Document ID: EPA-HQ-OPP-2004-0395-0038
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2005-02-16T05:00Z

40
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.:
453584­
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
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:
R.
A.
Herman
Testing
Facility:
Global
Environmental
Chemistry
Laboratory
­
Indianapolis
Lab,
Dow
Agrosciences
LLC,
9330
Zionsville
Rd.,
Indianapolis,
IN
46268­
1054
Study
Titles:
Thermolability
of
PS149B1
binary
delta­
endotoxin.
Study
Date:
November
16,
2000
Study
No.:
001041
Conclusion:
Results
indicate
that
the
SP149B1
proteins,
or
at
least
one
of
them,
is
inactivated
after
exposure
to
60

C
for
30
minutes,
based
upon
activity
against
the
southern
corn
rootworm.
It
is
possible
that
shorter
times
or
lower
temperatures
may
have
also
denatured
the
protein(
s),
but
these
were
not
tested.
With
the
current
protocol
design,
it
is
not
possible
to
determine
if
one
or
both
proteins
are
inactivated
following
heat
treatment.
To
address
this
issue,
the
registrant
should
consider
repeating
the
study
under
a
method
that
would
allow
this
distinction
to
be
made
as
part
of
the
submission
for
Section
3
registration.

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
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
ECL
GLP
laboratory.

Purpose:
To
measure
the
lability
of
the
two
PS149B1
ICP
proteins
to
heat.

METHODS
Test
substances
­
PS149B1
proteins
used
in
this
study
were
produced
in
Pseudomonas
fluorescens.
The
resulting
powders
contained
either
54
%
14
kDa
protein
(
TSN102172)
or
37
%
44
kDa
protein
(
TSN102171).
ELISA
was
performed
before
and
after
the
assays
in
this
study
to
confirm
that
the
proteins
were
stable
over
the
duration
of
the
study.
41
Heat
treatment
and
bioassay
of
southern
corn
rootworm
(
SCR)
­
A
1:
1
active
ingredient
(
AI)
mass
ratio
of
the
14
kDa
and
44
kDa
ICP
proteins
were
added
to
the
surface
of
insect
diets
and
allowed
to
dry
to
the
surface
of
the
agar.
Treated
diets
were
infested
with
neonate
larvae
of
the
southern
corn
rootworm
(
Diabrotica
undecimpunctata
howardi).

The
test
substance
was
formulated
in
10
mM
potassium
phosphate
(
pH
7.5)
and
was
prepared
on
each
test
date
for
the
assays.
The
concentration
was
chosen
to
target
a
final
concentration
on
the
insect
diet
of
10
µ
g
AI/
cm2
of
diet.
Four
aliquots
of
the
test
formulation
were
placed
at
different
temperatures
(
60

C,
75

C,
90

C)
for
heat
treatments
or
in
the
refrigerator
for
the
4

C
positive
control.
after
30
minutes
at
temperature,
the
vials
were
placed
on
ice.
A
negative
control
treatment
was
also
included
which
consisted
of
10
mM
potassium
phosphate
(
pH
7.5)
alone.

Approximately
500
mL
of
diet
was
added
to
each
of
128
wells
in
a
bioassay
tray.
Surface
area
of
each
well
was
approximately
1.5
cm2.
The
test
substance
formulation
(
50
µ
L)
was
applied
to
each
of
16
wells,
except
for
the
negative
control
(
10
mM
potassium
phosphate)
where
three
replicates
of
16
insects
were
treated.
After
the
surface
of
the
medium
dried,
each
well
was
infested
with
a
single
neonate
larva
of
SCR.

Data
analysis
­
Each
insect
was
weighed
after
the
assay
and
the
average
weight
for
each
treatment
compared
to
the
average
weight
for
the
control
treatment
(
10
mM
potassium
phosphate).
No
statistical
analysis
was
performed.

RESULTS
Bioassay
­
Growth
inhibition
was
used
to
compare
treatments
as
the
mortality
in
the
4

C
treatment
was
low
(
6
%).

Southern
corn
rootworm
TREATMENT
%
GROWTH
INHIBITION
ICP
@
4

C
70
%

ICP
@
60

C
­
3
%

ICP
@
75

C
­
3
%

ICP
@
90

C
1
%

Purity,
solubility
and
stability
studies
were
not
carried
out
on
the
test
substances
for
this
study
or
on
the
treated
test
substrates.
ELISA
was
used
to
demonstrate
the
stability
of
the
test
substance
proteins
at
the
beginning
and
end
of
the
study
to
indicate
a
lack
of
significant
change
over
time.
Since
comparisons
of
growth
inhibition
were
made
with
test
substance
formulations
made
fresh
for
each
assay,
the
relative
growth
inhibition
is
indicative
of
the
potency
of
the
formulation
following
treatment
at
the
given
temperature.
42
Results
indicate
that
the
SP149B1
proteins,
or
at
least
one
of
them,
is
inactivated
after
exposure
to
60

C
for
30
minutes.
It
is
possible
that
shorter
times
or
lower
temperatures
may
have
also
denatured
the
protein(
s),
but
these
were
not
tested.
With
the
current
protocol
design,
it
is
not
possible
to
determine
if
one
or
both
proteins
are
inactivated
following
heat
treatment.
To
address
this
issue,
the
registrant
should
consider
repeating
the
study
under
a
method
that
would
allow
this
distinction
to
be
made
as
part
of
the
submission
for
Section
3
registration.
Inclusion
of
a
lower
temperature
treatment
is
also
advised
to
determine
the
lowest
temperature
at
which
the
protein(
s)
denature.