32
DATA
EVALUATION
REPORT
6/
7/
2001
Reviewed
by:
Chris
A.
Wozniak,
Ph.
D.,
Biologist,
OPP
/
BPPD
/
s/
Secondary
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist,
OPP
/
BPPD
/
s/
MRID
No.:
452422­
12
Active
Ingredients:
Bacillus
thuringiensis
strain
PS149B1
(
NRRL
B­
21553)
binary
insect
control
proteins
as
expressed
in
maize
Product
Name:
PS149B1
insect
control
proteins
as
expressed
in
maize
ID
No.:
068467­
EUP­
G,
068467­
EUP­
L,
029964­
EUP­
R,
022964­
EUP­
G
Submission
No.:
S590323,
S590337
Chemical
No.:
006430
Bacillus
thuringiensis
PS149B1
proteins
DP
Barcode:
D271578
Sponsor:
Dow
Agrosciences
LLC,
9330
Zionsville
Rd.,
Indianapolis,
IN
46268.
Authors:
V.
A.
Korjagin,
A.
D.
Ernest
Testing
Facility:
Global
Environmental
Chemistry
Laboratory
­
Indianapolis
Lab,
Dow
Agrosciences
LLC,
9330
Zionsville
Rd.,
Indianapolis,
IN
46268­
1054
Study
Titles:
In
vitro
digestibility
of
PS149B1
proteins
Study
Date:
October
6,
2000
Study
No.:
000302
Conclusion:
Pseudomonas
fluorescens
strains
MR1253
and
1256
were
used
to
prepare
the
14
kDa
and
44
kDa
PS149B1
proteins,
respectively,
microbially
as
inclusion
bodies.
A
simulated
gastric
fluid
was
produced
to
determine
the
lability
of
these
proteins
in
an
acid
environment
containing
pepsin.
The
BSA
positive
control
was
rapidly
digested
(
within
1
min)
and
the
 ­
lactoglobulin
remained
intact
for
60
minutes,
as
expected,
the
duration
of
the
experiment.
The
14
kDa
protein
was
visible
on
the
SDS­
PAGE
gel
at
the
15
minute
sample
point,
but
not
afterwards.
It
was
detected
on
the
Western
blot,
which
has
greater
sensitivity,
at
the
20
minute
time
point,
but
not
in
later
sample
points.
A
band
was
also
noted
on
the
Western
blot
at
approximately
30
kDa.
This
band
was
noted
in
an
earlier
study
and
considered
to
be
the
result
of
protein
aggregation.
For
the
44
kDa
protein
Western
blot,
bands
were
observed
at
approximately
42
kDa
and
14
kDa.
A
single
band
was
observed
on
the
44
kDa
SDS­
PAGE
at
approximately
15
to
16
kDa.
These
bands
were
only
observed
at
the
one
minute
time
point,
but
not
afterwards.
It
is
concluded
that
both
ICP
proteins
are
susceptible
to
degradation
in
the
simulated
gastric
environment
and
differ
in
their
lability
to
these
conditions
(
i.
e.,
the
44
kDa
protein
is
more
rapidly
degraded).

Classification:
Acceptable.

Good
Laboratory
Practices:
This
study
was
conducted
in
compliance
with
the
Certification
of
Good
Laboratory
Practices
as
outlined
in
40
CFR
160,
with
the
following
exception:
the
commercially
available
control
/
reference
substances,
bovine
serum
albumin,
 ­
lactoglobulin
and
protein
molecular
weight
markers
were
not
obtained
/
maintained
under
GLP
systems.
33
Purpose:
To
determine
the
stability
of
the
PS149B1
proteins
in
a
simulated
gastric
assay.

METHODS
Test
substances
­
PS149B1
14
kDa
­
TSN102172
(
54
%
AI
(
w/
w)),
44
kDa
­
TSN
102171,
(
37
%
AI
(
w/
w)).
Test
substances
were
obtained
from
the
Test
Substance
Coordinator
at
DAS,
9330
Zionsville
Rd.,
Indianapolis,
IN.

Pseudomonas
fluorescens
strains
MR1253
and
1256
were
used
to
prepare
the
14
kDa
and
44
kDa
PS149B1
proteins,
respectively,
microbially
as
inclusion
bodies.
The
extracted
inclusions
were
washed
and
lyophilized,
then
sent
to
the
DAS
Indianapolis
Test
Substance
Coordinator.
ELISA
assays
were
conducted
before
the
first
experiment
and
after
the
last
experiment
to
demonstrate
that
the
test
substances
were
stable
over
the
course
of
the
study.

Control
substances
­
Bovine
serum
albumin
was
used
as
a
positive
control
since
it
is
known
to
breakdown
readily
in
simulated
gastric
assays.
 ­
lactoglobulin
was
chosen
because
it
fails
to
degrade
rapidly
in
simulated
gastric
assays.

Reference
substances
­
Molecular
weight
markers
were
obtained
from
Bio­
Rad
(
cat.#
161­
0324)
and
Novex
(
cat.#
LC5677)
for
use
in
SDS­
PAGE.

Test
methods
­
Test
and
control
substance
solutions
were
prepared
in
equimolar
concentrations
(
0.074
M)
as
follows:
5
mg
of
powder
of
TSN102172,
the
14
kDa
protein,
was
dissolved
in
5
mL
of
sodium
citrate
buffer.
16.6
mg
of
powder
of
TSN102171,
the
44
kDa
protein,
was
dissolved
in
5
mL
of
sodium
citrate
buffer.
BSA
(
24.8
mg)
was
dissolved
in
5
mL
of
water.
 ­
lactoglobulin
(
13.6
mg)
was
dissolved
in
5
mL
of
water
also.
Simulated
gastric
fluid
with
a
pH
of
1.2
and
containing
0.3
%
pepsin
was
prepared
according
to
the
United
States
Pharmacopeia.

Control
substance
digestion
­
Time
sampling
of
digestion
occurred
at
1,
5,
7,
10,
15,
20,
30,
and
60
minutes
at
37

C.
After
the
95
µ
L
aliquots
of
simulated
gastric
fluid
(
SGF)
were
equilibrated
to
37

C,
24.8
µ
g
(
5
µ
L)
of
BSA
and
13.6
µ
g
(
5
µ
L)
of
 ­
lactoglobulin
were
added
to
the
tubes.
At
the
time
points
above,
10
µ
L
was
removed
from
each
tube
and
added
to
4
µ
L
of
sodium
carbonate
to
stop
the
digestion.

PS149B1
protein
digestion
­
Test
substance
digestion
was
carried
out
similarly
to
control
substances,
but
the
SGF
contained
190
µ
L
of
volume
and
10
µ
g
(
10
µ
L)
of
the
14
kDa
protein
or
33.2
µ
g
(
10
µ
L)
of
the
44
kDa
protein
were
added
to
the
tubes.
At
the
predetermined
interval,
20
µ
L
of
each
stock
was
removed
and
added
to
8
µ
L
of
sodium
carbonate.
For
each
set
of
reactions
above,
an
undigested
control
reaction
was
run
in
which
water
was
substituted
for
the
SGF.

For
BSA
and
 ­
lactoglobulin,
the
entire
sample
was
mixed
with
an
equal
volume
of
sample
buffer
to
which
fresh
 ­
mercaptoethanol
was
added.
For
the
14
kDa
and
the
44
kDa
proteins
and
the
undigested
controls,
samples
were
first
diluted
in
water
and
then
mixed
with
an
equal
volume
of
sample
buffer.
A
single
4­
20
%
polyacrylamide
gels
was
used
to
separate
the
BSA
and
 ­
lactoglobulin
samples
and
duplicate
gels
were
run
to
separate
the
14
kDa
and
44
kDa
protein
samples.
The
amount
of
protein
loaded
into
each
lane
for
SDS­
PAGE
was
as
follows:
BSA
­
2.5
34
µ
g,
 ­
lactoglobulin
­
1.5
µ
g,
14
kDa
­
1
µ
g,
44
kDa
­
2
µ
g.
The
gels
used
for
Western
blotting
were
loaded
with
approximately
0.04
µ
g
of
the
14
kDa
and
44
kDa
proteins
per
lane
based
upon
analysis
performed
prior
to
digestion.

Following
electrophoretic
separation,
one
gel
for
each
of
the
control
substances
and
the
two
test
substances
(
4
total)
was
stained
with
Coomassie
Brilliant
Blue.
The
other
two
gels,
one
for
the
14
kDa
protein
and
one
for
the
44
kDa
protein
were
transferred
to
nitrocellulose
membranes.
Blots
were
blocked
with
Tris
buffered
saline
containing
0.3
%
Tween­
20
and
5
%
powdered
milk.
Polyclonal
antibody
raised
in
rabbits
against
the
14
kDa
protein
(
Strategic
Diagnostics,
Inc.,
lot
#
200.526B­
5)
and
the
44
kDa
protein
(
Strategic
Diagnostics,
Inc.,
lot
#
200.526A­
5)
were
used
to
detect
the
PS149B1
proteins.
After
washing
the
membrane,
anti­
rabbit
antibodies
conjugated
to
horseradish
peroxidase
were
used
to
detect
the
position
of
the
immunoreactive
proteins
using
chemiluminescent
substrates
and
detection
film.

RESULTS
As
expected,
the
BSA
positive
control
was
rapidly
digested
(
within
1
min)
and
the
 ­
lactoglobulin
remained
intact
for
60
minutes,
the
duration
of
the
experiment.
The
14
kDa
protein
was
visible
on
the
SDS­
PAGE
gel
at
the
15
minute
sample
point,
but
not
afterwards.
It
was
detected
on
the
Western
blot,
which
has
greater
sensitivity,
at
the
20
minute
time
point,
but
not
in
later
sample
points.
A
band
was
also
noted
on
the
Western
blot
at
approximately
30
kDa.
This
band
was
noted
in
an
earlier
study
and
is
considered
to
be
the
result
of
protein
aggregation.
It
disappeared
after
the
1
minute
incubation
in
the
SGF
as
evidenced
by
a
lack
of
reaction
at
the
5
minute
sampling.

For
the
44
kDa
protein
Western
blot,
bands
were
observed
at
approximately
42
kDa
and
14
kDa.
A
single
band
was
observed
on
the
44
kDa
SDS­
PAGE
at
approximately
15
to
16
kDa.
These
bands
were
only
observed
at
the
one
minute
time
point,
but
not
afterwards.

The
concentrations
of
the
14
kDa
and
44
kDa
proteins
were
too
low
to
be
detectable
by
Coomassie
Blue
staining.
The
undigested
control
for
these
proteins
also
failed
to
show
on
the
Western
blots.
The
entire
study
was
repeated.
The
experiment
was
largely
successful
this
second
time,
except
that
the
14
kDa
undigested
controls
failed
to
show
up
in
Western
blotting.
Left
over
samples
were
used
from
the
freezer
storage
to
repeat
this
portion
of
the
study
and
the
Western
blotting
was
successful
(
i.
e.,
the
14
kDa
protein
was
visualized
by
reaction
to
the
antibody).
The
reasons
why
this
failed
to
occur
in
the
initial
experiments
is
unclear.
