Document ID: EPA-HQ-OPP-2006-0554-0002
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2006-07-17T04:00Z

1
UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
OFFICE
OF
PESTICIDES
AND
TOXIC
SUBSTANCES
JUN
14
2006
MEMORANDUM
SUBJECT:
Review
of
Human
Health
and
Product
Characterization
Data
for
a
Temporary
Tolerance
Exemption
for
Bt
Cry
1A.
105
and
Cry2Ab2
in
corn
and
an
EUP
Request
for
Insect­
Protected
Corn
MON
89034,
MON
89017,
and
MON
89034
x
MON
89017
TO:
Mike
Mendelsohn
Regulatory
Action
Leader
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511P)

FROM:
Rebecca
L.
Edelstein,
Ph.
D.,
Chemist
/
s/
Ibrahim
Barsoum,
Ph.
D.,
Biologist
/
s/
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511P)

THROUGH:
John
L.
Kough,
Ph.
D.,
Biologist
/
s/
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511P)

ACTION
REQUESTED:
To
review
the
human
health
and
product
characterization
data
for
a
temporary
tolerance
exemption
petitions
for
Bt
Cry1A.
105
and
EUP
request
for
MON
89034,
MON
89017,
and
MON
89034
x
MON
89017
corn
CONCLUSION:
Monsanto
has
submitted
an
EUP
request
for
events
MON
89034,
MON
88017,
and
MON
89034
x
MON
88017,
as
well
as
petitions
for
temporary
tolerance
exemptions
for
Cry1A.
105
and
Cry2Ab2
in
corn.
The
product
characterization
and
human
health
data
submitted
on
event
MON
89034
and
the
data
previously
submitted
on
event
MON
88017
are
sufficient
to
support
the
EUP
request.
In
2
addition,
the
product
characterization
and
human
health
data
submitted
on
Cry1A.
105
and
cited
for
Cry2Ab2
are
sufficient
to
support
the
temporary
tolerance
exemption
requests.
The
Cry3Bb1
and
CP4
EPSPS
proteins
are
already
covered
by
tolerance
exemptions
(
40
CFR
180.1214:
Cry3Bb1
protein
and
the
genetic
material
necessary
for
its
production
in
corn
when
used
as
plant­
incorporated
protectants
in
field
corn,
sweet
corn,
and
popcorn;
40
CFR
180.1174:
CP4
EPSPS
and
the
genetic
material
necessary
for
its
production
in
all
plants).

MON
89034,
which
was
developed
by
Agrobacterium­
mediated
transformation
of
corn
using
the
2T­
DNA
plasmid
vector
PV­
ZMIR245,
produces
two
Bacillus
thuringiensis
proteins,
Cry1A.
105
and
Cry2Ab2.
These
proteins
are
intended
to
provide
protection
from
feeding
damage
caused
by
a
number
of
lepidopteran
pests.
Cry1A.
105
is
a
chimeric
protein
composed
of
portions
of
Cry1Ab,
Cry1Ac,
and
Cry1F
proteins.
MON
88017,
developed
by
Agrobacterium­
mediated
transformation
of
corn
with
plasmid
vector
PV­
ZMIR39,
produces
Cry3Bb1
and
CP4
EPSPS
proteins.
The
Cry3Bb1
protein
is
intended
to
provide
protection
from
feeding
damage
by
corn
rootworm
larvae,
and
the
CP4
EPSPS
protein
confers
tolerance
to
the
Roundup
®
family
of
herbicides.

Characterization
of
the
DNA
isolated
from
event
MON
89034
corn
using
restriction
enzyme
digests
and
Southern
blot
analysis
indicated
that
the
expected
DNA
inserted
in
the
corn
genome
at
a
single
locus
and
contains
one
copy
each
of
the
cry1A.
105
and
cry2Ab2
expression
cassettes.
There
were
no
other
elements
detectable
other
than
those
associated
with
the
respective
cassettes.
No
backbone
sequences
from
plasmid
PV­
ZMIR245
or
nptII
coding
sequences
were
detected
in
the
corn
genome.

Protein
characterization
data
demonstrate
that
the
plant­
produced
Cry1A.
105
has
characteristics
and
activities
that
are
similar
to
those
of
the
E.
coli­
produced
Cry1A.
105
used
in
several
safety
studies.
The
following
techniques
were
used
to
characterize
and
compare
the
plant­
produced
and
the
E.
coliproduced
proteins:
sodium
dodecyl
sulfate
polyacrylamide
gel
electrophoresis
(
SDS­
PAGE),
western
blot
analysis,
densitometry,
matrix
assisted
laser
desorption
time
of
flight
(
MALDI­
TOF)
mass
spectrometry,
glycosylation
analysis,
and
a
Cry1A.
105
insecticidal
activity
assay.
Glycoslyation
analysis
indicated
that
the
proteins
are
not
glycoslyated.
These
analyses
demonstrated
the
structural
and
functional
similarity
between
the
plant­
produced
and
the
E.
coli­
produced
Cry1A.
105
proteins
and
justified
the
use
of
E.
coli­
produced
protein
in
toxicity
studies.
Monsanto
also
provided
information
showing
the
similarity
between
Cry1A.
105,
Cry1Ab,
and
Cry1Ac.

In
support
of
the
safety
of
the
Cry1A.
105
protein,
Monsanto
submitted
an
acute
oral
toxicity
study,
which
demonstrated
the
lack
of
mammalian
toxicity
at
high
levels
of
exposure
to
the
pure
Cry1A.
105
protein.
An
in
vitro
digestibility
study
demonstrated
that
the
protein
digested
rapidly
(
within
~
30
seconds
in
simulated
gastric
fluid).
In
addition,
amino
acid
sequence
comparisons
showed
no
similarities
between
Cry1A.
105
and
known
allergens
or
mammalian
toxins.
A
heat
stability
study
showed
that
Cry1A.
105
is
no
longer
detectable
by
Western
blot
after
heating
at
204
º
C
for
20
minutes.

In
support
of
the
safety
of
the
Cry2Ab2
protein,
Monsanto
cited
previously
submitted
data
demonstrating
the
rapid
digestion
of
Cry2Ab2
in
simulated
digestive
fluid,
lack
of
mammalian
toxicity,
and
lack
of
amino
acid
similarities
between
Cry2Ab2
and
known
allergens
or
mammalian
3
toxins.
Monsanto
did
not
submit
protein
characterization
data
on
the
plant­
produced
Cry2Ab2
from
event
MON
89034
to
demonstrate
equivalence
with
the
B.
thuringiensis­
produced
protein
used
in
several
of
the
safety
studies.
However,
Monsanto
previously
submitted
data
demonstrating
similarity
of
the
protein
produced
in
event
MON
840
and
the
B.
thuringiensis­
produced
protein,
and
Monsanto
provided
information
indicating
that
the
Cry2Ab2
coding
sequence
(
including
the
transit
peptide
sequence
SSU­
CTP
targeting
sequence)
inserted
in
event
MON
89034
is
identical
to
the
coding
sequence
inserted
in
event
MON
840
corn:
In
an
attachment
to
an
email
sent
by
Natalia
Bogdanova
of
Monsanto
on
June
1,
2006,
Monsanto
states
"
The
cry2Ab2
expression
cassette
in
vector
PV­
ZMIR245
that
was
used
to
produce
MON
89034
contains
the
same
elements
as
the
cry2Ab2
cassette
in
vector
ZMBK28
with
one
exception.
The
e35S
promoter
that
was
used
to
regulate
expression
of
the
cry2Ab2
in
MON
84006
was
replaced
by
the
FMV
promoter
in
PVZMIR245
In
the
EUP
data
package
reviewed
here,
Monsanto
also
submitted
data
demonstrating
that
the
Cry2Ab2
protein
is
no
longer
detectable
by
western
blot
after
heating
at
204
º
C
for
20
minutes.

Expression
level
data
were
provided
for
Cry1A.
105,
Cry2Ab2,
and
Cry3Bb1
in
the
individual
events
and
in
the
conventional
cross.
All
three
proteins
are
expressed
at
relatively
low
levels.
The
data
were
produced
using
ELISA
methods
specific
for
each
protein.
DATA
REVIEW
RECORD
Active
Ingredient:
Bacillus
thuringiensis
Cry1A.
105,
Cry2Ab2,
and
Cry3Bb1
insecticidal
proteins
and
the
genetic
material
necessary
for
their
production
in
corn
Product
Name:
MON
89034,
MON
88017,
MON
89034
x
MON
88017
Company
Name:
Monsanto
Company
ID
No:
Chemical
Number:
006498,
006514,
006515,
006502
Decision
Number:
353287
DP
Barcode:
324861
MRID/
Study
Titles:
46471601 
Data
in
Support
of
an
Application
for
an
Experimental
Use
Permit
to
Test
ZMIR245xMON
88017
Combined
Corn
Trait
along
with
ZMIR245
and
MON
88017
46694501 
Summary
of
Southern
Blot
Analyses
of
MON
89034
and
MON
89697
Corn
46694502 
Assessment
of
the
Cry1A.
105,
Cry2Ab2,
and
Cry3Bb1
Protein
Levels
in
Tissues
of
Insect­
Protected
Corn
MON
89034,
MON
89034
x
MON
88017,
MON
89597,
and
MON
89597
x
MON
88017
46694503 
Qualitative
Detection
Method
for
the
Cry2Ab2
Protein
in
Corn
Leaf
and
Seed
of
MON
89034
and
MON
89597
46694601 
Structural
and
Functional
Similarity
of
the
Cry1A.
105
Protein
to
Cry1A
Class
of
Bacillus
thuringiensis
Proteins:
Final
Report
46694603 
Acute
Oral
Toxicity
Study
in
Mice
with
Cry1A.
105
Protein
46694604 
Characterization
of
the
Cry1A.
105
Protein
Purified
from
the
Corn
Grain
of
MON
89034
and
Comparison
of
the
Physiochemical
and
Functional
4
Properties
of
the
Plant­
Produced
and
E.
coli­
Produced
Cry1A.
105
Proteins
46694605 
Bioinformatics
Analysis
of
the
Cry1A.
105
Protein
Utilizing
the
AD6,
Toxin5,
and
Allpeptides
Databases
46694606 
Assessment
of
the
In
Vitro
Digestibility
of
the
Cry1A.
105
Protein
in
Simulated
Gastric
Fluid
46694607 
Immunodetection
of
Cry2Ab2
and
Cry1A.
105
Proteins
in
Corn
Grain
from
MON
89034
Following
Heat
Treatment
45086305 
Assessment
of
the
Equivalence
of
Proteins
Expressed
in
Corn
Event
MON
840
BACKGROUND:
Monsanto
has
submitted
an
EUP
request
for
events
MON
89034,
MON
88017,
and
MON
89034
x
MON
88017.
MON
89034,
which
was
developed
by
Agrobacterium­
mediated
transformation
of
corn
using
the
2T­
DNA
plasmid
vector
PV­
ZMIR245,
produces
two
Bacillus
thuringiensis
proteins,
Cry1A.
105
and
Cry2Ab2.
These
proteins
are
intended
to
provide
protection
from
feeding
damage
caused
by
a
number
of
lepidopteran
pests.
Cry1A.
105
is
a
chimeric
protein
composed
of
portions
of
Cry1Ab,
Cry1Ac,
and
Cry1F
proteins.
EPA
granted
a
temporary
tolerance
exemption
for
Cry2Ab2
in
corn
and
cotton
in
2001,
and
removed
the
time
limitation
for
cotton
in
2004;
however,
the
exemption
has
expired
for
Cry2Ab2
in
corn.
Monsanto
has
submitted
petitions
to
establish
temporary
tolerance
exemptions
for
both
Cry1A.
105
and
Cry2Ab2
in
corn.
Monsanto
is
relying
on
previously
submitted
data
to
support
the
tolerance
exemption
for
Cry2Ab2
but
has
submitted
new
data,
reviewed
in
this
memorandum,
to
support
the
tolerance
exemption
for
Cry1A.
105.
Safety
data
on
Cry2Ab2
were
reviewed
previously
in
a
memorandum
from
M.
Watson
to
M.
Mendelsohn
dated
Feb.
12,
2001
(
MRIDs
44966602
Acute
Oral
Toxicity
Study;
44966603
In
vitro
Digestibility;
44966604
Bioinformatics
Analysis 
Allergen
Database;
44966605
Bioinformatics
Analysis 
Toxin
and
Public
Domain
Genetic
Databases).
Monsanto
is
also
relying
on
previously
submitted
data
to
demonstrate
equivalence
of
the
plant­
produced
Cry2Ab2
protein
and
the
microbially­
produced
protein,
which
was
used
in
some
of
the
safety
studies.
Monsanto
had
previously
submitted
the
protein
characterization
data
to
support
an
EUP
request
for
MON
840;
however,
the
data
were
never
reviewed
since
the
EUP
request
was
withdrawn.
The
data
are
therefore
reviewed
in
this
memorandum.

MON
88017,
developed
by
Agrobacterium­
mediated
transformation
of
corn
with
plasmid
vector
PV­
ZMIR39,
produces
Cry3Bb1
and
CP4
EPSPS
proteins.
The
Cry3Bb1
protein
is
intended
to
provide
protection
from
feeding
damage
by
corn
rootworm
larvae,
and
the
CP4
EPSPS
protein
confers
tolerance
to
the
Roundup
®
family
of
herbicides.
On
March
31,
2004,
EPA
established
a
tolerance
exemption
for
Bt
Cry3Bb1
protein
and
the
genetic
material
necessary
for
its
production
in
corn
when
used
as
plant­
incorporated
protectants
in
field
corn,
sweet
corn,
and
popcorn
(
40
CFR
180.1214).
In
addition,
EPA
has
established
a
tolerance
exemption
for
CP4
EPSPS
and
the
genetic
material
necessary
for
its
production
in
all
plants
(
40
CFR
180.1174).
Bt
Cry3Bb1
protein
and
the
genetic
material
necessary
for
its
production
in
event
MON
88017
corn
is
currently
registered
(
EPA
Reg.
No.
524­
LLR).
The
human
health
and
product
characterization
data
submitted
for
registration
of
the
product
(
reviewed
in
a
memorandum
dated
July
25,
2005
from
A.
Fellman
to
M.
Mendelsohn)
5
supports
this
EUP,
and
Bt
Cry3Bb1
produced
in
event
MON
88017
and
MON
88017
x
MON
89034
is
covered
by
the
tolerance
exemption
for
Bt
Cry3Bb1
(
40
CFR
180.1214).

This
memorandum
reviews
the
product
characterization
and
human
health
data
submitted
in
support
of
Monsanto's
EUP
request
and
petitions
for
establishing
tolerance
exemptions
for
Cry1A.
105
and
Cry2Ab2.

RECOMMENDATION:

The
submitted
human
health
data
are
sufficient
to
support
an
EUP
and
temporary
exemptions
from
the
requirement
of
a
food
tolerance.
However,
the
following
are
needed
for
section
3
registration:
 
Validated
detection
method
for
Cry1A.
105
and
Cry2Ab2
in
corn
 
Southern
blot
analysis
(
or
other
appropriate
molecular
characterization)
of
the
event(
s)
and
conventional
crosses
(
stacks)
being
registered
 
Protein
characterization
of
plant­
expressed
Cry2Ab2
from
event
MON
89034
to
show
equivalence
with
the
microbial
protein
used
in
safety
testing
 
Expression
levels
for
EPSPS
in
the
stacked
product
 
Plant­
produced
Cry1A.
105
shows
both
a
higher­
molecular
weight
immunoreactive
band
and
lower
molecular
weight
immunoreactive
bands
in
the
western
blot
analysis
in
addition
to
the
full­
length
protein.
These
additional
bands
appear
to
be
absent
in
the
E.
coli­
produced
protein
preparation.
Monsanto
should
address
whether
these
differences
are
a
result
of
protein
purification
or
whether
these
different
molecular
weight
proteins
are
likely
present
in
the
plant.
Monsanto
should
also
address
whether
use
of
the
E.
coli
protein
as
a
surrogate
for
safety
testing
is
appropriate,
given
the
differences
in
the
protein
preparations.
 
Provide
cited
protocols
for
Southern
blot
analysis
 
Discuss
determination
of
equivalence
between
microbially­
produced
and
plant­
produced
proteins
using
bioactivity
assays.
Are
statistics
used?
Why
was
the
acceptance
criteria
determined
to
be
<
4
fold
difference
between
the
mean
EC50
values
in
the
corn
earworm
dietincorporation
insect
biassay.
 
Provide
the
following
SOPs:
SOP
BR­
ME­
0044­
03,
SOP
BR­
ME­
0044­
02,
and
BR­
ME­
0956­
02.

HUMAN
HEALTH
ASSESSMENT
Cry1A.
105
6
Section
408(
c)(
2)(
A)(
i)
of
the
FFDCA
allows
EPA
to
establish
an
exemption
from
the
requirement
for
a
tolerance
(
the
legal
limit
for
a
pesticide
chemical
residue
in
or
on
a
food)
only
if
EPA
determines
that
the
exemption
is
"
safe."
Section
408(
c)(
2)(
A)(
ii)
of
the
FFDCA
defines
"
safe"
to
mean
that
"
there
is
a
reasonable
certainty
that
no
harm
will
result
from
aggregate
exposure
to
the
pesticide
chemical
residue,
including
all
anticipated
dietary
exposures
and
all
other
exposures
for
which
there
is
reliable
information."
This
includes
exposure
through
drinking
water
and
in
residential
settings,
but
does
not
include
occupational
exposure.
Pursuant
to
section
408(
c)(
2)(
B),
in
establishing
or
maintaining
in
effect
an
exemption
from
the
requirement
of
a
tolerance,
EPA
must
take
into
account
the
factors
set
forth
in
section
408(
b)(
2)(
C),
which
require
EPA
to
give
special
consideration
to
exposure
of
infants
and
children
to
the
pesticide
chemical
residue
in
establishing
a
tolerance
and
to
"
ensure
that
there
is
a
reasonable
certainty
that
no
harm
will
result
to
infants
and
children
from
aggregate
exposure
to
the
pesticide
chemical
residue...."
Additionally,
section
408(
b)(
2)(
D)
of
the
FFDCA
requires
that
the
Agency
consider
"
available
information
concerning
the
cumulative
effects
of
a
particular
pesticide's
residues"
and
"
other
substances
that
have
a
common
mechanism
of
toxicity."
EPA
performs
a
number
of
analyses
to
determine
the
risks
from
aggregate
exposure
to
pesticide
residues.
First,
EPA
determines
the
toxicity
of
pesticides.
Second,
EPA
examines
exposure
to
the
pesticide
through
food,
drinking
water,
and
through
other
exposures
that
occur
as
a
result
of
pesticide
use
in
residential
settings.

Toxicological
Profile
Consistent
with
section
408(
b)(
2)(
D)
of
the
FFDCA,
EPA
has
reviewed
the
available
scientific
data
and
other
relevant
information
in
support
of
this
action
and
considered
its
validity,
completeness
and
reliability
and
the
relationship
of
this
information
to
human
risk.
EPA
has
also
considered
available
information
concerning
the
variability
of
the
sensitivities
of
major
identifiable
subgroups
of
consumers,
including
infants
and
children.

1.
Mammalian
Toxicity
and
Allergenicity
Assessment
Monsanto
has
submitted
acute
oral
toxicity
data
demonstrating
the
lack
of
mammalian
toxicity
at
high
levels
of
exposure
to
the
pure
Cry1A.
105
protein.
These
data
demonstrate
the
safety
of
the
product
at
a
level
well
above
maximum
possible
exposure
levels
that
are
reasonably
anticipated
in
the
crop.
Basing
this
conclusion
on
acute
oral
toxicity
data
without
requiring
further
toxicity
testing
and
residue
data
is
similar
to
the
Agency
position
regarding
toxicity
testing
and
the
requirement
of
residue
data
for
the
microbial
Bacillus
thuringiensis
products
from
which
this
plantincorporated
protectant
was
derived
(
See
40
CFR
Sec.
158.740(
b)(
2)(
i)).
For
microbial
products,
further
toxicity
testing
and
residue
data
are
triggered
by
significant
adverse
acute
effects
in
studies
such
as
the
mouse
oral
toxicity
study,
to
verify
the
observed
adverse
effects
and
clarify
the
source
of
these
effects
(
Tiers
II
&
III).

An
acute
oral
toxicity
study
in
mice
(
MRID
46694603)
indicated
that
Cry1A.
105
is
non­
toxic
to
humans.
Cry1A.
105
produced
from
microbial
culture
was
dosed
by
gavage
as
two
doses
separated
by
4
hours
(
±
20
minutes)
to
10
females
and
10
males
(
2072
mg/
kg
body
weight).
Two
control
groups
were
also
included
in
the
study:
a
bovine
serum
albumin
protein
control,
and
a
vehicle
control.
One
male
in
the
test
protein
group
was
moribund
and
sacrificed
on
day
1
due
to
a
7
mechanical
dosing
error;
this
death
was
not
attributed
to
the
test
material.
All
other
mice
survived
the
study.
There
were
no
significant
differences
in
body
weight
or
body
weight
change
among
the
three
groups
during
the
study,
and
no
treatment­
related
gross
pathological
findings
were
observed.
The
oral
LD50
for
males,
females,
and
combined
mice
was
greater
than
2072
mg/
kg.

When
proteins
are
toxic,
they
are
known
to
act
via
acute
mechanisms
and
at
very
low
dose
levels
(
Sjoblad,
Roy
D.,
et
al.,
"
Toxicological
Considerations
for
Protein
Components
of
Biological
Pesticide
Products,"
Regulatory
Toxicology
and
Pharmacology
15,
3­
9
(
1992)).
Therefore,
since
no
acute
effects
were
shown
to
be
caused
by
Cry1A.
105,
even
at
relatively
high
dose
levels,
the
Cry1A.
105
protein
is
not
considered
toxic.
Further,
amino
acid
sequence
comparisons
showed
no
similarities
between
the
Cry1A.
105
and
known
toxic
proteins
in
protein
databases
that
would
raise
a
safety
concern.

Since
Cry1A.
105
is
a
protein,
allergenic
potential
was
also
considered.
Currently,
no
definitive
tests
for
determining
the
allergenic
potential
of
novel
proteins
exist.
Therefore,
EPA
uses
a
weight­
ofevidence
approach
where
the
following
factors
are
considered:
source
of
the
trait;
amino
acid
sequence
similarity
with
known
allergens;
prevalence
in
food;
and
biochemical
properties
of
the
protein,
including
in
vitro
digestibility
in
simulated
gastric
fluid
(
SGF)
and
glycosylation.
Current
scientific
knowledge
suggests
that
common
food
allergens
tend
to
be
resistant
to
degradation
by
heat,
acid,
and
proteases,
may
be
glycosylated,
and
can
be
present
at
high
concentrations
in
the
food.

1.
Source
of
the
trait.
Bacillus
thuringiensis
is
not
considered
to
be
a
source
of
allergenic
proteins.
2.
Amino
acid
sequence.
A
comparison
of
the
amino
acid
sequence
of
Cry1A.
105
with
known
allergens
showed
no
overall
sequence
similarity
or
identity
at
the
level
of
eight
contiguous
amino
acid
residues.
3.
Prevalence
in
food.
Expression
level
analysis
indicated
that
the
protein
is
present
at
relatively
low
levels
in
corn:
approximately
3
µ
g/
g
in
grain
on
a
dry
weight
basis.
Thus,
the
expression
has
been
shown
to
be
in
the
parts
per
million
range.
4.
Digestibility.
The
Cry1A.
105
protein
was
digested
within
30
seconds
in
simulated
gastric
fluid
containing
pepsin.
5.
Glycosylation.
Cry1A.
105
expressed
in
corn
was
shown
to
have
not
to
be
glycosylated
6.
Conclusion.
Considering
all
of
the
available
information,
EPA
has
concluded
that
the
potential
for
Cry1A.
105
to
be
a
food
allergen
is
minimal.

2.
Aggregate
Exposures
Pursuant
to
FFDCA
section
408(
b)(
2)(
D)(
vi),
EPA
considers
available
information
concerning
aggregate
exposures
from
the
pesticide
residue
in
food
and
all
other
non­
occupational
exposures,
including
drinking
water
from
ground
water
or
surface
water
and
exposure
through
pesticide
use
in
gardens,
lawns,
or
buildings
(
residential
and
other
indoor
uses).

The
Agency
has
considered
available
information
on
the
aggregate
exposure
levels
of
consumers
8
(
and
major
identifiable
subgroups
of
consumers)
to
the
pesticide
chemical
residue
and
to
other
related
substances.
These
considerations
include
dietary
exposure
under
the
tolerance
exemption
and
all
other
tolerances
or
exemptions
in
effect
for
the
plant­
incorporated
protectants
chemical
residue,
and
exposure
from
non­
occupational
sources.
Exposure
via
the
skin
or
inhalation
is
not
likely
since
the
plant­
incorporated
protectant
is
contained
within
plant
cells,
which
essentially
eliminates
these
exposure
routes
or
reduces
these
exposure
routes
to
negligible.
In
addition,
even
if
exposure
can
occur
through
inhalation,
the
potential
for
Cry1A.
105
to
be
an
allergen
is
low,
as
discussed
above.
Although
the
allergenicity
assessment
focuses
on
potential
to
be
a
food
allergen,
the
data
also
indicate
a
low
potential
for
Cry1A.
105
to
be
an
inhalation
allergen.
Exposure
via
residential
or
lawn
use
to
infants
and
children
is
also
not
expected
because
the
use
sites
for
the
Cry1A.
105
protein
is
agricultural.
Oral
exposure,
at
very
low
levels,
may
occur
from
ingestion
of
processed
corn
products
and,
theoretically,
drinking
water.
However
oral
toxicity
testing
showed
no
adverse
effects.
Furthermore,
the
expression
of
the
Cry1A.
105
protein
in
corn
kernels
has
been
shown
to
be
in
the
parts
per
million
range,
which
makes
the
expected
dietary
exposure
several
orders
of
magnitude
lower
than
the
amount
of
Cry1A.
105
shown
to
have
no
toxicity.
Therefore,
even
if
negligible
aggregate
exposure
should
occur,
the
Agency
concludes
that
such
exposure
would
result
in
no
harm
due
to
the
lack
of
mammalian
toxicity
and
low
potential
for
allergenicity
demonstrated
for
the
Cry1A.
105
protein.

3.
Cumulative
Effects
Pursuant
to
FFDCA
section
408(
b)(
2)(
D)(
v),
EPA
has
considered
available
information
on
the
cumulative
effects
of
such
residues
and
other
substances
that
have
a
common
mechanism
of
toxicity.
These
considerations
included
the
cumulative
effects
on
infants
and
children
of
such
residues
and
other
substances
with
a
common
mechanism
of
toxicity.
Because
there
is
no
indication
of
mammalian
toxicity
from
the
plant­
incorporated
protectant,
we
conclude
that
there
are
no
cumulative
effects
for
the
Cry1A.
105
protein.

4.
Determination
of
Safety
for
U.
S.
Population,
Infants
and
Children
a)
Toxicity
and
Allergenicity
Conclusions
The
data
submitted
and
cited
regarding
potential
health
effects
for
the
Cry1A.
105
protein
includes
the
characterization
of
the
expressed
Cry1A.
105
protein
in
corn,
as
well
as
the
acute
oral
toxicity
study,
amino
acid
sequence
comparisons
to
known
allergens
and
toxins,
and
in
vitro
digestibility
of
the
protein.
The
results
of
these
studies
were
used
to
evaluate
human
risk,
and
the
validity,
completeness,
and
reliability
of
the
available
data
from
the
studies
were
also
considered.

Adequate
information
was
submitted
to
show
that
the
Cry1A.
105
test
material
derived
from
microbial
culture
was
biochemically
and
functionally
equivalent
to
the
protein
produced
by
the
plantincorporated
protectant
ingredient
in
corn.
Microbially
produced
protein
was
used
in
the
safety
studies
so
that
sufficient
material
for
testing
was
available.

The
acute
oral
toxicity
data
submitted
support
the
prediction
that
the
Cry1A.
105
protein
would
be
9
non­
toxic
to
humans.
As
mentioned
above,
when
proteins
are
toxic,
they
are
known
to
act
via
acute
mechanisms
and
at
very
low
dose
levels
(
Sjoblad,
Roy
D.,
et
al.,
"
Toxicological
Considerations
for
Protein
Components
of
Biological
Pesticide
Products,"
Regulatory
Toxicology
and
Pharmacology
15,
3­
9
(
1992)).
Since
no
treatment­
related
adverse
effects
were
shown
to
be
caused
by
the
Cry1A.
105
protein,
even
at
relatively
high
dose
levels,
the
Cry1A.
105
protein
is
not
considered
toxic.
Basing
this
conclusion
on
acute
oral
toxicity
data
without
requiring
further
toxicity
testing
and
residue
data
is
similar
to
the
Agency
position
regarding
toxicity
and
the
requirement
of
residue
data
for
the
microbial
Bacillus
thuringiensis
products
from
which
this
plant­
incorporated
protectant
was
derived
(
See
40
CFR
158.740(
b)(
2)(
i)).
For
microbial
products,
further
toxicity
testing
and
residue
data
are
triggered
when
significant
adverse
effects
are
seen
in
studies
such
as
the
mouse
oral
toxicity
study.
Further
studies
verify
the
observed
adverse
effects
and
clarify
the
source
of
these
effects
(
Tiers
II
and
III).

Residue
chemistry
data
were
not
required
for
a
human
health
effects
assessment
of
the
subject
plantincorporated
protectant
ingredients
because
of
the
lack
of
mammalian.
However,
data
submitted
demonstrated
low
levels
of
the
Cry1A.
105
in
corn
tissues.

Since
Cry1A.
105
is
a
protein,
potential
allergenicity
is
also
considered
as
part
of
the
toxicity
assessment.
Considering
all
of
the
available
information
(
1)
Cry1A.
105
originates
from
a
nonallergenic
source;
(
2)
Cry1A.
105
has
no
sequence
similarities
with
known
allergens;
(
3)
Cry1A.
105
is
not
glycosylated;
(
4)
Cry1A.
105
will
only
be
present
at
low
levels
in
food;
and
(
5)
Cry1A.
105
is
rapidly
digested
in
simulated
gastric
fluid;
EPA
has
concluded
that
the
potential
for
Cry1A.
105
to
be
a
food
allergen
is
minimal.

Neither
available
information
concerning
the
dietary
consumption
patterns
of
consumers
(
and
major
identifiable
subgroups
of
consumers
including
infants
and
children)
nor
safety
factors
that
are
generally
recognized
as
appropriate
for
the
use
of
animal
experimentation
data
were
evaluated.
The
lack
of
mammalian
toxicity
at
high
levels
of
exposure
to
the
Cry1A.
105
protein,
as
well
as
the
minimal
potential
to
be
a
food
allergen,
demonstrate
the
safety
of
the
product
at
levels
well
above
possible
maximum
exposure
levels
anticipated
in
the
crop.

The
genetic
material
necessary
for
the
production
of
the
plant­
incorporated
protectant
active
ingredient
include
the
nucleic
acids
(
DNA,
RNA)
that
encode
these
proteins
and
regulatory
regions.
The
genetic
material
(
DNA,
RNA),
necessary
for
the
production
of
the
Cry1A.
105
protein
has
been
exempted
under
the
blanket
exemption
for
all
nucleic
acids
(
40
CFR
174.475).

b)
Infants
and
Children
Risk
Conclusions
FFDCA
section
408(
b)(
2)(
C)
provides
that
EPA
shall
assess
the
available
information
about
consumption
patterns
among
infants
and
children,
special
susceptibility
of
infants
and
children
to
pesticide
chemical
residues
and
the
cumulative
effects
on
infants
and
children
of
the
residues
and
other
substances
with
a
common
mechanism
of
toxicity.
In
addition,
FFDCA
section
408(
b)(
2)(
C)
also
provides
that
EPA
shall
apply
an
additional
tenfold
margin
of
safety
for
infants
and
children
in
the
case
of
threshold
effects
to
account
for
prenatal
and
postnatal
toxicity
and
the
completeness
of
10
the
database
unless
EPA
determines
that
a
different
margin
of
safety
will
be
safe
for
infants
and
children.

In
this
instance,
based
on
all
the
available
information,
the
Agency
concludes
that
there
is
a
finding
of
no
toxicity
for
the
Cry1A.
105
protein
and
the
genetic
material
necessary
for
its
production.
Thus,
there
are
no
threshold
effects
of
concern
and,
as
a
result,
the
provision
requiring
an
additional
margin
of
safety
does
not
apply.
Further,
the
considerations
of
consumption
patterns,
special
susceptibility,
and
cumulative
effects
do
not
apply.

c)
Overall
Safety
Conclusion
There
is
a
reasonable
certainty
that
no
harm
will
result
from
aggregate
exposure
to
the
U.
S.
population,
including
infants
and
children,
to
the
Cry1A.
105
protein
and
the
genetic
material
necessary
for
its
production.
This
includes
all
anticipated
dietary
exposures
and
all
other
exposures
for
which
there
is
reliable
information.
The
Agency
has
arrived
at
this
conclusion
because,
as
discussed
above,
no
toxicity
to
mammals
has
been
observed,
nor
any
indication
of
allergenicity
potential
for
the
plant­
incorporated
protectant.

5.
Other
Considerations
a)
Endocrine
Disruptors
The
pesticidal
active
ingredient
is
a
protein,
derived
from
a
source
that
is
not
known
to
exert
an
influence
on
the
endocrine
system.
Therefore,
the
Agency
is
not
requiring
information
on
the
endocrine
effects
of
the
plant­
incorporated
protectant
at
this
time.

b)
Analytical
Method(
s)

A
short
description
of
an
enzyme­
linked
immunosorbent
assay
for
the
detection
and
quantification
of
Cry1A.
105
in
corn
tissue
has
been
submitted.

c)
Codex
Maximum
Residue
Level
No
Codex
maximum
residue
level
exists
for
the
plant­
incorporated
protectant
Bacillus
thuringiensis
Cry1A.
105
protein
and
the
genetic
material
necessary
for
its
production
in
corn.

HUMAN
HEALTH
ASSESSMENT
Cry2Ab2
11
Section
408(
c)(
2)(
A)(
i)
of
the
FFDCA
allows
EPA
to
establish
an
exemption
from
the
requirement
for
a
tolerance
(
the
legal
limit
for
a
pesticide
chemical
residue
in
or
on
a
food)
only
if
EPA
determines
that
the
exemption
is
"
safe."
Section
408(
c)(
2)(
A)(
ii)
of
the
FFDCA
defines
"
safe"
to
mean
that
"
there
is
a
reasonable
certainty
that
no
harm
will
result
from
aggregate
exposure
to
the
pesticide
chemical
residue,
including
all
anticipated
dietary
exposures
and
all
other
exposures
for
which
there
is
reliable
information."
This
includes
exposure
through
drinking
water
and
in
residential
settings,
but
does
not
include
occupational
exposure.
Pursuant
to
section
408(
c)(
2)(
B),
in
establishing
or
maintaining
in
effect
an
exemption
from
the
requirement
of
a
tolerance,
EPA
must
take
into
account
the
factors
set
forth
in
section
408(
b)(
2)(
C),
which
require
EPA
to
give
special
consideration
to
exposure
of
infants
and
children
to
the
pesticide
chemical
residue
in
establishing
a
tolerance
and
to
"
ensure
that
there
is
a
reasonable
certainty
that
no
harm
will
result
to
infants
and
children
from
aggregate
exposure
to
the
pesticide
chemical
residue...."
Additionally,
section
408(
b)(
2)(
D)
of
the
FFDCA
requires
that
the
Agency
consider
"
available
information
concerning
the
cumulative
effects
of
a
particular
pesticide's
residues"
and
"
other
substances
that
have
a
common
mechanism
of
toxicity."
EPA
performs
a
number
of
analyses
to
determine
the
risks
from
aggregate
exposure
to
pesticide
residues.
First,
EPA
determines
the
toxicity
of
pesticides.
Second,
EPA
examines
exposure
to
the
pesticide
through
food,
drinking
water,
and
through
other
exposures
that
occur
as
a
result
of
pesticide
use
in
residential
settings.

Toxicological
Profile
Consistent
with
section
408(
b)(
2)(
D)
of
the
FFDCA,
EPA
has
reviewed
the
available
scientific
data
and
other
relevant
information
in
support
of
this
action
and
considered
its
validity,
completeness
and
reliability
and
the
relationship
of
this
information
to
human
risk.
EPA
has
also
considered
available
information
concerning
the
variability
of
the
sensitivities
of
major
identifiable
subgroups
of
consumers,
including
infants
and
children.

1.
Mammalian
Toxicity
and
Allergenicity
Assessment
Monsanto
has
submitted
acute
oral
toxicity
data
demonstrating
the
lack
of
mammalian
toxicity
at
high
levels
of
exposure
to
the
pure
Cry2Ab2
protein.
These
data
demonstrate
the
safety
of
the
product
at
a
level
well
above
maximum
possible
exposure
levels
that
are
reasonably
anticipated
in
the
crop.
Basing
this
conclusion
on
acute
oral
toxicity
data
without
requiring
further
toxicity
testing
and
residue
data
is
similar
to
the
Agency
position
regarding
toxicity
testing
and
the
requirement
of
residue
data
for
the
microbial
Bacillus
thuringiensis
products
from
which
this
plantincorporated
protectant
was
derived
(
See
40
CFR
Sec.
158.740(
b)(
2)(
i)).
For
microbial
products,
further
toxicity
testing
and
residue
data
are
triggered
by
significant
adverse
acute
effects
in
studies
such
as
the
mouse
oral
toxicity
study,
to
verify
the
observed
adverse
effects
and
clarify
the
source
of
these
effects
(
Tiers
II
&
III).

An
acute
oral
toxicity
study
in
mice
(
MRID
44966602)
indicated
that
Cry2Ab2
is
non­
toxic
to
humans.
Three
groups
of
ten
male
and
ten
female
mice
were
dosed
by
oral
gavage
with
30,
300,
or
1000
mg/
kg
bodyweight
of
microbially­
produced
Cry2Ab2
protein.
Two
negative
control
groups
were
also
included
in
the
study:
bovine
serum
albumin
protein
control,
and
a
vehicle
control
(
purified
water).
Two
deaths
occurred
in
control
group
animals;
both
deaths
were
attributed
to
gavage
injury.
12
All
other
mice
survived
the
study.
Several
animals
in
both
the
control
and
test
groups
lost
weight
during
the
study,
and
several
abnormalities
were
observed
by
gross
necropsy
in
several
animals
in
both
the
test
and
control
groups.
There
were
no
significant
differences
between
the
test
and
control
groups;
therefore,
the
Cry2Ab2
protein
does
not
appear
to
cause
any
significant
adverse
effects
at
an
exposure
level
of
up
to
1000
mg/
kg
bodyweight.

When
proteins
are
toxic,
they
are
known
to
act
via
acute
mechanisms
and
at
very
low
dose
levels
(
Sjoblad,
Roy
D.,
et
al.,
"
Toxicological
Considerations
for
Protein
Components
of
Biological
Pesticide
Products,"
Regulatory
Toxicology
and
Pharmacology
15,
3­
9
(
1992)).
Therefore,
since
no
acute
effects
were
shown
to
be
caused
by
Cry2Ab2,
even
at
relatively
high
dose
levels,
the
Cry2Ab2
protein
is
not
considered
toxic.
Further,
amino
acid
sequence
comparisons
showed
no
similarities
between
the
Cry2Ab2
protein
and
known
toxic
proteins
in
protein
databases
that
would
raise
a
safety
concern.

Since
Cry2Ab2
is
a
protein,
allergenic
potential
was
also
considered.
Currently,
no
definitive
tests
for
determining
the
allergenic
potential
of
novel
proteins
exist.
Therefore,
EPA
uses
a
weight­
ofevidence
approach
where
the
following
factors
are
considered:
source
of
the
trait;
amino
acid
sequence
similarity
with
known
allergens;
prevalence
in
food;
and
biochemical
properties
of
the
protein,
including
in
vitro
digestibility
in
simulated
gastric
fluid
(
SGF)
and
glycosylation.
Current
scientific
knowledge
suggests
that
common
food
allergens
tend
to
be
resistant
to
degradation
by
heat,
acid,
and
proteases,
may
be
glycosylated,
and
can
be
present
at
high
concentrations
in
the
food.

1.
Source
of
the
trait.
Bacillus
thuringiensis
is
not
considered
to
be
a
source
of
allergenic
proteins.
2.
Amino
acid
sequence.
A
comparison
of
the
amino
acid
sequence
of
Cry2Ab2
with
known
allergens
showed
no
significant
overall
sequence
similarity
or
identity
at
the
level
of
eight
contiguous
amino
acid
residues.
3.
Prevalence
in
food.
Expression
level
analysis
indicated
that
the
protein
is
present
at
relatively
low
levels
in
corn:
approximately
2.3
µ
g/
g
in
grain
on
a
dry
weight
basis.
Thus,
the
expression
has
been
shown
to
be
in
the
parts
per
million
range.
4.
Digestibility.
The
Cry2Ab2
protein
was
digested
within
15
seconds
in
simulated
gastric
fluid
containing
pepsin.
5.
Glycosylation.
Cry2Ab2
expressed
in
corn
was
shown
not
to
be
glycosylated.
6.
Conclusion.
Considering
all
of
the
available
information,
EPA
has
concluded
that
the
potential
for
Cry2Ab2
to
be
a
food
allergen
is
minimal.

2.
Aggregate
Exposures
Pursuant
to
FFDCA
section
408(
b)(
2)(
D)(
vi),
EPA
considers
available
information
concerning
aggregate
exposures
from
the
pesticide
residue
in
food
and
all
other
non­
occupational
exposures,
including
drinking
water
from
ground
water
or
surface
water
and
exposure
through
pesticide
use
in
gardens,
lawns,
or
buildings
(
residential
and
other
indoor
uses).
13
The
Agency
has
considered
available
information
on
the
aggregate
exposure
levels
of
consumers
(
and
major
identifiable
subgroups
of
consumers)
to
the
pesticide
chemical
residue
and
to
other
related
substances.
These
considerations
include
dietary
exposure
under
the
tolerance
exemption
and
all
other
tolerances
or
exemptions
in
effect
for
the
plant­
incorporated
protectants
chemical
residue,
and
exposure
from
non­
occupational
sources.
Exposure
via
the
skin
or
inhalation
is
not
likely
since
the
plant­
incorporated
protectant
is
contained
within
plant
cells,
which
essentially
eliminates
these
exposure
routes
or
reduces
these
exposure
routes
to
negligible.
In
addition,
even
if
exposure
can
occur
through
inhalation,
the
potential
for
Cry2Ab2
to
be
an
allergen
is
low,
as
discussed
above.
Although
the
allergenicity
assessment
focuses
on
potential
to
be
a
food
allergen,
the
data
also
indicate
a
low
potential
for
Cry2Ab2
to
be
an
inhalation
allergen.
Exposure
via
residential
or
lawn
use
to
infants
and
children
is
also
not
expected
because
the
use
sites
for
the
Cry2Ab2
protein
is
agricultural.
Oral
exposure,
at
very
low
levels,
may
occur
from
ingestion
of
processed
corn
products
and,
theoretically,
drinking
water.
However,
oral
toxicity
testing
showed
no
adverse
effects.
Furthermore,
the
expression
of
the
Cry2Ab2
protein
in
corn
kernels
has
been
shown
to
be
in
the
parts
per
million
range,
which
makes
the
expected
dietary
exposure
several
orders
of
magnitude
lower
than
the
amount
of
Cry2Ab2
shown
to
have
no
toxicity.
Therefore,
even
if
negligible
aggregate
exposure
should
occur,
the
Agency
concludes
that
such
exposure
would
result
in
no
harm
due
to
the
lack
of
mammalian
toxicity
and
low
potential
for
allergenicity
demonstrated
for
the
Cry2Ab2
protein.

3.
Cumulative
Effects
Pursuant
to
FFDCA
section
408(
b)(
2)(
D)(
v),
EPA
has
considered
available
information
on
the
cumulative
effects
of
such
residues
and
other
substances
that
have
a
common
mechanism
of
toxicity.
These
considerations
included
the
cumulative
effects
on
infants
and
children
of
such
residues
and
other
substances
with
a
common
mechanism
of
toxicity.
Because
there
is
no
indication
of
mammalian
toxicity
from
the
plant­
incorporated
protectant,
we
conclude
that
there
are
no
cumulative
effects
for
the
Cry2Ab2
protein.

4.
Determination
of
Safety
for
U.
S.
Population,
Infants
and
Children
a)
Toxicity
and
Allergenicity
Conclusions
The
data
submitted
and
cited
regarding
potential
health
effects
for
the
Cry2Ab2
protein
includes
the
characterization
of
the
expressed
Cry2Ab2
protein
in
corn,
as
well
as
the
acute
oral
toxicity
study,
amino
acid
sequence
comparisons
to
known
allergens
and
toxins,
and
in
vitro
digestibility
of
the
protein.
The
results
of
these
studies
were
used
to
evaluate
human
risk,
and
the
validity,
completeness,
and
reliability
of
the
available
data
from
the
studies
were
also
considered.

Adequate
information
was
submitted
to
show
that
the
Cry2Ab2
test
material
derived
from
microbial
culture
was
biochemically
and
functionally
equivalent
to
the
protein
produced
by
the
plant­
incorporated
protectant
ingredient
in
corn.
Microbially
produced
protein
was
used
in
the
safety
studies
so
that
sufficient
material
for
testing
was
available.
14
The
acute
oral
toxicity
data
submitted
support
the
prediction
that
the
Cry2Ab2
protein
would
be
non­
toxic
to
humans.
As
mentioned
above,
when
proteins
are
toxic,
they
are
known
to
act
via
acute
mechanisms
and
at
very
low
dose
levels
(
Sjoblad,
Roy
D.,
et
al.,
"
Toxicological
Considerations
for
Protein
Components
of
Biological
Pesticide
Products,"
Regulatory
Toxicology
and
Pharmacology
15,
3­
9
(
1992)).
Since
no
treatment­
related
adverse
effects
were
shown
to
be
caused
by
the
Cry2Ab2
protein,
even
at
relatively
high
dose
levels,
the
Cry2Ab2
protein
is
not
considered
toxic.
Basing
this
conclusion
on
acute
oral
toxicity
data
without
requiring
further
toxicity
testing
and
residue
data
is
similar
to
the
Agency
position
regarding
toxicity
and
the
requirement
of
residue
data
for
the
microbial
Bacillus
thuringiensis
products
from
which
this
plantincorporated
protectant
was
derived
(
See
40
CFR
158.740(
b)(
2)(
i)).
For
microbial
products,
further
toxicity
testing
and
residue
data
are
triggered
when
significant
adverse
effects
are
seen
in
studies
such
as
the
mouse
oral
toxicity
study.
Further
studies
verify
the
observed
adverse
effects
and
clarify
the
source
of
these
effects
(
Tiers
II
and
III).

Residue
chemistry
data
were
not
required
for
a
human
health
effects
assessment
of
the
subject
plant­
incorporated
protectant
ingredients
because
of
the
lack
of
mammalian
toxicity.
However,
data
submitted
demonstrated
low
levels
of
the
Cry1A.
105
in
corn
tissues.

Since
Cry2Ab2
is
a
protein,
potential
allergenicity
is
also
considered
as
part
of
the
toxicity
assessment.
Considering
all
of
the
available
information
(
1)
Cry2Ab2
originates
from
a
nonallergenic
source;
(
2)
Cry2Ab2
has
no
sequence
similarities
with
known
allergens;
(
3)
Cry2Ab2
is
not
glycosylated;
(
4)
Cry2Ab2
will
only
be
present
at
low
levels
in
food;
and
(
5)
Cry2Ab2
is
rapidly
digested
in
simulated
gastric
fluid;
EPA
has
concluded
that
the
potential
for
Cry2Ab2
to
be
a
food
allergen
is
minimal.

Neither
available
information
concerning
the
dietary
consumption
patterns
of
consumers
(
and
major
identifiable
subgroups
of
consumers
including
infants
and
children)
nor
safety
factors
that
are
generally
recognized
as
appropriate
for
the
use
of
animal
experimentation
data
were
evaluated.
The
lack
of
mammalian
toxicity
at
high
levels
of
exposure
to
the
Cry2Ab2
protein,
as
well
as
the
minimal
potential
to
be
a
food
allergen,
demonstrate
the
safety
of
the
product
at
levels
well
above
possible
maximum
exposure
levels
anticipated
in
the
crop.

The
genetic
material
necessary
for
the
production
of
the
plant­
incorporated
protectant
active
ingredient
include
the
nucleic
acids
(
DNA,
RNA)
that
encode
these
proteins
and
regulatory
regions.
The
genetic
material
(
DNA,
RNA),
necessary
for
the
production
of
the
Cry2Ab2
protein
has
been
exempted
under
the
blanket
exemption
for
all
nucleic
acids
(
40
CFR
174.475).

b)
Infants
and
Children
Risk
Conclusions
FFDCA
section
408(
b)(
2)(
C)
provides
that
EPA
shall
assess
the
available
information
about
consumption
patterns
among
infants
and
children,
special
susceptibility
of
infants
and
children
to
pesticide
chemical
residues
and
the
cumulative
effects
on
infants
and
children
of
the
residues
and
other
substances
with
a
common
mechanism
of
toxicity.
In
addition,
FFDCA
section
408(
b)(
2)(
C)
also
provides
that
EPA
shall
apply
an
additional
tenfold
margin
of
safety
for
infants
and
children
in
the
case
of
threshold
effects
to
account
for
prenatal
and
postnatal
toxicity
and
the
completeness
of
the
database
unless
EPA
determines
that
a
different
margin
of
safety
will
be
safe
for
infants
and
children.
15
In
this
instance,
based
on
all
the
available
information,
the
Agency
concludes
that
there
is
a
finding
of
no
toxicity
for
the
Cry2Ab2
protein
and
the
genetic
material
necessary
for
its
production.
Thus,
there
are
no
threshold
effects
of
concern
and,
as
a
result,
the
provision
requiring
an
additional
margin
of
safety
does
not
apply.
Further,
the
considerations
of
consumption
patterns,
special
susceptibility,
and
cumulative
effects
do
not
apply.

c)
Overall
Safety
Conclusion
There
is
a
reasonable
certainty
that
no
harm
will
result
from
aggregate
exposure
to
the
U.
S.
population,
including
infants
and
children,
to
the
Cry2Ab2
protein
and
the
genetic
material
necessary
for
its
production.
This
includes
all
anticipated
dietary
exposures
and
all
other
exposures
for
which
there
is
reliable
information.
The
Agency
has
arrived
at
this
conclusion
because,
as
discussed
above,
no
toxicity
to
mammals
has
been
observed,
nor
any
indication
of
allergenicity
potential
for
the
plant­
incorporated
protectant.

5.
Other
Considerations
a)
Endocrine
Disruptors
The
pesticidal
active
ingredient
is
a
protein,
derived
from
a
source
that
is
not
known
to
exert
an
influence
on
the
endocrine
system.
Therefore,
the
Agency
is
not
requiring
information
on
the
endocrine
effects
of
this
plant­
incorporated
protectant
at
this
time.

b)
Analytical
Method(
s)

A
short
description
of
an
enzyme­
linked
immunosorbent
assay
for
the
detection
and
quantification
of
Cry2Ab2
in
corn
tissue
has
been
submitted,
and
a
commercially
available
qualitative
immunochromatographic
test
strip
was
shown
to
detect
the
Cry2Ab2
protein
in
corn
tissues.

c)
Codex
Maximum
Residue
Level
No
Codex
maximum
residue
level
exists
for
the
plant­
incorporated
protectant
Bacillus
thuringiensis
Cry2Ab2
protein
and
the
genetic
material
necessary
for
its
production
in
corn.

References
Sjoblad,
R.
D.,
McClintock,
J.
T.,
and
Engler,
R.,
"
Toxicological
Considerations
for
Protein
Components
of
Biological
Pesticide
Products,"
Reg.
Toxicol.
Pharmacol.
15(
1),
1992,
3­
9.

SUMMARY
OF
DATA
SUBMITTED:

MRID
46471601 
Data
in
Support
of
an
Application
for
an
Experimental
Use
Permit
to
Test
ZMIR245xMON
88017
Combined
Corn
Trait
along
with
ZMIR245
and
MON
88017 
This
MRID
presents
summary
information
for
events
MON
89034,
89597
and
89771.
Only
brief
summary
information,
figures
and
tables
are
presented
for
the
genetic
constructs,
molecular
16
characterization
and
protein
(
Cry1A.
105,
Cry2Ab2,
Cry3Bb1)
expression
levels
for
each
hybrid.
No
detailed
information
is
presented
for
materials,
methods
or
results.
It
is
noted
that
detailed
data
for
MON
89034
and
89597
were
submitted
to
EPA
by
Monsanto
in
November
2005
(
MRIDs
46694501
­
04
and
MIRDs
46494601
­
07)
which
appears
to
replace
this
earlier
submission.
The
detailed
data
in
the
November
2005
MRIDs
for
MON
89034
and
89597
provides
a
full
suite
of
data
in
support
of
the
EUP
application
and
are
reviewed
in
this
memorandum.

MRID
46694501 
Summary
of
Southern
Blot
Analyses
of
MON
89034
and
MON
89697
Corn 
Southern
blot
analyses
indicate
that
MON
89034
and
MON
89597
have
the
introduced
DNA
inserted
in
the
corn
genome
at
a
single
locus
and
contain
one
copy
each
of
the
cry1A.
105
and
cry2Ab2
expression
cassettes.
All
expression
elements
are
shown
to
be
present
in
each
of
the
inserts,
and
there
are
no
other
elements
detectable
other
than
those
associated
with
the
respective
cassettes.
No
backbone
sequences
from
plasmid
PV­
ZMIR245
or
nptII
coding
sequences
were
detected
in
the
corn
genome.
Classification:
ACCEPTABLE 
however,
need
Southern
blot
data
for
MON89034
x
MON88017
(
or
whatever
event(
s)
and
cross(
es)
are
being
registered)
for
section
3
registration
MRID
46694502 
Assessment
of
the
Cry1A.
105,
Cry2Ab2,
and
Cry3Bb1
Protein
Levels
in
Tissues
of
Insect­
Protected
Corn
MON
89034,
MON
89034
x
MON
88017,
MON
89597,
and
MON
89597
x
MON
88017 
The
levels
of
Cry1A.
105,
Cry2Ab2
and
Cry3Bb1
proteins
in
corn
leaf
(
OSL1
and
OSL4),
grain,
forage
and
pollen
tissues
collected
from
MON
89034,
MON
89034
x
MON
88017,
MON
89597,
MON
89597
x
MON
88017
were
determined
using
ELISA
methods.
Corn
tissue
samples
were
collected
from
plants
grown
in
a
greenhouse
except
for
MON
89034
forage
samples
which
were
collected
from
the
field.
The
mean
Cry1A.
105
protein
levels
in
MON
89034
OSL1,
OSL4,
grain,
forage
and
pollen
tissues
were
72,
37,
3.2,
30,
and
15
µ
g/
g
dwt,
respectively.
The
mean
Cry2Ab2
protein
levels
in
MON
89034
OSL1,
OSL4,
grain
and
forage
tissues
were
49,
84,
1.9,
and
29
µ
g/
g
dwt,
respectively.
The
mean
Cry1A.
105
protein
levels
in
MON
89034
x
MON
88017
OSL1,
OSL4,
grain,
forage
and
pollen
tissues
were
71,
60,
3.1,
83,
and
7.6
µ
g/
g
dwt,
respectively.
The
mean
Cry2Ab2
protein
levels
in
MON
89034
x
MON
88017
in
OSL1,
OSL4,
grain
and
forage
tissues
were
50,
74,
2.5,
and
53
µ
g/
g
dwt,
respectively.

The
mean
Cry1A.
105
protein
levels
in
MON
89597
OSL1,
OSL4,
grain,
forage,
and
pollen
tissues
were
78,
29,
2.3,
36,
and
9.4
µ
g/
g
dwt,
respectively.
The
mean
Cry2Ab2
protein
levels
in
MON
89597
OSL1,
OSL4,
grain
and
forage
tissues
were
33,
41,
2.1,
and
29
µ
g/
g
dwt,
respectively.
The
mean
Cry1A.
105
protein
levels
in
MON
89597
x
MON
88017
OSL1,
OSL4,
grain,
forage,
and
pollen
tissues
were
81,
14,
1.6,
17,
and
4.3
µ
g/
g
dwt,
respectively.
The
mean
Cry2Ab2
protein
levels
in
MON
89597
x
MON
88017
in
OSL1,
OSL4,
grain
and
forage
tissues
were
32,
36,
1.9,
and
26
µ
g/
g
dwt,
respectively.
Cry2Ab2
protein
levels
were
less
than
the
assay
LOQ
(
0.625
µ
g/
g)
for
pollen
from
all
lines
analyzed.

Classification:
ACCEPTABLE.

MRID
46694503 
Qualitative
Detection
Method
for
the
Cry2Ab2
Protein
in
Corn
Leaf
and
Seed
of
MON
89034
and
MON
89597 
A
commercially
available
qualitative
immunochromatographic
test
strip
(
QuickStix
 
kit
AS
005
LS)
was
obtained
from
EnviroLogix
Inc.
to
determine
if
the
strips
can
detect
the
Cry2Ab2
protein
produced
in
MON
89034
and
MON
17
89597.
The
QuickStix
 
kit
AS
005
LS
detected
the
presence
of
Cry2Ab2
in
MON
89034
and
89597.
It
was
demonstrated
that
extracts
of
leaves
or
seed
from
MON
89034
or
MON
89597
(
both
expressing
Cry2Ab2)
can
be
distinguished
from
corn
plants
that
do
not
express
the
Cry2Ab2
protein.

MRID
46694601 
Structural
and
Functional
Similarity
of
the
Cry1A.
105
Protein
to
Cry1A
Class
of
Bacillus
thuringiensis
Proteins:
Final
Report 
A
summary
of
current
information
about
the
structural
and
functional
similarities
of
the
Cry1A.
105
protein
to
other
Bt
Cry1
proteins
is
presented
in
this
submission.
The
Cry1A.
105
protein
is
a
chimeric
protein
with
overall
amino
acid
sequence
identity
to
the
Cry1Ac,
Cry1Ab
and
Cry1F
proteins
of
93.6,
90.0
and
76.7%,
respectively.
A
structure
model
of
the
Cry1A.
105
protein
was
developed
using
the
X­
ray
crystal
structure
of
the
Cry1Aa
protein.
The
model
demonstrated
high
overall
main
chain
structural
similarity
with
Cry1Aa.
Models
of
Cry1Ab
and
Cry1Ac
were
also
prepared
using
the
Cry1A.
105
model.
Comparison
of
the
aligned
folds
of
all
three
proteins
showed
that
Cry1Ab
and
Cry1A.
105
have
essentially
the
same
main
chain
structure
and
that
Cry1Ac
differs
slightly
in
its
main
chain
structure
from
the
other
two
in
domain
III.
Thus,
comparison
of
the
modeled
crystal
structures
of
the
Cry1A.
105,
Cry1Ab,
and
Cry1Ac
with
the
experimental
Cry1Aa
X­
ray
crystal
structure
demonstrated
high
structure
similarity
between
the
four
proteins.

Monsanto
also
summarized
results
from
bioactivity
assays
using
Cry1A.
105,
Cry1Ab,
and
Cry1Ac
in
this
submission
and
indicates
that
complete
study
reports
will
be
submitted
for
registration.
Monsanto
states
that
purified
E.
coli­
produced
Cry1A.
105
protein
had
significant
activity
against
each
representative
lepidopteran
insect
larvae
in
laboratory
diet
bioassays.
Tests
species
included;
black
cutworm
(
Agrotis
ipsilon),
corn
earworm
(
Helicoverpa
zea),
fall
armyworm
(
Spodoptera
frugiperda)
and
European
corn
borer
(
Ostrinia
nubilalis).
Cry1A.
105
insecticidal
activity
was
similar
to
other
Cry1
proteins
(
i.
e.,
Cry1Ac,
Cry1F,
Cry1Ab).
Coleopteran
and
heteropteran
larvae
showed
no
indication
of
sensitivity
to
the
Cry1A.
105
protein.
Results
from
testing
purified
Cry1A.
105
protein
against
non­
target
invertebrates
from
different
orders
such
as
honey
bee,
minute
pirate
bug,
earthworms,
parasitic
hymenoptera
and
ladybird
beetle
demonstrated
no
meaningful
activity.
Corn
tissues
from
MON
89034
were
tested
in
a
bioassay
to
assess
potential
activity
of
the
Cry1A.
105
and
Cry2Ab2
proteins
against
Collembola
(
Folsomia
candida),
Daphnia
magna
and
bobwhite
quail
with
results
indicating
no
effect
on
the
tested
non­
target
organisms.

MRID
46694603 
Acute
Oral
Toxicity
Study
in
Mice
with
Cry1A.
105
Protein 
The
Cry1A.
105
test
protein
(
2072
mg/
kg
body
weight)
was
dosed
by
gavage
as
two
doses
separated
by
4
hours
(
±
20
minutes).
The
BSA
protein
control
(
1998
mg/
kg
body
weight)
was
dosed
using
the
same
procedure
as
for
the
test
protein
group.
The
vehicle
control
group
was
dosed
with
carbonatebicarbonate
with
reduced
glutathione.
Body
weight
was
recorded
prior
to
fasting,
prior
to
dosing,
and
on
days
7
and
14.
The
test
animals
were
observed
for
clinical
signs
of
toxicity
two
times
postdosing
and
for
14
days.
A
general
health/
mortality
check
was
done
twice
daily.
All
animals
were
necropsied.
One
male
in
the
test
protein
group
was
moribund
and
sacrificed
on
day
1
due
to
a
mechanical
dosing
error,
which
resulted
in
a
perforated
esophagus.
All
other
mice
survived
the
study.
There
were
no
significant
differences
in
body
weight
or
body
weight
change
among
the
three
groups
during
the
study.
The
oral
LD50
for
males,
females,
and
combined
mice
was
greater
than
2072
mg/
kg.
This
places
Cry1A.
105
Protein
in
TOXICITY
CATEGORY
III
due
to
dose
amounts
only;
no
signs
of
toxicity
were
observed.
Classification:
ACCEPTABLE.
18
MRID
46694604 
Characterization
of
the
Cry1A.
105
Protein
Purified
from
the
Corn
Grain
of
MON
89034
and
Comparison
of
the
Physiochemical
and
Functional
Properties
of
the
Plant­
Produced
and
E.
coli­
Produced
Cry1A.
105
Proteins 
The
physicochemical
properties
and
functional
properties
of
the
plant­
produced
Cry1A.
105
were
analyzed
and
compared
with
the
properties
of
the
E.
coli
produced
Cry1A.
105
using
sodium
dodecyl
sulfate
polyacrylamide
gel
electrophoresis
(
SDS­
PAGE),
western
blot
analysis,
densitometry,
matrix
assisted
laser
desorption
time
of
flight
(
MALDI­
TOF)
mass
spectrometry,
glycosylation
analysis,
and
a
Cry1A.
105
insecticidal
activity
assay.
Similar
immunoreactive
bands
migrating
between
approximately
85
and
130
kDa
were
observed
in
the
plant­
produced
Cry1A.
105
and
E.
coli­
produced
reference
samples,
and
the
full­
length
Cry1A.
105
protein
(~
130
kDa)
was
observed
in
both
the
plant­
produced
and
E.
coli­
produced
protein
samples.
MALDI­
TOF
mass
spectrometry
analysis
of
the
~
130
kDa
band
after
trypsin
digestion
yielded
peptide
masses
consistent
with
peptide
masses
of
the
predicted
sequence
of
the
Cry1A.
105
protein.
The
identified
peptide
masses
yielded
43.8%
overall
coverage
of
the
expected
peptide
sequence
(
516
of
the
1177
amino
acids).
Immunoreactivity
with
the
Nterminal
peptide
antibody
demonstrated
that
the
N­
terminus
in
the
plant­
produced
full­
length
Cry1A.
105
protein
was
intact.
Glycosylation
analysis
demonstrated
that
neither
the
plant­
produced
nor
the
E.
coli­
produced
Cry1A.
105
protein
is
glycosylated.
The
plant­
produced
and
E.
coliproduced
proteins
gave
similar
results
in
the
corn
earworm
diet­
incorporation
bioactivity
assay:
the
mean
EC50
values
for
the
plant­
produced
Cry1A.
105
protein
and
E.
coli­
produced
reference
standard
were
determined
to
be
0.0074
and
0.012
µ
g
Cry1A.
105
per
mL
diet,
respectively.
The
results
of
this
study
demonstrate
the
structural
and
functional
similarity
between
the
plant­
produced
and
the
E.
coli­
produced
Cry1A.
105
proteins.
Classification:
ACCEPTABLE.

MRID
46694605 
Bioinformatics
Analysis
of
the
Cry1A.
105
Protein
Utilizing
the
AD6,
Toxin5,
and
Allpeptides
Databases 
Bioinformatic
analyses
were
used
to
search
for
sequence
similarities
between
the
Cry1A.
105
protein
and
toxins
and
allergens.
The
FASTA
alignment
tool
and
the
allergen
(
AD5),
toxin
(
TOXIN5),
and
public
domain
(
ALLPEPTIDES)
database
sequences
were
used
to
assess
structural
similarity.
No
significant
similarities
were
found,
other
than
with
the
Cry1Ac
protein;
this
alignment
is
not
surprising,
since
the
Cry1A.
105
protein
contains
a
significant
portion
of
the
Cry1Ac
protein.
The
Cry1A.
105
protein
sequence
was
also
screened
against
the
AD5
sequence
database
using
a
pair­
wise
comparison
algorithm.
No
matches
of
8
amino
acids
or
more
were
found
for
the
Cry1A.
105
protein
in
the
AD5
database.
No
similarities
between
Cry1A.
105
protein
and
known
allergens,
human
or
animal
toxins,
or
pharmacologically
active
proteins
were
found
in
the
study.
Classification:
ACCEPTABLE.

MRID
46694606 
Assessment
of
the
In
Vitro
Digestibility
of
the
Cry1A.
105
Protein
in
Simulated
Gastric
Fluid 
No
bands
representative
of
intact
Cry1A.
105
protein
were
identified
by
SDS­
PAGE
after
 
30
seconds
incubation
with
simulated
gastric
fluid
containing
pepsin.
A
very
faint
band
of
4.5
kDaltons
was
observed
between
the
30
second
and
20
minute
digestions
but
was
not
observed
after
20
minutes.
The
limit
of
detection
for
the
SDS­
PAGE
method
was
determined
to
be
5
ng
for
the
full­
length
Cry1A.
105
protein.
Both
the
pepsin
stability
and
test
material
stability
controls
gave
appropriate
responses.
In
the
Western
Blot
assay,
Cry1A.
105
was
not
19
immunologically
identifiable
within
30
seconds
of
incubation.
The
limit
of
detection
for
the
method
was
determined
to
be
1
ng
for
the
full­
length
Cry1A.
105
protein.
The
pepsin
stability
and
test
material
stability
controls
gave
appropriate
responses.
Classification:
ACCEPTABLE.

MRID
46694607 
Immunodetection
of
Cry2Ab2
and
Cry1A.
105
Proteins
in
Corn
Grain
from
MON
89034
Following
Heat
Treatment 
The
immunodetectability
of
the
Cry1A.
105
and
Cry2Ab2
proteins
in
corn
grain
from
MON
89034
following
heat
treatment
was
assessed.
MON
89034
and
conventional
grain
were
ground,
mixed
with
water,
and
then
heated
in
an
oven
at
204
º
C
for
20
minutes
to
simulate
the
heating
process
used
commercially
to
process
grain.
Heated
and
unheated
grain
was
extracted
with
two
buffers:
50
mM
CAPS
and
50
mM
NLS
(
CAPS
containing
2%
N­
Lauroyl
sarcosine).
The
extracts
were
analyzed
using
western
blot
to
detect
the
presence
of
the
Cry1A.
105
and
Cry2Ab2
proteins.
The
amount
of
immunodetectable
Cry2Ab2
protein
in
either
CAPS
or
NLS
buffer
extracts
of
MON
89034
after
heating
was
below
the
lower
LOD.
Based
on
the
LOD
and
the
estimated
amount
of
protein
in
the
unheated
extract,
it
was
determined
that
the
Cry2Ab2
protein
decreased
at
least
77%
and
70%,
respectively
following
heat
treatment,
relative
to
their
original
values.
Likewise,
the
amount
of
immunodetectable
Cry1A.
105
protein
in
either
CAPS
or
NLS
buffer
extracts
of
MON
89034
after
heating
was
below
the
limit
of
detection
(
LOD)
and
had
decreased
by
at
least
94%
and
78%,
respectively,
relative
to
their
original
values.
This
loss
is
likely
due
to
protein
degradation
or
aggregation
into
an
insoluble
complex
as
a
result
of
heat
treatment.
Classification:
ACCEPTABLE.

MRID
45086305 
Assessment
of
the
Equivalence
of
Proteins
Expressed
in
Corn
Event
MON
840 
The
physicochemical
properties
and
functional
properties
of
the
plant­
produced
Cry2Ab2
were
analyzed
and
compared
with
the
properties
of
the
B.
thuringiensis­
produced
Cry2Ab2
using
sodium
dodecyl
sulfate
polyacrylamide
gel
electrophoresis
(
SDS­
PAGE),
western
blot
analysis,
matrix
assisted
laser
desorption
time
of
flight
(
MALDI­
TOF)
mass
spectrometry
(
conducted
prior
to
this
study;
results
summarized
in
appendix),
glycosylation
analysis,
and
a
Cry2Ab2
insecticidal
activity
assay.
In
the
SDS­
PAGE
analysis,
the
plant­
produced
and
B.
thuringiensis­
produced
proteins
both
migrated
at
an
approximate
molecular
weight
of
64
kDa.
In
the
western
blot
analysis,
Monsanto
states
that
both
proteins
comigrated
and
had
similar
immunoreactivity
However,
the
bands
for
the
plant­
produced
protein
appear
to
be
fainter
compared
to
the
B.
t.­
produced
protein,
although
the
same
amounts
of
protein
were
reportedly
loaded
on
the
gel.
MALDI­
TOF
mass
spectrometry
analysis
of
the
plant­
produced
protein
allowed
>
58%
of
the
Cry2Ab2
protein
to
be
identified
using
observed
masses
that
correspond
to
the
expected
masses.
Glycosylation
analysis
demonstrated
that
neither
the
plant­
produced
nor
the
B.
t.­
produced
Cry2Ab2
protein
is
glycosylated.
The
plant­
produced
and
B.
t.­
produced
proteins
gave
similar
results
in
the
corn
earworm
bioactivity
assay:
the
plant­
produced
protein
had
2­
fold
more
activity
than
the
microbially­
produced
protein,
which
falls
within
the
range
of
assay
variability.
The
results
of
these
analyses
demonstrate
the
structural
and
functional
similarity
between
the
plant­
produced
and
the
B.
thuringiensis­
produced
Cry2Ab2
proteins.
The
plant­
produced
NPT
II
protein
was
also
analyzed
and
compared
with
the
bacterially­
produced
protein
to
demonstrate
equivalence.
Classification:
ACCEPTABLE.
For
section
3
registration,
Cry2Ab2
produced
in
the
event
being
registered
should
be
characterized.

Comparison
of
the
genetic
elements
that
regulate
expression
of
the
cry2Ab2
genes
as
20
produced
in
MON
840
and
MON
89034 
This
submission
provides
summary
information
on
the
expression
cassettes
used
to
produce
MON
840
and
MON
89034
corn.
Monsanto
previously
submitted
product
characterization
data
for
MON
840
and
compares
MON
840
with
MON
89034
to
support
bridging
protein
characterization
data
from
MON
840
to
support
the
EUP
for
MON
89034
and
a
temporary
tolerance
exemption
for
Cry2Ab2.
Monsanto
states
that
vector
PVZMIR245
used
to
produce
MON
89034
contains
the
same
cry2Ab2
cassette
as
that
in
vector
ZMBK28,
used
to
produce
MON
840,
except
that
the
e35S
promoter
that
regulates
cry2Ab2
expression
in
MON
840
was
replaced
by
the
FMV
promoter
in
PV­
ZMIR245.
Since
the
cry2Ab2
expression
cassettes
are
so
similar,
data
on
the
plant­
expressed
protein
from
event
MON
840
showing
equivalence
with
the
microbially­
expressed
Cry2Ab2
protein
can
be
used
in
support
of
the
EUP
for
MON
89034
and
MON
89045
x
MON
88017
and
the
temporary
tolerance
exemption.