:
Fontezuela
1,
Fontezuela
2,
Salto
and
Rojas.
Young
leaves,
pollen,
mature
root,
over­
season
root,
grain
and
forage
samples
were
taken
for
ELISA
analysis.
For
each
protein,
a
validated
double
antibody
sandwich
ELISA
has
been
developed.
Antibodies
to
these
proteins
were
raised
in
rabbits
and
Protein
A
selected.
The
trypsinized
core
of
Cry1Ab
was
used
rather
than
the
full
length
protein
for
antibody
generation.
For
Cry3Bb1
and
NPTII
protein
levels,
a
tissue
specific
correction
factor
was
used
to
optimize
the
accuracy
and
reduce
bias
in
the
estimation
of
protein
content
of
samples
based
upon
extraction
efficiency
and
recovery.
Samples
for
forage,
grain
and
root
analysis
of
Cry3Bb1
protein
content
were
stored
longer
than
their
intended
period
of
known
stability
in
some
instances.
An
increase
in
apparent
Cry3Bb1
protein
levels
resulted
from
the
estimated
10
to
20
%
instability
evidenced
in
these
samples
stored
beyond
their
intended
dates
of
analysis.
The
net
result
is
that
the
Cry3Bb1
levels
reported
may
be
overestimated
by
as
much
as
20
%.
Data
reported
from
forage
samples
were
not
adjusted
as
presented
for
this
instability
factor.
Root
tissues
were
estimated
to
exhibit
a
20
to
30
%
instability
which
resulted
in
a
decrease
in
apparent
Cry3Bb1
levels
in
roots.
Hence,
the
Cry3Bb1
levels
reported
may
be
underestimated
by
up
to
30
%.
Data
from
grain
samples
were
estimated
to
be
overestimated
by
up
to
20
%
due
to
instability
in
stored
samples.
The
reported
root
and
grain
data
were
not
adjusted,
as
reported,
for
this
instability
factor.
Data
regarding
the
stability
or
instability
of
extracts
stored
beyond
their
intended
expiration
dates
were
not
presented.
The
ranges
of
protein
levels
observed
for
Cry3Bb1,
Cry1Ab
and
NPTII
were
similar
across
all
four
sites
for
the
MON
863
x
MON
810
hybrid
and
the
MON
863
or
MON
810
hybrids
as
appropriate
for
the
4
transgene
in
question.
Averages
for
the
Cry3Bb1
and
Cry1Ab
proteins
were,
however,
somewhat
higher
in
the
MON
863
x
MON
810
hybrid
versus
the
single
trait
hybrids.
NPTII
protein
levels
and
ranges
in
the
MON
863
x
MON
810
hybrid
and
the
MON
863
hybrid
were
similar.
The
highest
levels
of
expression
of
Cry3Bb1
or
Cry1Ab
were
found
to
occur
in
pollen
for
Cry3Bb1
(
79.6
µ
g/
g
FWT)
and
in
leaf
(
17.9
µ
g/
g
FWT)
for
Cry1Ab.

Data
Waiver
Request:
Acute
toxicity
and
irritation
data
requirements
for
the
end­
use
product,
YieldGard
®
Plus
Corn
seed
containing
Cry1Ab
and
Cry3Bb1
proteins
from
Bacillus
thuringiensis
­
The
registrant
requests
a
waiver
for
the
following
studies:
Acute
Oral
Toxicity
(
Guideline
Reference
No.
152­
30);
Acute
Dermal
Toxicity
(
Guideline
Reference
No.
152­
31);
Acute
Inhalation
Toxicity
(
Guideline
Reference
No.
152­
32);
Primary
Dermal
Irritation
(
Guideline
Reference
No.
152­
34);
Primary
Eye
Irritation
(
Guideline
Reference
No.
152­
35);
Hypersensitivity
(
Guideline
Reference
No.
152­
36).
The
combination
of
a
lack
of
human
exposure
to
these
proteins
as
expressed
in
maize
and
a
safe
history
for
 ­
endotoxins
derived
from
Bacillus
thuringiensis
are
the
main
reasons
that
waivers
were
requested.
All
of
the
above
studies
which
are
required
for
registration
of
a
biological
pesticide
may
be
waived
due
to
lack
of
human
exposure
to
the
insecticidal
proteins,
or
in
the
case
of
the
acute
oral
toxicity
test,
previous
evaluation
of
these
proteins.
Since
the
primary
route
of
exposure
for
these
proteins,
Cry1Ab
and
Cry3Bb1,
is
through
consumption
of
the
maize
grain
or
forage
expressing
these
proteins,
the
acute
oral
toxicity
tests
previously
reviewed
for
these
proteins
will
be
sufficient
to
conclude
that
there
is
no
acute
oral
toxicity
to
mammals
associated
with
consumption
of
these
proteins.
These
proteins
are
known
to
act
individually
to
effect
a
typical
midgut
pathology
in
susceptible
insects
which
is
characteristic
of
previously
studied
 ­
endotoxins
of
Bacillus
thuringiensis.
No
synergistic
action
or
interaction
of
these
proteins
is
known
or
expected
to
occur.
5
DATA
EVALUATION
REPORT
Reviewed
by:
Chris
A.
Wozniak,
Ph.
D.,
Biologist,
OPP
/
BPPD
Secondary
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist,
OPP
/
BPPD
MRID
No.:
457917­
01
Active
Ingredients:
Bacillus
thuringiensis
Cry1Ab
/
Cry3Bb1
protein
as
expressed
in
maize
Product
Name:
Event
MON
810
x
MON
863,
Cry1Ab
/
Cry3Bb1as
expressed
in
maize
Confidentiality:
No
claim
of
confidentiality
is
made
under
FIFRA
10(
d)(
1)(
A),
(
B)
or
(
C).
ID
No.:
000524­
LEI
Submission
No.:
S600950
Chemical
No.:
006484
Bacillus
thuringiensis
Cry3Bb
protein
and
genetic
material
for
its
production.
DP
Barcode:
D276610
Sponsor:
Monsanto
Corporation,
700
Chesterfield
Parkway,
St.
Louis,
MO
63198.
Authors:
I.
G.
Borovkov,
T.
A.
Cavato,
R.
P.
Lirette
Testing
Facility:
Monsanto
Corporation,
Product
Characterization
Center,
Biotechnology
Regulatory
Services,
700
Chesterfield
Parkway
North,
St.
Louis,
MO
63198
Study
Titles:
Confirmation
of
the
molecular
identity
of
YieldGard
®
and
corn
rootworm
protected
combines
trait
corn
hybrid
MON
810
x
MON
863
by
Southern
blot
analysis.
Study
Date:
October
19,
2001
Study
No.:
01­
01­
39­
39;
MSL
17466
Conclusion:
The
corn
hybrid
MON
810
x
MON
863
containing
the
two
transformation
events,
MON
810
(
cry1Ab)
and
MON
863
(
cry3Bb1)
was
examined
for
the
presence
of
these
two
genes
encoding
 ­
endotoxins
in
the
resulting
hybrid.
Probes
for
the
cry1Ab
and
cry3Bb1
genes
were
obtained
from
previous
studies
and
corresponded
to
the
first
900
bp
(
cry1Ab)
or
the
entire
1.96
kbp
length
of
the
gene
(
cry3Bb1).
The
radiolabelled
(
32P)
probe
for
cry1Ab
hybridized
to
DNA
samples
on
nylon
membranes
and
resulted
in
a
lack
of
any
signal
detection
for
the
samples
from
MON
846
(
non­
transgenic)
and
MON
863
samples,
but
did
detect
the
presence
of
cry1Ab
in
the
MON
810
and
MON
810
x
MON
863
hybrid
plant
samples.
When
DNA
samples
were
probed
with
the
cry3Bb1
sequence,
hybridization
confirmed
the
presence
of
this
gene
in
MON
863
plants
and
the
MON
810
x
MON
863
hybrid,
but
again
failed
to
detect
the
presence
of
this
gene
in
the
negative
control
MON
846.
Plasmid
DNA
from
plasmids
containing
either
gene
separately
did
react
positively
with
the
appropriate
probes
when
the
restricted
plasmid
DNA
was
co­
electrophoresed
with
MON
846
DNA
and
hybridized
with
the
respective
probe.
From
these
results
it
is
evident
that
the
hybrid
MON
810
x
MON
863
contains
the
cry1Ab
and
cry3Bb1
genes.
Additionally,
the
restriction
patterns
noted
on
the
Southern
blot
provided
6
suggest
that
there
have
been
no
major
alterations
or
rearrangements
in
the
conventional
cross
of
these
two
events
(
lines)
for
these
two
gene
inserts.
Classification:
Acceptable.

Good
Laboratory
Practices:
This
submission
was
conducted
in
compliance
with
the
Certification
of
Good
Laboratory
Practices
as
outlined
in
40
CFR
160.

Purpose:
To
describe
and
confirm
the
presence
of
the
combined
trait
genotype
(
i.
e.,
cry1Ab
and
cry3Bb1)
in
the
MON
810
x
MON
863
cross,
known
as
YieldGard
®
Plus.

METHODS:
Test
substance
­
The
corn
hybrid
MON
810
x
MON
863
(
lot#
TII­
0108­
11672­
S)
containing
the
two
transformation
events,
MON
810
(
cry1Ab)
and
MON
863
(
cry3Bb1).
Grain
was
from
Production
Plan
#
99­
04­
39­
01
and
was
stored
at
­
80

C
after
grinding.

Control
substances
­
Both
single
transformation
event
maize
lines,
MON
810
(
lot#
TIO­
0108­
11670­
S)
and
MON
863
(
lot#
TIO­
0108­
11669­
S),
as
well
as
a
non­
transformed
control
hybrid
MON
846
(
lot#
TNT­
0108­
11671­
S)
were
included
as
controls
for
comparison.
Grain
from
these,
as
produced
under
Production
Plan
#
99­
04­
39­
01,
were
ground
and
stored
at
­
80

C
until
needed.

Reference
substances
­
Plasmid
PV­
ZMBK07
was
included
as
a
positive
control
for
Southern
blotting
as
it
contains
the
cry1Ab
gene
as
present
in
MON
810.
Similarly,
plasmid
PV­
ZMIR13
was
used
in
Southern
blotting
as
a
positive
control
for
cry3Bb1,
as
found
in
event
MON
863.
Molecular
weight
markers
from
Roche
Diagnostics
(
Indianapolis,
IN)
were
used
to
estimate
DNA
size
following
hybridization.

All
control
and
test
substances
were
verified
by
event­
specific
PCR
for
MON
810
and
MON
863
prior
to
use.
Data
supporting
this
evidence
were
not
provided
in
this
submission.

DNA
extraction
­
Approximately
6
g
of
ground
grain
sample
was
added
to
a
conical
tube.
CTAB
extraction
buffer
(
1.5
%
CTAB,
75
mM
Tris
pH
8.0,
100
mM
EDTA
pH
8.0,
1.05
M
NaCl,
0.75
%
PVP
[
MW
40000];
16
mL)
was
added
to
each
tube
and
these
were
allowed
to
incubate
at
65

C
for
approximately
30
minutes.
Chloroform:
Isoamyl
alcohol
(
24:
1)
was
added
to
the
sample
after
allowing
5
minutes
for
sample
cooling.
Following
mixing
and
centrifugation
(
5
min.,
16,139
x
g),
the
upper
phase
(
aqueous)
was
transferred
to
a
new
conical
tube
and
combined
with
1.6
mL
of
CTAB
buffer.
An
equal
volume
of
Chloroform:
Isoamyl
alcohol
(
24:
1)
was
added
to
the
separated
phase
and
centrifugation
repeated.
The
upper
phase
was
removed
to
a
new
tube
and
DNA
precipitated
with
CTAB
precipitation
buffer.
DNA
was
pelleted
at
10,329
x
g
and
7
resuspended
in
2
mL
of
high
salt
TE
(
10
mM
Tris,
pH
8.0;
10
mM
EDTA,
pH
8.0,
1
M
NaCl)
and
incubated
for
2
h
at
37

C.
The
suspended
DNA
sample
was
then
centrifuged
(
23,240
x
g)
and
the
supernatant
transferred
to
new
tube.
Sodium
acetate
(
3
M)
and
ethanol
were
added
to
the
supernatant
to
reprecipitate
the
DNA.
Following
inversion
the
DNA
was
spooled
out
and
transferred
to
two
ethanol
washes,
then
centrifuged
to
pellet
and
vacuum
dried.
The
pellet
was
dissolved
in
TE
and
stored
at
4

C
.

DNA
digestion
­
Approximately
10
µ
g
of
genomic
DNA
was
digested
overnight
at
37

C
with
one
or
more
restriction
enzyme(
s).
Following
digestion,
the
DNA
was
precipitated
with
sodium
acetate
and
ethanol,
then
suspended
in
water.

DNA
probes
­
Probes
for
the
cry1Ab
and
cry3Bb1
genes
were
obtained
from
previous
studies
and
corresponded
to
the
first
900
bp
(
cry1Ab)
or
the
entire
length
(
1.96
kbp)
of
the
gene
(
cry3Bb1).
Each
probe
template
was
labeled
with
32P­
dCTP
(
6000
Ci/
mmol)
using
a
random
priming
kit
(
Gibco
BRL).

Southern
blot
analysis
­
DNA
samples
from
ground
grain
were
electrophoresed
on
an
0.8
%
agarose
gel
and
then
stained
with
ethidium
bromide.
The
samples
were
depurinated
with
a
0.125
N
HCl
solution
for
10
minutes,
then
denatured
with
a
0.5
M
NaOH,
1.5
M
NaCl
solution
for
30
minutes.
The
gel
was
then
neutralized
with
0.5
M
Tris­
HCl,
pH
7.0,
1.5
M
NaCl
for
30
minutes
and
eventually
equilibrated
in
20
X
SSC
transfer
buffer.
DNA
samples
were
then
transferred
to
Hybond­
N
membrane
in
20
x
SSC
for
20
hours.
Transferred
DNA
fragments
were
crosslinked
to
the
membrane
with
an
ultraviolet
light
treatment.

Pre­
hybridization
treatment
of
the
membrane
was
carried
out
for
3
hours
in
500
mM
disodium
phosphate
pentahydrate,
7
%
sodium
dodecyl
sulfate,
and
10
mg/
mL
tRNA
from
E.
coli.
Hybridization
was
performed
in
fresh
pre­
hybridization
solution
with
radiolabelled
probe
for
17
to
20
hours
at
65

C.
Membranes
were
subsequently
washed
in
0.1
%
SDS,
0.1
%
SSC
for
two
15
min
periods
and
two
20
min
periods
at
65

C.
Autoradiography
was
performed
using
a
Kodak
Biomax
intensifying
screen
and
Kodak
Biomax
MS­
2
film.

RESULTS:
MON
810
and
MON
863
event
specific
fingerprints
­
Restriction
enzymes
NcoI
and
EcoRI
were
used
to
digest
test
and
control
DNA
samples.
DNA
from
MON
846,
the
non­
transgenic
control,
was
mixed
with
plasmid
PV­
ZMBK07
and
digested
similarly.
This
plasmid
is
cleaved
at
three
sites
by
the
two
restriction
enzymes
and
one
of
the
fragments
produced
(
3.5
kbp)
would
be
detected
by
the
900
bp
probe
for
cry1Ab
coding
region.
The
radiolabelled
(
32P)
900
bp
probe
was
hybridized
to
DNA
samples
on
a
nylon
membrane
and
resulted
in
a
lack
of
any
signal
detection
for
the
samples
from
MON
846
or
MON
863
plants.
This
was
expected
since
neither
of
these
genotypes
carry
the
cry1Ab
gene.
The
primary
signal
detected
was
from
a
3.5
kbp
band
present
in
the
lane
containing
the
restricted
PV­
ZMB07
DNA,
which
does
contain
the
cry1Ab
gene.
Less
intense
signal
was
detected
in
bands
hybridizing
with
the
probe
at
9.0,
6.5
and
6.0
kbp.
It
is
8
suggested
that
these
results
are
a
reflection
of
star
activity
by
the
restriction
enzymes
(
i.
e.,
nonspecific
cutting
by
enzymes
due
to
chemical
or
physical
conditions
present
at
the
time
of
digestion)
or
an
incomplete
digestion
by
the
enzymes.
Some
non­
specific
hybridization
was
reported
as
well
around
4.4
kbp
in
between
lanes
1
and
2
of
the
Southern
blot.
The
shape
of
the
signal
as
it
appears
following
autoradiography
indicates
that
it
is
not
due
to
direct
hybridization
with
an
electrophoresed
band
or
fragment
of
DNA.
The
most
likely
explanation
for
this
aberrant
signal
is
a
non­
specific
binding
of
the
probe
to
the
membrane
where
incomplete
blocking
of
the
surface
occurred.

DNA
samples
from
MON
810
and
MON
810
x
MON
863
plants
produced
the
predicted
3.1
kbp
fragment
as
a
hybridization
signal.
The
size
of
this
fragment
for
the
cry1Ab
coding
region
differs
from
that
expected
from
the
digestion
of
the
plasmid
PV­
ZMB07
due
to
the
loss
of
an
EcoRI
site
on
the
integrated
gene
or
construct.
This
was
previously
noted
for
MON
810
according
to
internal
Monsanto
documents.
As
a
result,
the
EcoRI
enzyme
cleaves
the
DNA
adjacent
to
the
insert
in
the
host
flanking
chromosomal
DNA.

EcoRV
was
used
to
test
for
the
presence
of
the
MON
863
event
in
test
and
control
samples.
DNA
from
MON
846
was
mixed
with
plasmid
DNA
PV­
ZMIR13
and
restricted
with
EcoRV.
This
enzyme
cleaves
PV­
ZMIR13
once
producing
a
7.3
kb
fragment
which
will
hybridize
with
the
probe
for
the
coding
region
of
MON
863
gene
cry3Bb1.
When
the
nylon
membrane
was
probed
using
a
1.96
kbp
radiolabelled
(
32P)
probe
comprising
the
cry3Bb1
coding
region,
hybridization
signal
was
detected
in
a
band
at
9.6
kbp
in
lanes
containing
DNA
from
MON
863
and
MON
810
x
MON
863
plants.
This
fragment
size
differs
from
that
predicted
by
EcoRV
digestion
of
the
plasmid
PV­
ZMBIR13
(
7.3
kbp)
as
the
second
EcoRV
site
which
results
in
fragment
production
from
digestion
of
the
genomic
DNA
must
come
from
the
flanking
DNA
near
the
inserted
cry3Bb1
gene
(
i.
e.,
the
plasmid
contains
only
a
single
EcoRV
site).
DNA
samples
from
MON
810
or
the
nontransgenic
MON
846
did
not
produce
any
detectable
hybridization
signal
when
probed
similarly.
This
was
expected
from
previous
studies
conducted
in­
house
by
Monsanto
and
is
predictable
from
the
plasmid
restriction
map
provided
with
this
submission.

From
these
results
it
is
evident
that
the
hybrid
MON
810
x
MON
863
contains
the
cry1Ab
and
cry3Bb1
genes.
Additionally,
the
restriction
patterns
noted
on
the
Southern
blot
provided
suggest
that
there
have
been
no
major
alterations
or
rearrangements
in
the
conventional
cross
of
these
two
events
(
hybrids)
for
these
two
gene
inserts.

DATA
EVALUATION
REPORT
9
Reviewed
by:
Chris
A.
Wozniak,
Ph.
D.,
Biologist,
OPP
/
BPPD
Secondary
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist,
OPP
/
BPPD
MRID
No.:
457917­
02
Active
Ingredients:
Bacillus
thuringiensis
Cry1Ab
/
Cry3Bb1
protein
as
expressed
in
maize
Product
Name:
Event
MON863
x
MON
810,
Cry3Bb1
/
Cry1Ab
as
expressed
in
maize
Confidentiality:
No
claim
of
confidentiality
is
made
under
FIFRA
10(
d)(
1)(
A),
(
B)
or
(
C).
ID
No.:
000524­
LEI
Submission
No.:
S600950
Chemical
No.:
006484
Bacillus
thuringiensis
Cry3Bb
protein
and
genetic
material
for
its
production.
DP
Barcode:
D276610
Sponsor:
Monsanto
Corporation,
700
Chesterfield
Parkway,
St.
Louis,
MO
63198.
Authors:
Y.
A.
Dudin,
B­
P
Tonnu,
R.
P.
Lirette
Testing
Facility:
Monsanto
Corporation,
Product
Characterization
Center,
Biotechnology
Regulatory
Services,
700
Chesterfield
Parkway
North,
St.
Louis,
MO
63198
Study
Titles:
Cry3Bb1,
Cry1Ab
and
NPTII
protein
levels
in
the
dual
trait
maize
hybrid
MON
863
x
MON
810
produced
in
Argentina
field
trials
conducted
during
the
1999­
2000
growing
season.
Study
Date:
December
12,
2001
Study
No.:
00­
01­
39­
44;
MSL
17266
Conclusion:
ELISA
values
for
field
grown
maize
samples
(
grain,
forage,
root,
leaf
and
pollen)
were
examined
with
a
double
antibody
sandwich
technique
and
Cry3Bb1
and
NPTII
protein
values
adjusted
for
method
bias
to
optimize
the
accuracy
based
upon
extraction
efficiency,
and
for
variance
in
storage
stability
of
extracts
in
some
instances.
The
ranges
of
protein
levels
observed
for
Cry3Bb1,
Cry1Ab
and
NPTII
were
similar
across
all
four
field
sites
for
the
MON
863
x
MON
810
hybrid
and
the
MON
863
or
MON
810
hybrids
as
appropriate
for
the
transgene
in
question.
Averages
for
the
Cry3Bb1
and
Cry1Ab
proteins
were,
however,
somewhat
higher
in
the
MON
863
x
MON
810
hybrid
versus
the
single
trait
hybrids.
NPTII
protein
levels
and
ranges
in
the
MON
863
x
MON
810
hybrid
and
the
MON
863
hybrid
were
similar.
The
highest
average
levels
of
expression
of
Cry3Bb1
or
Cry1Ab
were
found
to
occur
in
pollen
for
Cry3Bb1
(
79.6
µ
g/
g
FWT)
and
in
leaf
(
17.9
µ
g/
g
FWT)
for
Cry1Ab.
Classification:
Acceptable.

Good
Laboratory
Practices:
This
submission
was
conducted
in
compliance
with
the
Certification
of
Good
Laboratory
Practices
as
outlined
in
40
CFR
160.
10
Purpose:
To
describe
the
levels
of
Cry3Bb1,
Cry1Ab
and
NPTII
proteins
in
various
tissues
collected
from
the
insect­
protected
MON
863
x
MON
810
maize
hybrid.
The
plants
tested
resulted
from
a
multi­
site
field
trial
in
Argentina
during
the
1999­
2000
growing
season.

METHODS:
Test
substance
­
The
hybrid
maize
resulting
from
the
MON
863
x
MON
810
cross
represents
the
test
substance
for
this
study.
This
dual
trait
or
stacked
trait
hybrid
(
also
known
as
MT­
02)
results
from
the
contribution
of
the
cry3Bb1
and
nptII
genes
from
MON
863
and
the
cry1Ab
gene
from
MON
810.
The
Cry3Bb1
protein
from
MON
863
is
also
known
as
Cry3Bb1.11098,
but
will
be
referred
to
in
this
review
as
simply
Cry3Bb1.

Control
substances
­
MON
863,
MON
810
and
MON
846
represent
the
hybrids
used
as
controls
for
this
analysis.
The
last
hybrid
has
a
genetic
background
similar
to
that
of
the
other
hybrids,
but
does
not
contain
the
cry3Bb1,
cry1Ab
or
nptII
genes.
Event
specific
PCR
was
conducted
to
ascertain
the
presence
of
the
transgenes
in
MON
863,
MON
810
and
to
confirm
the
absence
of
transgenes
in
MON
846.
Additionally,
other
events
which
were
planted
in
the
field
at
the
time
of
hybrid
production
were
screened
for
in
these
hybrids
to
ensure
their
absence
in
the
test
and
control
substances
(
hybrids).

Reference
substances
­
Purified
Cry3Bb1,
Cry1Ab
and
NPTII
were
used
as
protein
standards
in
the
ELISA
assays.

Cry3Bb1
protein
standard
­
Variant
Cry3Bb1.11231
(
lot#
6312812)
protein
was
used
as
a
standard
in
the
Cry3Bb1
ELISA.
This
variant
differs
from
the
wild
type
Cry3Bb1
by
four
amino
acids
from
the
wild
type
form.
This
variant
was
used
as
a
standard
in
the
ELISA
assays.
The
variant
of
the
Cry3Bb1
protein
used
in
the
MON
863
event
differs
from
the
Cry3Bb1.11231
form
by
3
amino
acids
and
from
the
wild
type
by
seven
amino
acids.
The
variant
in
MON
863
(
Cry3Bb1.11098)
and
the
Cry3Bb1.11231
proteins
share
>
99%
homology.
The
polyclonal
antibodies
used
in
the
ELISA
were
produced
against
the
wild
type
form
of
Cry3Bb1.

Reference
protein
standard
for
the
ELISA
was
produced
by
fermentation
of
Bacillus
thuringiensis
encoding
Cry3Bb1.11231
and
stored
as
a
lyophilized
powder.
This
variant
and
the
one
found
in
MON
863
were
previously
shown
to
be
physicochemically
and
functionally
equivalent.
This
lyophilized
powder
was
prepared
in
50
mM
sodium
bicarbonate
with
0.05
%
(
v/
v)
Tween­
20,
pH
9.6
and
assigned
lot
#
6307780.
Storage
was
at
­
80
C.
Amino
acid
composition
analysis
was
used
to
estimate
the
concentration
of
this
protein
to
be
0.291
mg/
mL
and
purity
was
found
to
be
93.2
%
by
SDS­
PAGE
and
densitometry.
Adjusting
for
the
purity
of
the
preparation,
the
final
concentration
of
the
Cry3Bb1
protein
in
the
assay
was
0.271
mg/
mL.

Cry1Ab
protein
standard
­
Cry1Ab
protein
(
lot#
6700409),
produced
in
E.
coli,
was
trypsinized
and
established
as
the
reference
standard.
Purity
was
determined
as
94
%
by
SDS­
PAGE
and
densitometry.
Total
protein
concentration
was
determined
to
be
3.41
mg/
mL
by
amino
acid
11
composition
analysis,
but
following
adjustment
for
purity,
the
final
concentration
is
3.21
mg/
mL.
Aliquots
of
this
protein
were
stored
at
­
80

C
in
50
mM
carbonate­
bicarbonate
(
pH
10.25)
buffer
and
50
mM
sodium
chloride.

NPTII
protein
standard
­
The
coding
sequence
for
nptII
(
neomycin
phosphotransferase)
was
cloned
and
transferred
to
E.
coli
for
fermentation.
Lyophilized
protein
powder
(
lot#
4821020)
was
solubilized
in
50
mM
carbonate­
bicarbonate
buffer
and
assigned
lot
#
6340505
and
stored
at
­
80

C.
Amino
acid
composition
analysis
was
used
to
estimate
the
protein
concentration
at
1.15
mg/
mL
and
the
purity
was
determined
as
99.5
%
based
upon
SDS­
PAGE
and
subsequent
densitometry.
No
adjustment
for
purity
was
performed.

Generation
of
plant
samples
for
analysis
­
Four
field
sites
within
the
province
of
Buenos
Aires
(
Argentina)
were
used
to
culture
plants
for
sample
generation:
Fontezuela
1,
Fontezuela
2,
Salto
and
Rojas.
Agronomic
practices
and
field
conditions
were
typical
for
maize
production
at
the
four
sites.
A
randomized
complete
block
design
was
used
with
two
row
plots
and
four
replications
per
site
All
samples
collected
were
maintained
on
dry
ice
within
30
minutes
of
collection.
Grain,
however,
was
handled
at
ambient
temperatures.

Young
leaf
­
Young
leaves
(
YL)
were
pooled
from
15
plants
at
the
V­
4
growth
stage
(
approximately
18
days
post
planting)
for
each
of
the
four
replications.

Forage
­
Two
entire
plants,
including
both
aerial
and
root
portions,
were
harvested
from
each
of
the
four
replicates
for
forage
analysis.
Plants
were
taken
at
the
early
dent
stage,
approximately
90
days
post
planting.
Plant
material
was
cut
into
10
to
15
cm
segments
and
stored
frozen
on
dry
ice.

Mature
root
and
over­
season
root
­
The
root
mass
from
two
plants
collected
at
the
same
time
as
the
forage
samples
to
represent
the
mature
root
samples.
Only
one
sample
of
over­
season
roots
was
collected
at
46
days
post
planting
for
Cry3Bb1
analysis.

Pollen
­
Pollen
samples
were
collected
at
all
sites
approximately
60
days
post
planting
which
represents
50
%
pollen
shed.
Test
and
control
samples
were
prepared
by
collecting
20
to
25
g
of
pollen
from
10
to
15
plants
for
each
of
the
four
replicated
plots
at
each
of
the
four
sites.

Grain
­
Self­
pollinated
primary
ears
were
collected
from
all
four
sites
(
four
replicates
/
site)
and
air
dried
to
moisture
below
15
%.
Grain
was
collected
at
maturity
(
approximately
117
days
post
planting).

Sample
storage,
processing
and
shipping
­
Storage
and
processing
of
plant
samples
occurred
at
both
Fontezuela,
Argentina
and
St.
Louis,
MO,
USA.
Tissue
samples
were
handled
on
dry
ice
and
stored
frozen,
however,
grain
samples
were
handled
at
ambient
temperatures.
Samples
shipped
to
St.
Louis
were
shipped
on
dry
ice
and
stored
at
­
80

C
upon
receipt.
All
tissues,
except
pollen,
were
processed
prior
to
extraction.
Samples
processed
in
Argentina
were
stored
at
­
80

C
and
12
then
shipped
on
dry
ice
to
St.
Louis
for
further
analysis.

ELISA
analytical
methods
­
Extraction
of
protein
from
maize
tissues
­
Control
samples
were
extracted
prior
to
test
samples
to
minimize
contamination.
Forage
tissues
were
extracted
with
triborate
buffer
containing
0.2
%
(
w/
v)
ascorbic
acid
(
TBA)
at
a
1:
50
(
g/
mL)
ratio.
Grain
was
extracted
at
a
1:
100
ratio
(
g/
mL)
in
phosphate
buffered
saline
with
Tween­
20
(
PBST).
Pollen
samples
are
extracted
at
a
1:
40
ratio
using
TBA
for
Cry1Ab
and
PBST
with
0.1
%
BSA
(
bovine
serum
albumin)
for
Cry3BB1
analysis.
Leaf
samples
analyzed
for
NPTII
and
Cry3Bb1
proteins
were
processed
in
PBST
with
0.1
%
BSA
at
a
1:
100
(
g/
mL)
ratio
and
in
PBST
at
a
1:
50
ratio
for
CryAb1.
Mature
root
and
over­
season
root
were
extracted
in
PBST
with
0.1
%
(
w/
v)
BSA
at
a
1:
100
(
g/
mL)
ratio
for
Cry3Bb1
using
a
Polytron
®
homogenizer
(
Brinkmann
Instruments).
Pollen
was
extracted
with
a
Wheaton
2000
homogenizer
for
the
smaller
sample
sizes
associated
with
this
tissue.
Insoluble
materials
were
removed
by
use
of
a
Serum
Filter
System
(
Fisher
Scientific)
or
centrifugation.
Clarified
extracts
were
stored
at
­
80

C.

Cry3Bb1,
Cry1Ab
and
NPTII
protein
ELISA
­
For
each
protein,
a
validated
double
antibody
sandwich
ELISA
has
been
developed.
The
validation
data
is
presented
in
the
appendix
2
of
this
volume.
For
Cry3Bb1
protein
the
following
LODs
are:
0.087
µ
g/
g
FWT
for
leaf,
0.22
µ
g/
g
FWT
for
forage,
0.096
µ
g/
g
FWT
for
grain
and
0.76
µ
g/
g
FWT
for
roots.
The
LOD
for
pollen
was
estimated
from
non­
transgenic
field
samples
to
be
0.7
µ
g/
g.
For
Cry1Ab
protein
the
following
LODs
were
established:
0.55
µ
g/
g
FWT
for
leaf,
0.26
µ
g/
g
FWT
for
forage,
0.13
µ
g/
g
FWT
for
grain,
and
0.08
µ
g/
g
FWT
for
pollen.
The
LODs
for
NPTII
protein
were
established
at:
0.093
µ
g/
g
FWT
for
leaf,
0.075
µ
g/
g
FWT
for
forage,
and
0.076
µ
g/
g
FWT
for
grain.

Reagents
for
Cry3Bb1
ELISA
­
A
rabbit
anti­
Cry3Bb1
antibody
was
purified
by
Protein
A
selection
and
used
as
the
capture
antibody.
Two
lots
of
coating
antibody
were
using
during
the
testing:
#
6199830A
and
#
6199830B,
at
concentrations
of
3.95
mg/
mL
and
3.21
mg/
mL,
respectively.
The
rabbit
polyclonal
antibody
was
also
conjugated
to
horseradish
peroxidase
(
HRP)
to
serve
as
the
detection
antibody.
The
final
concentration
of
this
antibody
was
1.1
mg/
mL
and
it
was
stored
in
a
buffer
containing
0.02
M
potassium
phosphate,
0.15
M
sodium
chloride,
and
0.01
%
thimerosal.

Reagents
for
Cry1Ab
ELISA
­
A
rabbit
anti­
Cry1Ab
antibody
was
purified
by
Protein
A
selection
and
used
as
the
capture
antibody.
One
lot
of
coating
antibody
was
using
during
the
testing:
#
6178568A
at
a
concentration
of
7.7
mg/
mL.
The
rabbit
polyclonal
antibody
was
also
conjugated
to
alkaline
phosphatase
(
AP)
to
serve
as
the
detection
antibody.
The
final
concentration
of
this
antibody
was
4.5
mg/
mL
and
it
was
stored
in
a
buffer
containing
50
mM
Tris­
HCl,
pH
8.0,
1
mM
magnesium
chloride,
1
%
BSA
and
0.1
%
sodium
azide.

Reagents
for
the
NPTII
ELISA
­
A
rabbit
anti­
NPTII
antibody
was
purified
by
Protein
A
selection
and
used
as
the
capture
antibody.
Two
lots
of
coating
antibody
were
using
during
the
13
testing:
#
6549463A
at
concentration
of
4.7
mg/
mL
and
3.21
mg/
mL,
respectively.
The
rabbit
polyclonal
antibody
was
also
conjugated
to
horseradish
peroxidase
to
serve
as
the
detection
antibody.
The
final
concentration
of
this
antibody
was
3.0
mg/
mL
and
it
was
stored
in
a
buffer
containing
0.02
M
potassium
phosphate,
0.15
M
sodium
chloride,
and
0.01
%
thimerosal.

Cry3Bb1
and
NPTII
ELISA
­
Polyclonal
antibody
preparations
for
these
two
proteins
were
diluted
in
carbonate­
bicarbonate
buffer
at
a
final
concentration
of
5.0
mg/
mL.
Microtiter
plate
wells
were
coated
overnight
at
4

C
with
100
µ
L
of
the
diluted
antibody
solutions
(
separately).
Plates
were
washed
in
PBST
three
times
prior
to
assay.
Antibody
binding
was
carried
out
at
37

C
for
60
to
70
minutes
using
50
µ
L
of
the
antibody
preparation
and
50
µ
L
of
the
secondary
conjugated
antibody
simultaneously.
Plates
were
then
washed
again
with
PBST
three
times.
Tetramethylbenzidine
(
TMB)
was
used
as
the
development
agent
for
these
reactions
at
room
temperature
for
10
to
15
minutes.
Reactions
were
terminated
with
6
M
phosphoric
acid.
Absorbance
readings
were
taken
at
450
nm
and
650
nm
as
reference
wavelengths
and
a
correlation
between
color
development
and
degree
of
secondary
antibody
binding
was
established
using
standard
calibration
curves.
A
seven
point
standard
curve,
ranging
from
1
to
64
ng/
mL,
for
each
ELISA
was
used
to
estimate
the
concentration
of
protein
in
the
ELISA
by
interpolation.

Cry1Ab
ELISA
­
Antibody
to
Cry1Ab
was
raised
in
rabbits
using
the
tryptic
core
of
the
protein
as
an
antigen.
The
antibody
was
diluted
in
plate
coating
buffer
and
add
to
wells
at
2.0
µ
g/
mL
in
250
µ
L
/
well
and
plates
were
incubated
overnight
at
4

C.
Prior
to
assay
plates
were
washed
two
times
with
PBST.
Tissue
extracts
were
digested
with
trypsin
prior
to
assay
(
175
µ
g/
mL)
and
the
reaction
terminated
by
the
addition
of
50
mM
phenylmethylsulfonyl
fluoride
to
a
final
concentration
of
1.21
mM.
Trypsinized
sample
extracts
were
incubated
(
200
µ
L)
overnight
at
4

C
with
50
µ
L
of
rabbit
anti­
Cry1Ab
antibody
conjugated
to
AP.
Plates
were
washed
three
times
with
PBST.
Wells
were
subsequently
developed
using
p­
nitrophenyl
phosphate
(
pNPP)
in
ethanolamine
buffer,
pH
9.7
for
20
to
25
minutes
at
room
temperature.
Absorbance
readings
were
taken
at
450
nm
and
650
nm
as
reference
wavelengths
and
a
correlation
between
color
development
and
degree
of
secondary
antibody
binding
was
established
using
standard
calibration
curves.
A
seven
point
standard
curve,
ranging
from
1
to
64
ng/
mL,
for
each
ELISA
was
used
to
estimate
the
concentration
of
protein
in
the
ELISA
by
interpolation.
The
standard
curve
ranged
from
0.4
to
16
ng/
mL.

To
estimate
the
final
concentration
of
Cry1Ab,
it
has
historically
required
a
multiplication
by
a
factor
of
2
to
account
for
the
difference
in
reaction
of
the
trypsinized
core
of
the
protein
versus
the
full
length
moiety.
No
data
was
presented
to
corroborate
this
statement.

Control
of
bias
­
A
tissue
specific
correction
factor
was
used
to
optimize
the
accuracy
and
reduce
bias
in
the
estimation
of
protein
content
of
samples.
The
following
equation
describes
the
calculations:
14
Bias
Correction
Factor
=
(%
extraction
efficiency)
x
(%
spike
and
recovery)
(
100)
(
100)

In
order
to
maintain
consistency
with
previously
reported
data,
the
Cry1Ab
data
were
not
adjusted
for
method
bias.
For
Cry3Bb1
and
NPTII
an
estimation
of
protein
content
in
a
sample
was
obtained
by
using
the
observed
µ
g/
g
FWT
divided
by
the
tissue
specific
method
bias
correction
factor
as
follows:

Observed
µ
g/
g
FWT
=
Corrected
µ
g/
g
FWT
Bias
Correction
Factor
Data
reduction
and
statistical
analysis
­
ELISA
plates
were
measured
for
absorbance
on
a
microplate
reader.
HRP
readings
were
taken
at
450
nm,
whereas
the
AP
absorbance
readings
were
taken
at
405
nm.
A
reference
wavelength
of
650
nm
was
used
in
both
cases;
this
value
was
then
subtracted
from
the
sample
reading
at
405
nm
or
450
nm
as
appropriate.
Data
reduction
analysis
was
performed
using
SOFTmax
PRO
version
2.4.1
(
Molecular
Devices,
Sunnyvale,
CA).
For
all
three
ELISA
methods,
a
four
parameter
logistic
curve
was
used
to
fit
the
data
for
absorbance
readings
of
standard
curve
measurements
and
sample
data.
Interpolating
from
the
standard
curve,
the
amount
of
protein
in
the
tissue
extracts
was
converted
to
µ
g/
g
FWT
using
dilution
factors,
tissue
to
buffer
ratios,
and
the
bias
correction
factors
described
above.

RESULTS:
Test
and
control
substance
characterization
­
Event
specific
PCR
was
used
to
characterize
grain
samples
harvested
from
all
sites.
PCR
was
used
to
identify
MON
863
and
MON
810
events
in
samples
as
well
as
other
events
present
in
the
fields
but
not
part
of
this
submission.
Grain
identity
was
confirmed
in
all
sites
with
one
exception
where
in
a
sample
from
the
MON
863
plants
and
one
from
the
MON
863
x
MON
810
hybrid
were
excluded
due
to
the
presence
of
a
contaminating
event.
For
the
control
hybrid,
MON
846,
13
of
16
grain
samples
were
determined
to
be
`
event
free'.
In
the
three
control
samples
excluded,
the
Cry3Bb1
and
NPTII
protein
levels
were
below
their
respective
LODs,
they
were
still
excluded
from
analysis.

Test
and
control
substance
stability
­
Some
of
the
Cry3Bb1
samples
(
tissues
and
extracts)
were
stored
beyond
their
recommended
date
of
storage.
From
previous
work
on
the
stability
of
Cry3Bb1
at
­
80

C,
it
was
possible
to
determine
the
method
bias
using
experimental
time
points
which
covered
the
time
frame
considered.
This
was
also
true
for
some
NPTII
sample
extracts.
All
tissues
and
extracts
were
stored
at
­
80

C
to
prevent
any
further
degradation
throughout
the
study.

Cry3Bb1
protein
stability
­
Samples
for
forage,
grain
and
root
analysis
of
Cry3Bb1
protein
content
were
stored
longer
than
their
intended
period
of
known
stability
in
some
instances.
An
analysis
of
forage
tissue
stability
using
time
points
(
i.
e.,
storage
periods)
similar
to
those
present
in
15
this
analysis.
An
increase
in
apparent
Cry3Bb1
protein
levels
resulted
from
the
estimated
10
to
20
%
instability
evidenced
in
these
samples
stored
beyond
their
intended
dates
of
analysis.
The
net
result
is
that
the
Cry3Bb1
levels
reported
may
be
overestimated
by
as
much
as
20
%.
Data
reported
from
forage
samples
were
not
adjusted
as
presented
for
this
instability
factor.
Root
tissues
were
estimated
to
exhibit
a
20
to
30
%
instability
which
resulted
in
a
decrease
in
apparent
Cry3Bb1
levels
in
roots.
Hence,
the
Cry3Bb1
levels
reported
may
be
underestimated
by
up
to
30
%.
Data
from
grain
samples
were
estimated
to
be
overestimated
by
up
to
20
%
due
to
instability
in
stored
samples.
The
reported
root
and
grain
data
were
not
adjusted,
as
reported,
for
this
instability
factor.

NPTII
protein
extract
stability
­
Grain
extracts
analyzed
for
NPTII
levels
were
initially
analyzed
during
the
stability
period
and
then
again
following
expiration
of
that
period.
The
first
data
set
was
accepted
with
one
deviation
from
the
accepted
protocol
(
SOP)
in
that
blank
readings
were
slightly
above
the
SOP
specification.
The
positive
and
negative
control
samples
were
found
to
be
within
their
acceptable
ranges
as
defined
by
the
SOP.
This
deviation
was
not
considered
to
have
compromised
the
analysis.
When
this
analysis
was
repeated,
all
of
the
ELISA
criteria
were
met
for
the
SOP,
however,
the
extracts
were
then
outside
the
stability
period.
This
second
data
set
confirmed
the
initial
data
set
in
that
the
levels
of
NPTII
were
determined
to
be
below
the
quantifiable
NPTII
protein
level.
The
first
data
set
was
then
used
as
a
valid
assessment
of
the
NPTII
protein
levels
observed
in
grain.

Two
forage
extracts
were
analyzed
beyond
their
known
stability
periods
to
determine
that
the
NPTII
levels
were
below
the
LOD.
A
second
analysis
reached
the
same
conclusion;
NPTII
levels
reported
for
the
MON
863
samples
were
determined
to
be
valid
estimates
of
this
protein's
levels
in
forage.

Protein
levels
in
maize
tissues
­
Average
values
are
reported
for
the
levels
of
Cry3Bb1,
Cry1Ab
and
NPTII
as
tabulated
below.
These
are
reported
as
µ
g/
g
FWT
with
a
total
number
of
samples
analyzed
being
16,
except
where
noted.
Average
values
and
standard
deviations
reported
omit
the
samples
found
to
be
below
the
LOD,
however,
these
samples
were
included
in
the
ranges
reported.

For
the
dual
trait
samples
analyzed
for
Cry1Ab
in
grain,
15
samples
were
analyzed
since
one
was
discarded
due
to
contamination.
Grain
analyzed
for
Cry3Bb1
in
MON
863
consisted
of
14
samples
as
one
was
omitted
for
contamination
and
one
was
found
to
be
below
the
LOD.
For
the
MON
863
x
MON
810
hybrid,
15
samples
were
analyzed
as
one
was
discarded
for
contamination.
Root
and
forage
data
for
the
MON
863
hybrid
consisted
of
13
plant
samples
as
three
samples
were
below
the
LOD.
For
both
the
MON
863
and
MON
863
x
MON
810
hybrids,
the
NPTII
data
set
included
15
extracts
as
one
sample
was
excluded
in
each
case
due
contamination.

Pollen
analyses
for
Cry3Bb1
protein
levels
in
MON
863
contained
16
samples
and
levels
were
similar
across
all
four
field
sites.

MON
846
(
non­
transgenic)
samples
were
not
included
in
the
Cry1Ab
ELISA
as
required
by
the
16
SOP.
A
negative
control
(
QC)
sample
was
included
in
every
ELISA
to
detect
any
problems
with
the
assay.
This
negative
QC
sample
was
within
the
guidelines
established
in
the
SOP.

Cry3Bb1
protein
levels
­
Levels
in
the
MON
863
x
MON
810
hybrid
presented
below
are
overestimates
of
Cry3Bb1
protein
levels
since
some
of
the
samples
were
beyond
their
documented
stability
period.

Tissue
Type
and
Collection
Time
(
Days
post
planting*)
Average
Cry3Bb1
protein
levels
(
µ
g/
g
FWT)

MON
863
x
MON
810
Average
Cry3Bb1
protein
levels
(
µ
g/
g
FWT)

MON
863
Young
leaf
(
18)
46.7
(
35.5
­
53.2)
30.0
(
21.3
­
47.2)

Forage
(
90)
23.6
(
6.7
­
39.7)
12.8
(<
0.22
­
28.8)

Grain
(
117)
61.1
(
38.5
­
83.1)
43.7
(<
0.096
­
84.1)

Pollen
(
60)
79.6
(
65.1
­
96.5)
60.4
(
29.7
­
90.7)

Mature
root
(
90)
19.7
(
6.0
­
41.7)
16.2
(<
0.76
­
49.8)

Over­
season
root
(
46)
22.0
(
N/
A)
20.0
(
N/
A)

*
Days
are
approximate.

Cry1Ab
protein
levels
­
Levels
in
the
MON
863
x
MON
810
hybrid
presented
below
are
overestimates
of
Cry1Ab
protein
levels
since
some
of
the
samples
were
beyond
their
documented
stability
period.

Tissue
Type
and
Collection
Time
(
Days
post
planting*)
Average
CryAb1
protein
levels
(
µ
g/
g
FWT)

MON
863
x
MON
810
Average
CryAb1
protein
levels
(
µ
g/
g
FWT)

MON
810
Young
leaf
(
18)
17.9
(
14.1
­
27.5)
13.0
(
9.8
­
15.4)

Forage
(
90)
7.9
(
3.9
­
11.9)
5.6
(
3.0
­
8.2)

Grain
(
117)
0.84
(
0.63
­
1.2)
0.46
(
0.24
­
0.77)

Pollen
(
60)
<
0.08
(<
0.08
­
0.18)
<
0.08
(<
0.08)

*
Days
are
approximate.
17
NPTII
protein
levels
­
Similar
levels
of
NPTII
protein
were
found
in
MON
863
and
MON
863
x
MON
810.

Tissue
Type
and
Collection
Time
(
Days
post
planting*)
Average
NPTII
protein
levels
(
µ
g/
g
FWT)

MON
863
x
MON
810
Average
NPTII
protein
levels
(
µ
g/
g
FWT)

MON
810
Young
leaf
(
18)
1.6
(
0.53
­
2.3)
1.06
(
0.58
­
1.6)

Forage
(
90)
0.19
(
0.13
­
0.27)
0.17
(<
0.075
­
0.33)

Grain
(
117)
<
0.076
(<
0.076)
<
0.076
(<
0.1)

*
Days
are
approximate.

Conclusions
­
The
ranges
of
protein
levels
observed
for
Cry3Bb1,
Cry1Ab
and
NPTII
were
similar
across
all
four
sites
for
the
MON
863
x
MON
810
hybrid
and
the
MON
863
or
MON
810
hybrids
as
appropriate
for
the
transgene
in
question.
Averages
for
the
Cry3Bb1
and
Cry1Ab
proteins
were,
however,
somewhat
higher
in
the
MON
863
x
MON
810
hybrid
versus
the
single
trait
hybrids.
NPTII
protein
levels
and
ranges
in
the
MON
863
x
MON
810
hybrid
and
the
MON
863
hybrid
were
similar.
For
Cry3Bb1
and
NPTII
protein
levels,
a
tissue
specific
correction
factor
was
used
to
optimize
the
accuracy
and
reduce
bias
in
the
estimation
of
protein
content
of
samples
based
upon
extraction
efficiency
and
recovery.
The
highest
average
levels
of
expression
of
Cry3Bb1
or
Cry1Ab
were
found
to
occur
in
pollen
for
Cry3Bb1
(
79.6
µ
g/
g
FWT)
and
in
leaf
(
17.9
µ
g/
g
FWT)
for
Cry1Ab.

DATA
EVALUATION
REPORT
18
Reviewed
by:
Chris
A.
Wozniak,
Ph.
D.,
Biologist,
OPP
/
BPPD
Secondary
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist,
OPP
/
BPPD
MRID
No.:
Administrative
Materials
Active
Ingredients:
Bacillus
thuringiensis
Cry1Ab
/
Cry3Bb1
protein
as
expressed
in
maize
Product
Name:
Event
MON863
x
MON
810,
Cry3Bb1
/
Cry1Ab
as
expressed
in
maize
Confidentiality:
No
claim
of
confidentiality
is
made
under
FIFRA
10(
d)(
1)(
A),
(
B)
or
(
C).
ID
No.:
000524­
LEI
Submission
No.:
S600950
Chemical
No.:
006484
Bacillus
thuringiensis
Cry3Bb
protein
and
genetic
material
for
its
production.
DP
Barcode:
D276610
Sponsor:
Monsanto
Corporation,
700
Chesterfield
Parkway,
St.
Louis,
MO
63198.
Authors:
B.
G.
Hammond,
W.
F.
Heydens,
D.
P.
Ward
Testing
Facility:
Monsanto
Company,
600
13th
Street
N.
W.,
Suite
660,
Washington,
D.
C.
20005
Study
Titles:
Data
Waiver
Request:
Acute
toxicity
and
irritation
data
requirements
for
the
end­
use
product,
YieldGard
®
Plus
Corn
seed
containing
Cry1Ab
and
Cry3Bb1
proteins
from
Bacillus
thuringiensis.
Study
Date:
August
16,
2002
Study
No.:
MSL­
18126
Conclusion:
The
registrant
requests
a
waiver
for
the
following
studies:
Acute
Oral
Toxicity
(
Guideline
Reference
No.
152­
30);
Acute
Dermal
Toxicity
(
Guideline
Reference
No.
152­
31);
Acute
Inhalation
Toxicity
(
Guideline
Reference
No.
152­
32);
Primary
Dermal
Irritation
(
Guideline
Reference
No.
152­
34);
Primary
Eye
Irritation
(
Guideline
Reference
No.
152­
35);
Hypersensitivity
(
Guideline
Reference
No.
152­
36).
All
of
the
above
studies
which
are
required
for
registration
of
a
biological
pesticide
may
be
waived
due
to
lack
of
exposure
or
previous
evaluation
of
toxicity
in
the
case
of
oral
consumption
of
these
proteins.
Since
the
primary
route
of
exposure
for
these
proteins,
Cry1Ab
and
Cry3Bb1,
is
through
consumption
of
the
maize
expressing
these
proteins,
the
acute
oral
toxicity
tests
previously
reviewed
for
these
proteins
will
be
sufficient
to
conclude
that
there
is
no
acute
toxicity
associated
with
consumption
of
these
proteins.
These
proteins
are
known
to
act
individually
to
effect
a
typical
midgut
pathology
in
susceptible
insects
which
is
characteristic
of
previously
studied
 ­
endotoxins
of
Bacillus
thuringiensis.
No
synergistic
action
or
interaction
of
these
proteins
is
known
or
expected
to
occur.
Classification:
Acceptable.

Good
Laboratory
Practices:
This
submission
was
not
conducted
in
compliance
with
the
19
Certification
of
Good
Laboratory
Practices
as
outlined
in
40
CFR
160
as
it
falls
outside
the
scope
of
the
Good
Laboratory
Practice
Standards.
The
submission
represents
a
request
for
waiver
for
certain
toxicity
studies.

Purpose:
The
registrant
requests
a
waiver
for
the
following
studies:
Acute
Oral
Toxicity
(
Guideline
Reference
No.
152­
30);
Acute
Dermal
Toxicity
(
Guideline
Reference
No.
152­
31);
Acute
Inhalation
Toxicity
(
Guideline
Reference
No.
152­
32);
Primary
Dermal
Irritation
(
Guideline
Reference
No.
152­
34);
Primary
Eye
Irritation
(
Guideline
Reference
No.
152­
35);
Hypersensitivity
(
Guideline
Reference
No.
152­
36).

Data
Requirement:
Acute
Oral
Toxicity
(
Guideline
Reference
No.
152­
30)
­
In
previous
testing
of
the
Cry1Ab
and
Cry3Bb1
proteins
in
acute
oral
toxicity
studies
using
rodents
as
test
animals,
no
adverse
effects
were
noted
in
either
case.
The
maximum
dose
evaluated
was
4000
mg/
kg
body
weight
and
3200
mg/
kg
body
weight
for
the
Cry1Ab
and
Cry3Bb1
proteins,
respectively.
Neither
of
these
proteins
indicated
any
potential
for
adverse
effects
at
the
doses
tested,
which
far
exceed
those
found
in
a
plant­
incorporated
protectant.
The
probability
of
a
synergistic
reaction
between
the
two
proteins
as
it
relates
to
mammalian
toxicity
seems
very
low.
Since
the
primary
route
of
exposure
for
these
proteins,
Cry1Ab
and
Cry3Bb1,
is
through
consumption
of
the
maize
expressing
these
proteins,
the
acute
oral
toxicity
tests
previously
reviewed
for
these
proteins
will
be
sufficient
to
conclude
that
there
is
no
acute
toxicity
associated
with
consumption
of
these
proteins.
These
proteins
are
known
to
act
individually
to
effect
a
typical
midgut
pathology
in
susceptible
insects
which
is
characteristic
of
previously
studied
 ­
endotoxins
of
Bacillus
thuringiensis.
No
synergistic
action
or
interaction
of
these
proteins
is
known
or
expected
to
occur.
From
studies
on
other
 ­
endotoxins
from
Bacillus
thuringiensis
we
do
know
that
these
insecticidal
proteins
specifically
target
unique
receptors
to
effect
a
mid­
gut
pathology
typical
of
the
insecticidal
proteins
from
this
bacterium
and
that
competitive
binding
or
cross
resistance
from
these
two
classes
of
crystal
proteins
does
not
occur.

Acute
Dermal
Toxicity
(
Guideline
Reference
No.
152­
31)
­
The
proteins
Cry1Ab
and
Cry3Bb1
are
intended
to
be
produced
within
the
plant­
incorporated
protectant
YieldGard
®
Plus
maize.
As
such,
the
potential
for
exposure
of
the
skin
to
the
active
ingredient
proteins
is
negligible.
Additionally,
these
proteins
are
not
known
to
harbor
any
properties
which
would
suggest
that
they
represent
dermal
irritants
or
toxicants.

Acute
Inhalation
Toxicity
(
Guideline
Reference
No.
152­
32)
­
The
proteins
Cry1Ab
and
Cry3Bb1
are
intended
to
be
produced
within
the
plant­
incorporated
protectant
YieldGard
®
Plus
maize.
As
such,
the
potential
for
inhalation
exposure
to
the
active
ingredient
proteins
is
negligible.
Additionally,
these
proteins
are
not
known
to
harbor
any
properties
which
would
suggest
that
they
represent
pulmonary
irritants
or
toxicants.

Primary
Dermal
Irritation
(
Guideline
Reference
No.
152­
34)
­
The
proteins
Cry1Ab
and
Cry3Bb1
are
intended
to
be
produced
within
the
plant­
incorporated
protectant
YieldGard
®
Plus
20
maize.
As
such,
the
potential
for
exposure
of
the
skin
to
the
active
ingredient
proteins
is
negligible.
Additionally,
these
proteins
are
not
known
to
harbor
any
properties
which
would
suggest
that
they
represent
dermal
irritants
or
toxicants.

Primary
Eye
Irritation
(
Guideline
Reference
No.
152­
35)
­
The
proteins
Cry1Ab
and
Cry3Bb1
are
intended
to
be
produced
within
the
plant­
incorporated
protectant
YieldGard
®
Plus
maize.
As
such,
the
potential
for
exposure
of
the
eyes
to
the
active
ingredient
proteins
is
negligible.
Additionally,
these
proteins
are
not
known
to
harbor
any
properties
which
would
suggest
that
they
represent
ocular
irritants
or
toxicants.

Hypersensitivity
(
Guideline
Reference
No.
152­
36)
­
The
proteins
Cry1Ab
and
Cry3Bb1
are
intended
to
be
produced
within
the
plant­
incorporated
protectant
YieldGard
®
Plus
maize.
As
such,
the
potential
for
exposure
of
the
skin
to
the
active
ingredient
proteins
is
negligible.
Additionally,
these
proteins
are
not
known
to
harbor
any
properties
which
would
suggest
that
they
represent
dermal
irritants,
toxicants
or
sensitizers.