Document ID: EPA-HQ-OPP-2004-0395-0039
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2005-02-16T05:00Z

4
Reviewed
by:
Chris
A.
Wozniak,
Ph.
D.,
Biologist,
OPP
/
BPPD
9/
10/
2002
Secondary
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist,
OPP
/
BPPD
/
s/
MRID
No.:
455845­
01
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
Confidentiality:
No
claims
of
confidentiality
are
made
for
any
of
the
information
contained
in
this
study
on
the
basis
of
its
falling
within
the
scope
of
FIFRA
10
(
d)(
1)(
A)(
B),
or
(
C).
ID
No.:
1G06279;
68467­
EUP­
5
Submission
No.:
S610440
Chemical
No.:
006430
Bacillus
thuringiensis
PS149B1
proteins
DP
Barcode:
D281021
Sponsor:
Dow
AgroSciences
LLC,
9330
Zionsville
Rd.,
Indianapolis,
IN
46268.
Authors:
R.
A.
Herman
Testing
Facility:
Dow
AgroSciences,
5501
Oberlin
Drive,
San
Diego,
CA
92121
Study
Titles:
Heat
lability
of
individual
proteins
of
the
PS149B1
binary
ICP
Study
Date:
January
7,
2002
Study
No.:
010144
Conclusion:
The
decrease
in
growth
inhibition
activity
following
heating
of
the
14
kDa
and
44
kDa
mixture
suggests
that
heat
treatment
at
60

C
for
30
minutes
is
sufficient
to
denature
at
least
one
of
the
necessary
components
of
the
ICP.
No
mortality
effect
was
seen
with
the
Cry
protein
treatments,
as
expected
with
the
southern
corn
rootworm
larval
assay.
The
author
suggests
that
the
degree
of
inhibition
(
30
to
42
%
growth
inhibition)
seen
following
heat
treatment
of
the
mixture
is
typical
of
an
inactive
protein
preparation
as
observed
in
previous
assays.
This
difference
between
the
larval
weights
observed
with
the
ICP
­
temperature
treatments
and
the
buffer
control
was
termed
a
non­
specific
effect,
attributed
to
the
presence
of
proteins
in
the
diet.
The
44
kDa
protein
in
the
ICP
was
denatured
by
treatment
at
60

C
and
higher
temperatures
as
indicated
by
the
lack
of
response
to
a
14kDa
protein
spike.
The
decreased
activity
seen
in
the
44
kDa
spike
of
the
heated
ICP
as
temperature
of
incubation
was
increased
indicates
that
the
14
kDa
protein
was
more
heat
stable
than
the
44
kDa
protein.
The
relative
insect
activity
(
i.
e.,
growth
inhibition)
as
correlated
with
temperature
increase
shows
the
relative
stability
of
the
14
kDa
protein
at
60

C
and
75

C,
but
its
denaturation
to
background
levels
(
i.
e.,
36
%
growth
inhibition
compared
to
buffer
control)
at
90

C.
The
observed
growth
inhibitory
response
of
the
44
kDa
protein
alone
(
25
%
inhibition)
was
not
observed
previously
nor
expected.

Classification:
Supplemental.
The
registrant
needs
to
further
examine
or
explain
the
non­
specific
`
protein
effect'
seen
as
a
net
decrease
in
insect
larval
weight
of
30
%
to
42
%,
as
compared
to
the
negative
(
buffer)
control.
The
inhibitory
effect
of
5
the
44
kDa
protein
when
fed
alone
to
larvae
also
requires
some
further
inquiry.

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
exceptions:
purity,
solubility,
stability
and
uniformity
analyses
were
not
conducted
on
the
treatment
mixtures
applied
to
the
test
substrate
or
for
the
treated
test
substrates;
insect
source
of
supply
/
care
and
diet
preparation
/
delivery
were
conducted.
under
non­
GLP
conditions
prior
to
receipt
in
the
Regulatory
Laboratories
GLP
Laboratory.

Purpose:
To
examine
the
stability
of
the
two
insecticidal
crystal
proteins,
designated
as
Cry34Ab1
(
14
kDa)
and
Cry35Ab1
(
44
kDa),
to
heat
treatment,
as
measured
by
the
percent
growth
inhibition
observed
in
a
Southern
Corn
Rootworm
feeding
assay.

METHODS
Test
Substances
­
The
insecticidal
crystal
protein
found
in
Bacillus
thuringiensis
strain
PS149B1
consists
of
two
proteins
referred
to
as
Cry34Ab1
and
Cry35Ab1.
Their
molecular
masses
are
14
kDa
and
44
kDa,
respectively.
Powder
containing
54
%
of
the
14
kDa
 ­
endotoxin
(
Cry34Ab1)
and
a
separate
powder
containing
37
%
of
the
44
kDa
 ­
endotoxin
(
Cry35Ab1)
were
produced
in
Pseudomonas
fluorescens.
The
14
kDa
powder
is
referenced
as
TSN
102172
and
the
44
kDa
powder
as
TSN
102171.

Heat
treatment
and
bioassay
­
A
proprietary
diet
formulated
for
culture
of
the
southern
corn
rootworm
(
SCR),
Diabrotica
undecimpunctata
howardi,
was
used
as
the
delivery
medium
for
the
Cry34/
35
 ­
endotoxins.
Aqueous
formulations
of
these
two
insecticidal
crystal
proteins
(
ICP)
were
spread
onto
the
surface
of
the
agar­
based
diet
and
the
diet
infested
with
one
neonate
larva
of
the
SCR
per
well
of
a
128­
well
bioassay
tray.

To
provide
for
distinction
between
the
thermolability
of
the
two
proteins,
which
are
typically
both
needed
for
insecticidal
or
growth
inhibition
effects,
the
heated
ICP
(
combination
of
both
proteins)
was
spiked
with
non­
heated
samples
of
the
individual
proteins.
The
two
ICP
proteins
were
mixed
at
a
50:
1
(
14
kDa:
44kDa)
mass
ratio
to
avoid
suspending
excess
44
kDa
protein
in
the
formulation
since
this
has
been
shown
to
add
little
to
the
potency
when
applied
at
higher
ratios.

Each
of
the
two
proteins
was
formulated
on
the
day
of
testing
and
diluted
(
or
mixed)
as
needed
in
10
mM
potassium
phosphate
buffer
(
pH
7.5).
A
full
description
of
the
dilution
and
mixing
protocol
for
the
assay
is
attached
as
Figure
1.
A
solution
containing
a
mixture
of
the
two
proteins
was
divided
into
4
portions
and
held
for
30
minutes
at
4

C
(
refrigerated),
60

C,
75

C,
or
90
6

C
in
a
water
bath.
After
incubation
at
the
various
temperatures,
the
vials
containing
the
proteins
were
placed
on
ice.
The
solutions
were
then
either
diluted
further
or
spiked
with
individual
nonheated
protein
solutions
to
comprise
the
treatments
as
listed
in
Table
1
(
attached).

Controls
included
in
the
experiment
were
buffer
alone
(
10
mM
potassium
phosphate)
or
nonheated
individual
proteins
(
i.
e.,
Cry34Ab1,
Cry35Ab1).

Approximately
500
µ
L
of
a
proprietary
Western
corn
rootworm
diet
was
placed
into
each
well
of
a
128­
well
bioassay
tray.
The
protein
formulations
described
above
were
applied
to
each
of
16
wells
(
50
µ
L
to
each
well
of
surface
area
1.5
cm2)
per
treatment.
The
negative
control
treatment
(
phosphate
buffer)
was
applied
to
3
x
16
wells.
After
the
test
material
dried
to
the
diet
surface,
a
single
neonate
SCR
larva
was
added
to
each
well.
The
tray
was
covered
with
a
vented
lid
and
incubated
at
approximately
26

C.
Eggs
were
hatched
at
15
to
33

C
for
production
of
neonates.

Data
Analysis
­
Growth
inhibition
was
calculated
by
comparing
the
treatment
larval
weights
to
the
negative
control
(
phosphate
buffer)
weight
average.
No
further
statistical
analysis
was
performed.

RESULTS
AND
DISCUSSION
As
expected,
there
was
no
useful
mortality
data
in
that
the
positive
control
(
proteins
at
4

C)
showed
only
evidence
of
growth
inhibition
in
SCR
larvae.
The
treatments
resulted
in
growth
inhibition
responses
as
follows
in
Table
2.
Although
not
evidenced
in
previous
bioassays,
a
`
nonspecific
protein
effect'
was
suggested
as
an
explanation
for
the
observed
growth
inhibition
(
30
to
42
%)
of
SCR
larvae
and
for
the
failure
of
the
temperature
treatment
to
inactivate
the
growth
inhibiting
ability
of
the
protein
mixtures.
This
phenomenon
had
not
been
noted
previously
by
the
registrant.
The
apparent
growth
inhibitory
response
of
the
44
kDa
protein
alone
(
25
%
inhibition)
was
also
not
seen
previously
in
similar
experiments.

The
decrease
in
growth
inhibition
activity
following
heating
of
the
14
kDa
and
44
kDa
mixture
suggests
that
heat
treatment
at
60

C
for
30
minutes
is
sufficient
to
denature
at
least
one
of
the
necessary
components
of
the
ICP.
The
author
suggests
that
the
degree
of
inhibition
seen,
for
example,
following
60,
75
or
90

C
treatment
of
the
mixture,
is
typical
of
an
inactive
protein
preparation
(
e.
g.,
30
to
42
%
growth
inhibition).
The
difference
between
the
larval
weights
observed
with
the
ICP
­
temperature
treatments
and
the
buffer
control
was
due
to
a
non­
specific
effect
attributed
to
the
presence
of
proteins
in
the
diet
irrespective
of
the
amino
acid
sequence
and
conformation
of
the
ICPs.
Given
the
small
quantities
of
protein
loaded
along
with
the
treatments
and
the
observed
degradation
of
activity
with
heating,
it
is
not
clear
how
this
general
protein
effect
is
mediated.
Although
the
SCR
diet
is
proprietary,
it
is
assumed
that
the
formulation
must
include
a
protein
source
which
likely
dwarfs
the
amounts
of
protein
added
through
the
addition
of
Cry
protein
preparations.

Table
2.
7
Percent
SCR
Growth
Inhibition
(
compared
to
buffer
control
at
the
indicated
temperature)

Proteins
/
Temperature
4

C
60

C
75

C
90

C
14
kDa
+
44
kDa
(
ICP)
92
%
30
%
42
%
33
%

14
kDa
alone
68
%
 
 
­­

14
kDa
spike
of
ICP
 
a
72
%
71
%
71
%

44
kDa
alone
25
%
 
 
­­

44
kDa
spike
of
ICP
 
82
%
57
%
36
%

a
­
`
 
`
=
not
performed
ICP
=
insecticidal
crystal
proteins;
a
mixture
of
both
14
kDa
and
44
kDa
proteins
at
1:
50
ratio.

The
similar
values
of
growth
inhibition
noted
for
the
heat
treated
ICP
with
a
14
kDa
spike
and
the
14
kDa
protein
alone
(
4

C)
,
71
%
vs.
68
%,
suggests
that
the
44
kDa
protein
in
the
ICP
was
denatured
by
treatment
at
60

C
and
higher.
It
would
have
been
helpful
to
have
data
from
the
treatments
of
the
14
kDa
and
44
kDa
proteins
alone
at
60

C
and
higher
temperatures
for
comparison.
It
is
not
clear
why
these
treatments
were
not
included.

The
restoration
of
activity
with
the
44
kDa
spike
of
the
heated
ICP
indicates
that
the
14
kDa
protein
was
more
heat
stable
than
the
44
kDa
protein.
The
decrease
in
insect
activity
with
increased
incubation
temperature
shows
the
relative
stability
of
the
14
kDa
protein
at
60

C
and
75

C,
but
its
denaturation
to
background
levels
(
i.
e.,
36
%
growth
inhibition
compared
to
buffer
control)
at
90

C.
The
lack
of
increased
activity
following
addition
of
the
14
kDa
protein
to
the
heated
ICP
indicates
the
44
kDa
protein
is
significantly
affected
at
60

C.

CONCLUSIONS:
The
44
kDa
protein
of
the
PS149B1
ICP
(
Cry35A)
was
denatured
at
relatively
low
temperatures
(
i.
e.,
60

C
or
less),
while
the
14
kDa
protein
(
Cry34A)
is
moderately
heat
tolerant.
For
a
significant
effect
on
insect
growth
(
corn
rootworm
complex),
both
proteins
of
the
ICP
are
necessary
in
combination,
albeit
not
necessarily
in
equimolar
amounts.
The
effects
of
these
proteins
on
growth
of
SCR
after
heat
denaturation
of
the
proteins
is
unclear
as
to
mechanism
in
that
the
quantities
of
protein
presented
to
the
larvae
are
not
excessive
relative
to
normal
dietary
considerations.
Compared
to
control
treatment
(
i.
e.,
buffer),
the
30
to
42
%
decline
in
larval
weight
accumulation
seen
in
heat­
treated
protein
assays
suggests
that
there
is
either
some
residual
activity
present
within
the
test
substance
or
that
another
factor
present
is
responsible
for
the
activity.
A
decrease
in
growth
of
this
magnitude
is
still
biologically
significant
when
comparing
the
absence
of
treatment
(
i.
e.,
the
buffer
control)
with
the
Cry34/
35
ICP
treatment.
The
25
%
growth
inhibition
observed
in
the
treatment
containing
the
44
kDa
protein
alone
is
also
puzzling
in
that
the
binary
ICP
was
previously
considered
as
necessary
for
insecticidal
activity.
8
References:

1.)
Bacillus
thuringiensis
Toxin
Nomenclature:
www.
biols.
sussex.
ac.
uk/
home/
Neil_
Crickmore/
Bt/

2.)
Review
of
Product
Characterization,
Expression
Analysis
and
Acute
Oral
Toxicity
Studies
for
PS149B1
binary
insect
control
proteins
as
expressed
in
maize,
a
Bacillus
thuringiensis­
based
plantpesticide
for
an
experimental
use
permit;
review
by
Chris
A.
Wozniak,
Ph.
D.,
secondary
review
by
John
L.
Kough,
Ph.
D.,
Senior
Scientist,
through
Mike
Mendelsohn,
Regulatory
Action
Leader,
June
07,
2001.