JUN
19
2003
NOTE
TO
FILE
SUBJECT:
Assessment
of
Ribosomal
Inhibition
Protein
(
RIP)
activity
in
PS149B1
binary
insect
control
proteins
Cry34Ab1
and
Cry35Ab1,
as
expressed
in
maize
following
transformation
with
construct
PHP17662,
a
Bacillus
thuringiensis­
based
plant­
incorporated
protectant,
for
an
experimental
use
permit.

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
Summary
statements
without
supporting
data
in
product
characterization
submissions
by
the
registrants,
Mycogen
Seeds
/
Dow
Agrosciences
and
Pioneer
Hi­
Bred
International,
Inc.,
indicated
that
Cry34Ab1
and
Cry35Ab1
proteins
from
Bacillus
thuringiensis
strain
PS149B1
were
devoid
of
ribosomal
inhibitory
protein
(
RIP)
activity.
A
reassessment
of
those
studies
by
the
applicant
indicated
that
the
studies
were
equivocal
and
could
not
distinguish
RIP
activity
as
conducted.
The
company
therefore
wanted
to
remove
this
claim
from
the
submission.
This
review
evaluates
the
basis
for
that
assessment
by
the
company
and
its
relevance
to
the
EUP
and
tolerance
decisions.

BACKGROUND
Mycogen
Seeds
/
Dow
Agrosciences
and
Pioneer
Hi­
Bred
International,
Inc.,
have
submitted
a
request
for
an
experimental
use
permit
(
EUP)
for
their
product
as
a
means
of
corn
rootworm
2
control.
This
product
is
based
on
the
insecticidal
proteins
Cry34Ab1
and
Cry35Ab1
of
B.
thuringiensis
strain
PS149B1,
the
active
ingredient
of
this
plant­
incorporated
protectant.
These
two
proteins
work
in
concert
to
effect
a
midgut
pathology
similar
to
that
of
other
B.
thuringiensis
 ­
endotoxins.
This
EUP
is
intended
to
evaluate
plants
transformed
with
vector
PHP17662,
a
new
construct
from
this
applicant.
Events
intended
for
field
evaluation
under
the
guidelines
of
this
EUP
are
designated
as:
E4497.42.1.34,
E4497.45.2.16,
E4497.59.1.10,
E4497.66.1.27,
E4497.71.1.29,
and
E4497.71.1.33.
Maize
plants
are
also
intended
to
express
phosphinothricin
acetyl
transferase
for
tolerance
to
the
herbicide
glufosinate
ammonium.

Comment
to
File:
The
Agency's
assessment
of
data
submitted
on
RIP
activity
in
PS149B1
proteins,
("
Review
of
product
characterization
data
for
PS149B1
binary
insect
control
proteins
Cry34Ab1
and
Cry35Ab1,
as
expressed
in
maize
following
transformation
with
construct
PHP17662,
a
Bacillus
thuringiensis­
based
plant­
incorporated
protectant,
for
an
experimental
use
permit",
from
C.
Wozniak
to
M.
Mendelsohn,
June
12,
2003),
indicated
that
the
registrant
must
submit
a
revised
study
evaluating
the
RIP
activity
of
these
proteins
prior
to
Section
3
registration.
The
registrants
should
complete
this
study
since
initial
summary
statements
were
subsequently
found
to
be
based
upon
equivocal
data
and
the
registrants'
stated
desire
to
remove
the
statement
from
their
product
characterization
submission.
However,
the
requirement
for
this
study
is
not
based
upon
a
need
for
information
related
to
the
approval
of
the
requested
EUPs
or
a
pending
food
tolerance
action.
As
the
validity
of
information
was
raised,
it
is
appropriate
for
the
Agency
to
request
further
clarification
simply
for
the
sake
of
completing
the
file.
Previous
reviews
of
this
active
ingredient
contained
statements
indicating
a
lack
of
RIP
activity
(
MRID#
457906­
01
­
reviewed
in
Memorandum
from
C.
Wozniak,
to
M.
Mendelsohn,,
May
21,
2003).

The
initial
RIP
activity
experiments
conducted
by
Pioneer
Hi­
Bred,
Intl.,
Inc.,
were
performed
as
an
exploratory
procedure
to
elucidate
the
mode
of
action
for
Cry34Ab1
and
Cry35Ab1.
The
Agency
does
not
consider
these
types
of
studies
as
part
of
the
typical
characterization
assessment
for
insecticidal
plant­
incorporated
protectant
proteins.
Prior
to
the
final
characterization
of
the
PS149B1
proteins,
the
company
explored
many
aspects
and
avenues
of
these
proteins'
activity
until
is
was
clear
that
they
more
closely
fit
the
definition
and
expectations
of
insecticidal
crystal
proteins
typical
of
Bacillus
thuringiensis.
The
Agency
has
no
reason
to
suspect,
based
on
the
results
of
protein
characterization
and
oral
toxicity
studies,
that
either
of
these
proteins
in
any
way
resemble
proteins
with
RIP
activity.
However,
in
order
to
complete
the
actions
in
the
file
and
associated
jacket
prior
to
registration,
it
is
in
the
best
interest
of
the
company
to
repeat
the
study
with
appropriate
controls
so
that
the
question
of
RIP
activity
can
be
answered
without
equivocal
data.
