Document ID: EPA-HQ-OPP-2003-0299-0002
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-10-29T05:00Z

APR
10
2003
MEMORANDUM
SUBJECT:
Review
of
Section
G
Experimental
Plan
for
Maize­
optimized
Cry1F
insect
control
protein
as
expressed
in
maize,
a
Bacillus
thuringiensis
var.
aizawai­
based
plant­
incorporated
protectant.

TO:
Mike
Mendelsohn,
Product
Manager
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
FROM:
Chris
A.
Wozniak,
Ph.
D.,
Biologist
/
s/
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
THROUGH:
John
L.
Kough,
Ph.
D.,
Biologist
/
s/
Senior
Scientist,
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
ACTION
REQUESTED
To
review
the
experimental
design
for
the
field
testing
of
the
product
maize
optimized
Cry1F
insect
control
protein
from
Bacillus
thuringiensis
var
aizawai
strain
PS81l
(
NRRL
B­
18484)
as
modified
for
expression
in
maize
for
an
experimental
use
permit.
This
submission
requests
an
extension
of
the
previous
EUP,
which
expired
in
March,
2003,
until
March
2004.

BACKGROUND
Dow
AgroSciences
/
Mycogen
has
submitted
an
application
for
an
experimental
use
permit
to
evaluate
their
product
as
a
means
of
European
corn
borer
control.
These
products
are
based
on
the
insecticidal
protein
of
B.
thuringiensis
var.
aizawai,
Cry1F,
in
a
truncated
and
synthetic
(
modified)
form
which
represents
the
active
ingredient
of
this
plant­
pesticide.
The
coding
sequence
of
this
insecticidal
protein
gene
has
been
modified
to
express
the
same
protein
as
the
previously
registered
Cry1F,
however,
the
modifications
in
the
gene
sequence
will
provide
for
greater
stability
(
less
variation)
in
expression
levels;
there
is
no
change
in
the
sequence
or
structure
of
the
Cry1F
protein.
Maize
plants
are
also
intended
to
express
phosphinothricin
acetyl
transferase
for
tolerance
to
the
herbicide
glufosinate
ammonium.
2
EVALUATION
REPORT
Reviewed
by:
Chris
A.
Wozniak,
Ph.
D.,
Biologist,
OPP
/
BPPD
Secondary
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist,
OPP
/
BPPD
Active
Ingredients:
Bacillus
thuringiensis
var.
aizawai
 ­
endotoxin
(
synthetic
truncated
form)
as
optimized
for
and
expressed
in
maize
Product
Name:
Cry1F
insect
control
protein
as
expressed
in
maize
ID
No.:
068467­
EUP­
4
Chemical
No.:
006481
Bacillus
thuringiensis
Cry1F
protein
Confidentiality:
No
claim
of
confidentiality
is
made
for
information
contained
in
this
study
within
the
scope
of
FIFRA
10(
d)(
1)(
A),
(
B)
or
(
C).
Sponsor:
Mycogen
Seeds
/
Dow
AgroSciences
LLC,
9330
Zionsville
Rd.,
Indianapolis,
IN
46268
Authors:
P.
Hunst
Testing
Facility:
Various
field
sites
and
the
laboratories
of
Pioneer
Hi­
Bred
International,
Johnston,
IA,
and
Mycogen
Seeds
/
Dow
AgroSciences
LLC,
9330
Zionsville
Rd.,
Indianapolis,
IN
46268
Study
Titles:
Experimental
use
permit
extension
request
for
Bacillus
thuringiensis
var.
aizawai
mo
Cry1F
insecticidal
crystal
protein
as
expressed
in
maize.
Study
Date:
November
15,
2002
Introduction
­
A
previous
experimental
use
permit
(
68467­
EUP­
3)
evaluated
the
agronomic
characteristics,
breeding,
hybrid
production,
insect
resistance
management
trials,
herbicide
tolerance
and
efficacy
of
a
maize
optimized
Cry1F
maize
(
moCry1F)
for
control
of
the
European
Corn
Borer
(
ECB;
Ostrinia
nubilalis),
the
Fall
Armyworm
(
Spodoptera
frugiperda)
and
other
lepidopteran
pests
of
corn.
The
subject
of
the
present
EUP
request
is
the
same
modification
of
the
gene
sequence
(
cry1F)
designed
to
optimize
expression
in
maize
via
codon
usage
without
changing
the
amino
acid
sequence
of
the
Cry1F
protein.
This
maize
optimized
Cry1F
(
moCry1F)
corn
will
be
grown
under
an
experimental
program
to
evaluate
the
efficacy
of
this
plantincorporated
protectant
against
the
same
lepidopteran
pests
as
for
the
previously
examined
plant
optimized
poCry1F
maize.
Additionally,
agronomic
characteristics,
breeding
and
introgression
into
elite
corn
lines,
herbicide
tolerance
and
insect
resistance
management
concerns
will
also
be
addressed
during
this
field
study.
Plasmid
vector
PHP12537
was
used
to
create
Events
TC6228
and
TC6275.
These
events
will
be
the
subject
material
for
the
field
protocols
listed
below.

Since
there
are
no
feral
or
indigenous
relatives
of
maize
(
Zea
mays)
in
the
U.
S.,
its
territories
or
possessions,
and
there
is
an
established
food
tolerance
exemption
in
place
for
the
Cry1F
protein,
there
is
no
requirement
for
an
isolation
distance
from
other
crops
during
this
EUP.
3
Experimental
Design
­
The
following
protocols
will
be
evaluated
under
this
EUP:

EUP
Protocol
Acres
Lbs
of
Seed
B.
t.
mo
Cry1F
Maize
breeding
and
observation
nursery
44.0
733
B.
t.
mo
Cry1F
Maize
agronomic
observation
trial
172.0
2872
B.
t.
mo
Cry1F
Maize
demonstration
trial
10.5
175
Liberty
®
herbicide
tolerance
study
7.0
117
B.
t.
mo
Cry1F
Maize
efficacy
trial
53.0
885
B.
t.
mo
Cry1F
Maize
insect
resistance
management
trial
4.0
67
TOTALS
290.5
4849
The
experimental
program
is
reasonable
for
the
purpose
of
the
permit
and
the
requested
acreage
is
justified..
The
experimental
use
permit
will
be
used
to
conduct
field
testing
in
26
states
and
Puerto
Rico.

B.
t.
mo
Cry1F
Maize
breeding
and
observation
nursery
­
Standard
breeding
practices,
as
appropriate
for
maize
are
to
be
employed
to
achieve
the
production
of
moCry1F
corn
lines
suitable
for
further
study
and
incorporation
into
hybrid
seed
production
programs.
Maize
plants
are
to
be
crossed
with
genetically
engineered
and
non­
engineered
plants,
or
selfed,
as
necessary
for
production
of
breeding
lines
and
hybrids
of
moCry1F.
Treatment
with
Liberty
®
herbicide,
insect
bioassays
and
laboratory
analyses
will
be
performed
in
conjunction
with
this
protocol
to
assess
phenotype
and
gene
segregation.

Plots
will
be
located
at
12
different
locations
with
a
total
acreage
of
up
to
44.0
acres.
Planting
dates
are
to
run
from
04/
01/
03
until
03/
30/
04
and
harvest
from
06/
01/
03
to
07/
30/
04.
Since
the
previously
issued
food
tolerance
for
Cry1F
is
valid
for
Bacillus
thuringiensis
Cry1F
protein
and
the
genetic
material
necessary
for
its
production
(
from
the
insert
of
plasmid
PHP12537)
in
corn,
no
requirement
of
isolation
distance
is
mandated.
As
with
any
crop,
suitable
plot
design
and
timing
should
be
used
to
prevent
cross­
pollination
into
lines
which
are
not
intended
to
contain
the
genetic
material
carried
by
these
inbreds
and
hybrids
used
in
the
breeding
nursery.

Plots
are
to
be
marked
appropriately
to
indicate
the
field
boundaries
after
harvest
and
provide
for
an
effective
control
of
subsequent
volunteer
plants.
This
may
be
accomplished
with
stakes
or
other
physical
identifiers
and
volunteers
eradicated
with
herbicides
or
cultivation.
4
Samples
of
plant
material
and
seeds
produced
in
the
plots
may
be
used
for
research
purposes
and
saved
for
subsequent
planting
as
needed.
Remaining
plant
material
must
be
tilled
into
the
plot
or
properly
composted.
Composting
site(
s)
must
be
monitored
for
volunteer
plants
that
may
arise.
Any
volunteer
plants
must
be
destroyed.

B.
t.
mo
Cry1F
Maize
agronomic
observation
trial
­
Plants
will
be
assessed
for
agronomic
characteristics
and
phenotypic
effects
of
transgene
expression.
Treatment
with
Liberty
®
herbicide,
insect
bioassays,
and
other
laboratory
analyses
will
be
performed
in
conjunction
with
this
protocol
to
assess
phenotype
and
gene
segregation.

Plots
will
be
located
at
49
different
locations
with
a
total
acreage
of
up
to
172.0
acres.
Planting
dates
are
to
run
from
04/
01/
03
until
03/
30/
04
and
harvest
from
06/
01/
03
to
07/
30/
04.
Since
the
previously
issued
food
tolerance
for
Cry1F
is
valid
for
Bacillus
thuringiensis
Cry1F
protein
and
the
genetic
material
necessary
for
its
production
(
from
the
insert
of
plasmid
PHP12537)
in
corn,
no
requirement
of
isolation
distance
is
mandated.
As
with
any
crop,
suitable
plot
design
and
timing
should
be
used
to
prevent
cross­
pollination
into
lines
which
are
not
intended
to
contain
the
genetic
material
carried
by
these
inbreds
and
hybrids
used
in
the
breeding
nursery.

Plots
are
to
be
marked
appropriately
to
indicate
the
field
boundaries
after
harvest
and
provide
for
an
effective
control
of
subsequent
volunteer
plants.
This
may
be
accomplished
with
stakes
or
other
physical
identifiers
and
volunteers
eradicated
with
herbicides
or
cultivation.

Samples
of
plant
material
and
seeds
produced
in
the
plots
may
be
used
for
research
purposes
and
saved
for
subsequent
planting
as
needed.
Remaining
plant
material
must
be
tilled
into
the
plot
or
properly
composted.
Composting
site(
s)
must
be
monitored
for
volunteer
plants
that
may
arise.
Any
volunteer
plants
must
be
destroyed.

B.
t.
mo
Cry1F
Maize
demonstration
trial
­
Maize
plants
are
to
be
crossed
with
genetically
engineered
and
non­
engineered
plants,
or
selfed.

Treatment
with
Liberty
®
herbicide,
insect
bioassays
and
laboratory
analyses
will
be
performed
in
conjunction
with
this
protocol
to
assess
product
efficacy
against
the
European
Corn
Borer
and
other
lepidopteran
pests.

Plots
will
be
located
at
10
different
locations
with
a
total
acreage
of
up
to
10.5
acres.
Planting
dates
are
to
run
from
04/
01/
03
until
03/
30/
04
and
harvest
from
06/
01/
03
to
07/
30/
04.
Since
the
previously
issued
food
tolerance
for
Cry1F
is
valid
for
Bacillus
thuringiensis
Cry1F
protein
and
the
genetic
material
necessary
for
its
production
(
from
the
insert
of
plasmid
PHP12537)
in
corn,
no
requirement
of
isolation
distance
is
mandated.
As
with
any
crop,
suitable
plot
design
and
timing
should
be
used
to
prevent
cross­
pollination
into
lines
which
are
not
intended
to
contain
the
5
genetic
material
carried
by
these
inbreds
and
hybrids
used
in
the
breeding
nursery.

Transformation
events
TC6228
and
TC6275
will
be
planted
in
5
row
plots
30
feet
long.
Each
line
may
be
represented
by
a
segregant
without
the
cry1F
gene
and
one
segregant
with
the
cry1F
gene.
Rows
will
represent
experimental
units
and
may
contain
up
to
300
plants.

Plots
are
to
be
marked
appropriately
to
indicate
the
field
boundaries
after
harvest
and
provide
for
an
effective
control
of
subsequent
volunteer
plants.
This
may
be
accomplished
with
stakes
or
other
physical
identifiers
and
volunteers
eradicated
with
herbicides
or
cultivation.
Samples
of
plant
material
and
seeds
produced
in
the
plots
may
be
used
for
research
purposes
and
saved
for
subsequent
planting
as
needed.
Remaining
plant
material
must
be
tilled
into
the
plot
or
properly
composted.
Composting
site(
s)
must
be
monitored
for
volunteer
plants
that
may
arise.
Any
volunteer
plants
must
be
destroyed.

Liberty
®
herbicide
tolerance
study
­
B.
t.
mo
Cry1F
plants
will
be
tested
for
resistance
to
the
herbicide
Liberty
®
and
may
also
be
evaluated
for
yield,
herbicide
effects
on
plant
development
and
other
agronomic
traits.

Plots
will
be
located
at
8
different
locations
with
a
total
acreage
of
up
to
7.0
acres.
Planting
dates
are
to
run
from
04/
01/
03
until
03/
30/
04
and
harvest
from
06/
01/
03
to
07/
30/
04.
Since
the
previously
issued
food
tolerance
for
Cry1F
is
valid
for
Bacillus
thuringiensis
Cry1F
protein
and
the
genetic
material
necessary
for
its
production
(
from
the
insert
of
plasmid
PHP12537)
in
corn,
no
requirement
of
isolation
distance
is
mandated.
As
with
any
crop,
suitable
plot
design
and
timing
should
be
used
to
prevent
cross­
pollination
into
lines
which
are
not
intended
to
contain
the
genetic
material
carried
by
these
inbreds
and
hybrids
used
in
the
breeding
nursery.
Plants
may
be
naturally
or
artificially
infested
with
lepidopteran
pests,
such
as
the
ECB.

Plots
are
to
be
marked
appropriately
to
indicate
the
field
boundaries
after
harvest
and
provide
for
an
effective
control
of
subsequent
volunteer
plants.
This
may
be
accomplished
with
stakes
or
other
physical
identifiers
and
volunteers
eradicated
with
herbicides
or
cultivation.
Samples
of
plant
material
and
seeds
produced
in
the
plots
may
be
used
for
research
purposes
and
saved
for
subsequent
planting
as
needed.
Remaining
plant
material
must
be
tilled
into
the
plot
or
properly
composted.
Composting
site(
s)
must
be
monitored
for
volunteer
plants
that
may
arise.
Any
volunteer
plants
must
be
destroyed.

B.
t.
mo
Cry1F
Maize
efficacy
trial
­
The
ability
of
mo
Cry1F
maize
plants
to
resist
attack
from
the
European
Corn
Borer
(
ECB)
and
other
lepidopteran
insects
will
be
evaluated
in
this
protocol.
Genetically
modified
and
non­
modified
lines
will
be
sued
to
evaluate
the
effect
of
the
insecticidal
transgene
on
insect
feeding
and
damage.
6
Plots
will
be
located
at
58
different
locations
with
a
total
acreage
of
up
to
53.0
acres.
Planting
dates
are
to
run
from
04/
01/
03
until
03/
30/
04
and
harvest
from
06/
01/
03
to
7/
30/
04.
Since
the
previously
issued
food
tolerance
for
Cry1F
is
valid
for
Bacillus
thuringiensis
Cry1F
protein
and
the
genetic
material
necessary
for
its
production
(
from
the
insert
of
plasmid
PHP12537)
in
corn,
no
requirement
of
isolation
distance
is
mandated.
As
with
any
crop,
suitable
plot
design
and
timing
should
be
used
to
prevent
cross­
pollination
into
lines
which
are
not
intended
to
contain
the
genetic
material
carried
by
these
inbreds
and
hybrids
used
in
the
breeding
nursery.

Plots
are
to
be
marked
appropriately
to
indicate
the
field
boundaries
after
harvest
and
provide
for
an
effective
control
of
subsequent
volunteer
plants.
This
may
be
accomplished
with
stakes
or
other
physical
identifiers
and
volunteers
eradicated
with
herbicides
or
cultivation.
Samples
of
plant
material
and
seeds
produced
in
the
plots
may
be
used
for
research
purposes
and
saved
for
subsequent
planting
as
needed.
Remaining
plant
material
must
be
tilled
into
the
plot
or
properly
composted.
Composting
site(
s)
must
be
monitored
for
volunteer
plants
that
may
arise.
Any
volunteer
plants
must
be
destroyed.

B.
t.
mo
Cry1F
Maize
insect
resistance
management
trial
­
Maize
plants
expressing
the
Cry1F
transgene
and
those
segregants
not
expressing
Cry1F
will
be
tested
for
comparison
with
respect
to
infestation
and
damage
from
ECB
and
other
lepidopterans,
and
focus
on
determining
if
moCry1F
expresses
a
high
dose
against
the
ECB.

Plots
will
be
located
at
4
different
locations
with
a
total
acreage
of
up
to
4.0
acres.
Planting
dates
are
to
run
from
04/
01/
03
until
03/
30/
04
and
harvest
from
06/
01/
03
to
07/
30/
04.
Since
the
previously
issued
food
tolerance
for
Cry1F
is
valid
for
Bacillus
thuringiensis
Cry1F
protein
and
the
genetic
material
necessary
for
its
production
(
from
the
insert
of
plasmid
PHP12537)
in
corn,
no
requirement
of
isolation
distance
is
mandated.
As
with
any
crop,
suitable
plot
design
and
timing
should
be
used
to
prevent
cross­
pollination
into
lines
which
are
not
intended
to
contain
the
genetic
material
carried
by
these
inbreds
and
hybrids
used
in
the
breeding
nursery.

Plots
are
to
be
marked
appropriately
to
indicate
the
field
boundaries
after
harvest
and
provide
for
an
effective
control
of
subsequent
volunteer
plants.
This
may
be
accomplished
with
stakes
or
other
physical
identifiers
and
volunteers
eradicated
with
herbicides
or
cultivation.
Samples
of
plant
material
and
seeds
produced
in
the
plots
may
be
used
for
research
purposes
and
saved
for
subsequent
planting
as
needed.
Remaining
plant
material
must
be
tilled
into
the
plot
or
properly
composted.
Composting
site(
s)
must
be
monitored
for
volunteer
plants
that
may
arise.
Any
volunteer
plants
must
be
destroyed.

BPPD
Comment:
The
applicant
must
report
to
the
Agency
the
total
number
of
acres
of
maize
receiving
the
cry1F
gene
through
pollen
transfer
in
its
annual
report
regarding
the
EUP
progress.