­
1
­
UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
FEB
15
2006
OFFICE
OF
PREVENTION,
PESTICIDES
AND
TOXIC
SUBSTANCES
MEMORANDUM
SUBJECT:
Review
of
Product
Characterization
and
Human
Health
Data
for
Modified
Cry3A
(
mCry3A)
Bacillus
thuringiensis
insect
control
protein
and
maize
(
Corn)
Plants
Derived
from
Event
MIR604
(
EPA
Reg.
No.
67979­
L)
in
support
for
a
Permanent
Exemption
from
Tolerances
and
Section
3
Registration,
submitted
by
Syngenta
Seeds,
Inc.
 
Field
Crops­
NAFTA.

TO:
Mike
Mendelsohn,
Regulatory
Action
Leader
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

FROM:
Annabel
Fellman,
Environmental
Protection
Specialist
[
signed]
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

THROUGH:
John
L.
Kough,
Ph.
D.,
Biologist
[
signed]
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

ACTION
REQUESTED:
To
review
additional
product
characterization,
human
health
and
analytical
methods
data
submitted
by
Syngenta
Seeds,
Inc.­
Field
Crops
­
NAFTA
in
support
for
section
3
registration
and
a
permanent
exemption
from
tolerances
for
Modified
Cry3A
insect
control
protein
and
maize
(
Corn)
Plants
Derived
from
Event
MIR604.

CONCLUSION:
The
product
characterization,
protein
expression,
toxicological
and
allergenicity
data
support
the
finding
that
there
is
a
reasonable
certainty
of
no
harm
to
humans
from
the
aggregate
exposure
to
the
residues
of
the
mCry3A
protein,
including
all
anticipated
dietary
exposures
and
all
other
exposures
for
which
there
is
reliable
information.

*
REVIEW
DOES
NOT
CONTAIN
CONFIDENTIAL
BUSINESS
INFORMATION*
­
2
­
DATA
REVIEW
RECORD:
Active
Ingredient:
Modified
Cry3A
(
mCry3A)
Bacillus
thuringiensis
insecticidal
protein
and
the
genetic
material
necessary
for
their
production
(
via
pZM26)
in
transgenic
maize
(
corn)
plants
derived
from
Syngenta
Seeds'
transformation
Event
MIR604.
Product
Name:
Event
MIR604
Maize
Plants
Expressing
Modified
Cry3A
Bacillus
thuringiensis
Protein
Company
Name:
Syngenta
Seeds,
Inc.
 
Field
Corps­
NAFTA
ID
No:
67979
Chemical
Number:
006509
Decision
Number:
343070
DP
Barcode:
319746
MRID
No:

465974­
01
Independent
Laboratory
Validation
of
ELISA
method
for
the
Detection
of
mCry3A
in
Event
MIR604
corn
grain
BACKGROUND:
Syngenta
synthetically
modified
a
cry3A
gene
from
Bacillus
thuringiensis
subsp.
tenebrionis
to
optimize
the
gene
for
expression
in
maize
(
corn)
and
to
enhance
its
activity
against
the
western
corn
rootworm
(
WCRM;
Diabrotica
virgifera
vigifera)
and
northern
corn
rootworm
(
NCRM;
D.
longicornis
barberi)
(
Sekar
et
al.,
1987).
Syngenta's
maize
Event
MIR604
corn
plants
were
a
result
of
a
corn
plant
transformation
with
the
synthetic
modified
cry3A
gene,
which
provides
resistance
to
these
pests
(
Chen,
E.
and
Stacy,
C.,
2003).
Transformation
was
conducted
using
immature
maize
embryos
derived
from
a
proprietary
Zea
mays
line,
via
Agrobacterium­
mediated
transformation.
By
this
method,
genetic
elements
within
the
left
and
right
border
regions
of
the
transformation
vector
are
efficiently
transferred
and
integrated
into
the
genome
of
the
plant
cell.

Event
MIR604
maize
also
contains
the
pmi
gene,
which
was
introduced
along
with
the
mCry3A
protein
via
the
same
pZM26
transformation
vector.
The
gene
represents
the
manA
gene
from
Escherichia
coli
and
encodes
the
enzyme
phosphomannose
isomerase
(
PMI),
which
was
employed
as
a
selectable
marker
during
the
process
of
regenerating
plant
material
following
transformation
(
Negrotto,
et
al.,
2000).
The
PMI
protein
is
a
common
enzyme
involved
in
carbohydrate
metabolism
to
allow
for
selection
of
transformants
in
cell
culture,
by
only
allowing
transformed
corn
cells
to
utilize
mannose
as
a
sole
carbon
source,
while
corn
cells
lacking
the
pmi
gene
fail
to
grow.
An
existing
permanent
exemption
from
the
requirement
of
a
tolerance
has
been
established
for
PMI
in
all
crops
when
used
as
a
plant­
incorporated
protectant
inert
ingredient
(
see
40
CFR
180.1252,
effective
May
14,
2004).
This
regulation
eliminates
the
need
to
establish
a
maximum
permissible
level
for
residues
in
or
on
all
plant
commodities
of
phosphomannose
isomerase
and
the
genetic
material
necessary
for
its
production
in
all
plants
when
part
of
a
plant­
incorporated
protectant.

In
the
Federal
Register
of
April
6,
2005
(
70
FR
17323),
the
Agency
established
a
temporary
exemption
from
the
requirement
of
a
tolerance
for
Modified
Cry3A
and
the
genetic
material
­
3
­
necessary
for
their
production
in
corn
which
will
expire
October
15,
2006.
In
addition,
EPA
issued
an
experimental
permit
for
the
use
Bt
mCry3A
protein
and
the
genetic
material
necessary
for
their
production
(
via
plasmid
pZM26)
in
Event
MIR604
corn
and
associated
activities
(
such
as
collection
of
field
data;
harvesting
&
processing
of
seed
after
last
planting)
on
575
acres
of
field
corn.
To
support
their
requests
for
EUP
and
a
temporary
tolerance,
Syngenta
Seeds,
Inc.
submitted
product
characterization
and
protein
expression
analyses,
toxicological
and
allergenicity
data
[
see
memoranda
from
A.
Fellman
through
J.
Kough
to
M.
Mendelsohn,
dated
02/
11/
2005,
02/
23/
2005,
03/
03/
2005,
and
05/
31/
2005].
The
submitted
study
titles,
conclusions,
and
their
MRID
numbers
are
provided
(
see
Table
1)
in
this
report.
The
final
study
to
complete
data
requirements
for
Section
3
registration
for
Modified
Cry3A
protein
and
the
genetic
material
necessary
for
their
production
(
via
plasmid
pZM26)
in
Event
MIR604
corn
is
reviewed
in
this
report
as
well.

Permanent
Exemption
for
the
Requirement
of
a
Tolerance
(
4F6838)
EPA
has
established
an
exemption
from
tolerance
requirements
pursuant
to
FFDCA
section
408(
j)(
3)
for
Bacillus
thuringiensis
Cry3A
delta­
endotoxin
and
the
genetic
material
necessary
for
its
production
in
potatoes,
and
this
tolerance
exemption
has
been
reassessed
and
meets
the
408(
c)(
2)
standard
(
see
40
CFR
180.1147).
An
exemption
from
the
requirement
of
a
tolerance
has
been
established
for
PMI
in
all
crops
when
used
as
a
plant­
incorporated
protectant
inert
ingredient
(
see
40
CFR
180.1252,
effective
May
14,
2004).

In
the
Federal
Register
of
April
6,
2005
(
70
FR
17323),
the
Agency
established
a
temporary
exemption
from
the
requirement
of
a
tolerance
for
Modified
Cry3A
and
the
genetic
material
necessary
for
their
production
in
corn
which
will
expire
October
15,
2006.
In
addition,
EPA
issued
an
experimental
permit
for
the
use
Bt
mCry3A
protein
and
the
genetic
material
necessary
for
their
production
(
via
plasmid
pZM26)
in
Event
MIR604
corn
and
associated
activities
(
such
as
collection
of
field
data;
harvesting
&
processing
of
seed
after
last
planting)
on
575
acres
of
field
corn.

Syngenta
Seeds,
Inc.
 
Field
Crops­
NAFTA
has
submitted
a
petition
for
a
permanent
exemption
from
the
requirement
of
a
tolerance
pursuant
to
section
408(
d)(
1)
of
the
Federal
Food,
Drug,
and
Cosmetic
Act
with
respect
to
the
plant­
incorporated
protectant
modified
Cry3A
Bacillus
thuringiensis
insect
control
protein
and
the
genetic
material
necessary
for
its
production
in
all
corn.

Previously
submitted
studies
demonstrated
the
lack
of
toxicity
of
the
mCry3A
protein
following
acute
oral
high­
dose
exposure
to
mice,
rapid
degradation
of
mCry3A
upon
exposure
to
simulated
mammalian
gastric
fluid
and
the
lack
of
significant
amino
acid
sequence
homology
of
the
mCry3A
protein
to
proteins
known
to
be
mammalian
toxins
or
human
allergens.
Therefore,
dietary
exposure
to
mCry3A
in
corn
is
not
anticipated
to
pose
any
harm
for
the
U.
S.
population
as
stated
in
the
memorandum
from
A.
Fellman,
through
J.
Kough,
Ph.
D.,
to
M.
Mendelsohn,
dated
May
31,
2005.

The
only
outstanding
data
gap
identified
in
the
May
31,
2005
memorandum
was
the
submission
of
a
validated
analytical
method
for
detection
of
mCry3A
in
corn.
A
new
submission
for
the
analytical
method
employing
a
monoclonal
antibody
is
reviewed
(
MRID
No.
465974­
01)
in
this
report.
­
4
­
Preliminary
Safety
Assessment
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
A
safe.@
Section
408(
c)(
2)(
A)(
ii)
of
the
FFDCA
defines
A
safe
@

to
mean
that
A
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.
Section
408(
b)(
2)(
C)
of
the
FFDCA
requires
EPA
to
give
special
consideration
to
exposure
of
infants
and
children
to
the
pesticide
chemical
residue
in
establishing
a
tolerance
and
to
A
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
A
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.

Product
Characterization
Profile
A
mCry3A
Bacillus
thuringiensis
(
Bt)
insect
control
protein
is
produced
in
transgenic
corn
plants
derived
from
transformation
Event
MIR604.
A
cry3A
gene
from
Bt
subsp.
tenebrionis
was
recreated
synthetically
to
optimize
for
expression
in
corn.
Additional
changes
in
this
cornoptimized
gene
were
made,
such
that
the
encoded
mCry3A
protein
has
enhanced
activity
against
larvae
of
the
western
corn
rootworm
(
WCRM;
Diabrotica
virgifera
vigifera)
and
northern
corn
rootworm
(
NCRM;
D.
longicornis
barberi).
Introduced
via
transformation
vector
pZM26,
a
mcry3A
specific
probe,
consisting
of
1797
base
pairs
(
bp),
was
incorporated
between
an
MTL
promoter
(
2556
bp)
from
the
Zea
mays
metallothionein­
like
gene
and
a
terminator
sequence
from
the
nopaline
synthase
(
NOS)
gene
of
Agrobacterium
tumefaciens
used
to
provide
a
polyadenylation
site.
An
Escherichia
coli
manA
gene
encoding
a
phosphomannose
isomerase
pmi
gene
(
1176
bp)
was
incorporated
between
a
promoter
region
from
the
Zea
mays
polyubiquitin
gene
(
ZmUbInt
(
1993
bp))
and
the
same
NOS
terminator
sequence
described
above.
This
pmi
gene,
which
was
introduced
along
with
the
mCry3A
protein
via
the
same
pZM26
transformation
vector,
encodes
the
enzyme
phosphomannose
isomerase
(
PMI),
which
is
employed
as
a
selectable
marker
during
the
process
of
regenerating
plant
material
following
transformation.
The
PMI
protein
is
a
common
enzyme
involved
in
carbohydrate
metabolism
to
allow
for
selection
of
transformants
in
cell
culture,
by
only
allowing
transformed
corn
cells
to
utilize
mannose
as
a
sole
carbon
source,
while
corn
cells
lacking
the
pmi
gene
fail
to
grow.

Hybridization
patterns
indicate
that
one
full
length
copy
of
each
of
the
mcry3A
and
pmi
genes
were
integrated
into
the
maize
genome.
Moreover,
DNA
sequencing
revealed
that
there
was
a
44
and
43
bp
truncation
at
the
right
and
left
break
points
of
the
T­
DNA
insert,
respectively,
during
the
transformation
process
that
resulted
in
MIR604.
Therefore,
the
overall
integrity
of
the
insert
and
the
contiguousness
of
the
functional
elements
were
confirmed.
­
5
­
The
native
Cry3A
protein
of
Bt
subsp.
tenebrionis
is
a
ca.
73
kDa
polypeptide
of
644
amino
acids.
By
comparison,
the
mCry3A
protein
expressed
in
Event
MIR64
corn
is
a
ca.
67
kDa
polypeptide
of
598
amino
acids.
The
amino
acid
sequence
of
the
mCry3A
protein
corresponds
to
that
of
the
native
Cry3A
protein,
except:
1)
Its
N­
terminus
corresponds
to
methionine­
48
of
the
native
protein;
and
2)
A
cathepsin­
G
protease
recognition
site
has
been
introduced,
beginning
at
amino
acid
residue
155
of
the
native
protein.
This
cathepsin­
G
recognition
site
has
the
sequence
alanine­
alanine­
proline­
phenylalanie,
and
has
replaced
the
amino
acids
valine­
155,
serine­
156,
and
serine­
157
in
the
native
protein.

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.

Data
have
been
submitted
demonstrating
the
lack
of
mammalian
toxicity
at
high
levels
of
exposure
to
the
pure
mCry3A
protein.
These
data
demonstrate
the
safety
of
the
products
at
levels
well
above
maximum
possible
exposure
levels
that
are
reasonably
anticipated
in
the
crops.
This
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
by
significant
acute
effects
in
studies
such
as
the
mouse
oral
toxicity
study,
to
verify
the
observed
effects
and
clarify
the
source
of
these
effects
(
Tiers
II
and
III).

An
acute
oral
toxicity
study
was
submitted
for
the
mCry3A
protein.
The
acute
oral
toxicity
data
submitted
support
the
prediction
that
the
mCry3A
protein
would
be
non­
toxic
to
humans.
Male
and
female
mice
(
5
of
each)
were
dosed
with
2,377
milligrams/
kilograms
bodyweight
(
mg/
kg
bwt)
of
mCry3A
protein.
With
the
exception
of
one
female
in
the
test
group
that
was
euthanized
on
day
2
(
due
to
adverse
clinical
signs
consistent
with
a
dosing
injury),
all
other
mice
survived
the
study,
gained
weight,
had
no
test
material­
related
clinical
signs,
and
had
no
test
material­
related
findings
at
necropsy.

When
proteins
are
toxic,
they
are
known
to
act
via
acute
mechanisms
and
at
very
low
dose
levels
(
Sjoblad,
Roy
D.,
et
al.
1992).
Therefore,
since
no
effects
were
shown
to
be
caused
by
the
plant­
incorporated
protectants,
even
at
relatively
high
dose
levels,
the
mCry3A
protein
is
not
considered
toxic.
Amino
acid
sequence
comparisons
showed
no
similarity
between
the
mCry3A
protein
to
known
toxic
proteins
available
in
public
protein
data
bases.
According
to
the
Codex
Alimintarius
guidelines,
the
assessment
of
potential
toxicity
also
includes
stability
to
heat
(
FAO/
WHO
Standards
Programme,
2001).
Further
data
demonstrate
that
mCry3A
is
inactivated
against
WCRM,
when
heated
to
95
°
C
for
30
minutes.

Since
mCry3A
is
a
protein,
allergenic
sensitivities
were
considered.
Currently,
no
definitive
tests
for
determining
the
allergenic
potential
of
novel
proteins
exist.
Therefore,
EPA
uses
a
­
6
­
weight
of
the
evidence
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
acid
and
proteases;
may
be
glycosylated;
and
present
at
high
concentrations
in
the
food.

Data
have
been
submitted
that
demonstrate
that
the
mCry3A
protein
is
rapidly
degraded
by
gastric
fluid
in
vitro.
In
a
solution
of
simulated
gastric
fluid
1
mg/
mE
mCry3A
test
protein
mixed
with
simulated
gastric
fluid
(
pH
1.2,
containing
2
mg/
mL
NaCl,
14
pL
6
N
HC1,
and
2.7
mg/
mL
pepsin)
resulting
in
10
pepsin
activity
units/
jag
protein
(
complies
with
2000
US
Pharmacopoeia
recommendations),
complete
degradation
of
detectable
mCry3A
protein
occurred
within
2
minutes.
A
comparison
of
amino
acid
sequences
of
known
allergens
uncovered
no
evidence
of
any
homology
with
mCry3A,
even
at
the
level
of
8
contiguous
amino
acids
residues.
Further
data
demonstrate
that
mCry3A
is
not
glycoslylated,
is
inactivated
when
heated
to
95
°
C
for
30
minutes,
and
is
present
in
low
levels
in
corn
tissue.

Therefore,
the
potential
for
the
mCry3A
protein
to
be
a
food
allergen
is
minimal.
As
noted
above,
toxic
proteins
typically
act
as
acute
toxins
with
low
dose
levels.
Therefore,
since
no
effects
were
shown
to
be
caused
by
the
plant­
incorporated
protectant,
even
at
relatively
high
dose
levels,
the
mCry3A
protein
is
not
considered
toxic.

Aggregate
Exposures
In
examining
aggregate
exposure,
section
408
of
the
FFDCA
directs
EPA
to
consider
available
information
concerning
exposures
from
the
pesticide
residue
in
food
and
all
other
nonoccupational
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
(
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
protectant
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.
Exposure
via
residential
or
lawn
use
to
infants
and
children
is
also
not
expected
because
the
use
sites
for
the
mCry3A
protein
are
all
agricultural
for
control
of
insects.
Oral
exposure,
at
very
low
levels
may
occur
from
ingestion
of
processed
corn
products
and,
potentially,
drinking
water.

However,
oral
toxicity
testing
done
at
a
dose
in
excess
of
2
gm/
kg
showed
no
adverse
effects.
Furthermore,
the
expression
of
the
modified
Cry3A
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
amounts
of
mCry3A
protein
shown
to
have
no
toxicity.
Therefore,
­
7
­
even
if
negligible
aggregate
exposure
should
occur,
the
Agency
concludes
that
such
exposure
would
present
no
harm
due
to
the
lack
of
mammalian
toxicity
and
the
rapid
digestibility
demonstrated
for
the
mCry3A
protein.

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,
resulting
from
the
plant­
incorporated
protectant,
we
conclude
that
there
are
no
cumulative
effects
for
the
mCry3A
protein.

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
mCry3A
protein
include
the
characterization
of
the
expressed
mCry3A
protein
in
corn,
as
well
as
the
acute
oral
toxicity,
heat
stability,
and
in
vitro
digestibility
of
the
proteins.
The
results
of
these
studies
were
determined
applicable
to
evaluate
human
risk,
and
the
validity,
completeness,
and
reliability
of
the
available
data
from
the
studies
were
considered.

Adequate
information
was
submitted
to
show
that
the
mCry3A
protein
test
material
derived
from
microbial
cultures
was
biochemically
and
functionally
similar
to
the
protein
produced
by
the
plant­
incorporated
protectant
ingredients
in
corn.
Microbially
produced
protein
was
chosen
in
order
to
obtain
sufficient
material
for
testing.

The
acute
oral
toxicity
data
submitted
supports
the
prediction
that
the
mCry3A
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.
1992).
Since
no
effects
were
shown
to
be
caused
by
mCry3A
protein,
even
at
relatively
high
dose
levels
(
2,377
mg
mCry3A/
kg
bwt),
the
mCry3A
protein
is
not
considered
toxic.
This
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)).
Moreover,
mCry3A
showed
no
sequence
similarity
to
any
known
toxinand
was
inactivated
by
heat
against
WCRM.

Protein
residue
chemistry
data
for
mCry3A
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
mCry3A
in
corn
tissues
with
less
than
2
micrograms
mCry3A
protein/
gram
dry
weight
in
kernels
and
less
than
30
micrograms
mCry3A
protein/
gram
dry
weight
of
whole
corn
plant.

Since
modified
Cry3A
is
a
protein,
its
potential
allergenicity
is
also
considered
as
part
of
the
toxicity
assessment.
Data
considered
as
part
of
the
allergenicity
assessment
include
that
the
modified
Cry3A
protein
came
from
Bacillus
thuringiensis
which
is
not
a
known
allergenic
­
8
­
source,
showed
no
sequence
similarity
to
known
allergens,
was
readily
degraded
by
pepsin,
and
was
not
glycosylated
when
expressed
in
the
plant.
Therefore,
there
is
a
reasonable
certainty
that
modified
Cry3A
protein
will
not
be
an
allergen.

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
mCry3A
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
ingredients
are
the
nucleic
acids
(
DNA,
RNA)
which
comprise
genetic
material
encoding
these
proteins
and
their
regulatory
regions.
The
genetic
material
(
DNA,
RNA)
necessary
for
the
production
of
mCry3A
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(
bfl2)(
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
data
base
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
mCry3A
protein
and
the
genetic
material
necessary
for
their
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
provisions
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
mCry3A
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
previously
discussed,
no
toxicity
to
mammals
has
been
observed,
nor
any
indication
of
allergenicity
potential
for
the
plant­
incorporated
protectant.

Other
Considerations
A.
Endocrine
Disruptors
The
pesticidal
active
ingredient
is
a
protein,
derived
from
sources
that
are
not
known
to
exert
­
9
­
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
method
for
extraction
and
ELISA
analysis
of
mCry3A
protein
in
corn
has
been
submitted
and
found
acceptable
by
the
Agency.

C.
Codex
Maximum
Residue
Level
No
Codex
maximum
residue
levels
exist
for
the
plant­
incorporated
protectant
Bacillus
thuringiensis
mCry3A
protein
and
the
genetic
material
necessary
for
its
production
in
corn.

RECOMMENDATION:
As
previously
noted,
there
is
a
reasonable
certainty
that
no
harm
will
result
from
aggregate
exposure
to
the
U.
S.
population,
including
infants
and
children,
to
the
mCry3A
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.
Therefore,
the
product
characterization
and
human
health
data
submitted
are
sufficient
to
support
Section
3
registration
and
a
permanent
exemption
from
the
requirement
of
a
tolerance.

The
data
submitted
for
the
ELISA
method
for
determining
mCry3A
protein
in
Event
MIR604
grain
are
classified
as
ACCEPTABLE
and
satisfies
the
EPA
Residue
Chemistry
Guidelines
OPPTS
860.1340(
c)(
6)
Residue
Analytical
Methods
and
PR
Notice
96­
1.
However,
the
EPA's
Analytical
Method
Laboratory
located
in
Fort
Meade
(
Maryland)
will
have
to
independently
validate
Syngenta's
ELISA
protocol
for
accuracy,
precision,
and
sensitivity.
­
10
­
Summaries
of
each
review
supporting
the
safety
findings
in
the
areas
of
product
characterization,
human
toxicity,
and
allergenicity
for
this
product
are
provided
below.

Table
1.
Previously
submitted
product
characterization,
toxicity,
and
allergenicity
studies
MRID
Title
Summary
461556­
01
Review
of
Characterization
and
Safety
of
Modified
Cry3A
protein
and
maize
(
corn)
plants
derived
from
Event
MIR604
with
comparison
to
native
Cry3A
protein
The
mCry3A
protein
contains
598
amino
acids
(
ca.
67
kDa),
whereas
the
native
Cry3A
protein
is
ca.
73
kDa
polypeptide
of
644
amino
acids.
The
amino
acid
sequence
of
the
mCry3A
protein
corresponds
to
that
of
the
native
Cry3A
protein,
except:
1)
Its
Nterminus
corresponds
to
methionine­
48
of
the
native
protein;
and
2)
A
cathepsin­
G
protease
recognition
site
has
been
introduced,
beginning
at
amino
acid
residue
155
of
the
native
protein.
The
susceptibility
of
insect
pest
species
and
insecticidal
properties,
such
as
solubilization,
proteolytic
processing,
receptor
binding,
and
membrane
pore
forming
properties,
were
also
determined
for
mCry3A
protein
and
differentiated
with
native
Cry3A
protein.
The
mCry3A
protein
has
a
similar
spectrum
of
activity
to
the
native
Cry3A,
but
with
enhanced
toxicity
to
NCRW
and
WCRW.

Classification:
Acceptable
461556­
02
Molecular
characterization
of
event
MIR604
maize
(
corn)
expressing
a
modified
Cry3A
bacillus
thuringiensis
protein
Corn
Event
MIR604
does
not
contain
any
of
the
backbone
sequences
from
the
transforming
plasmid
pZM26.
Three
nucleotide
changes
were
identified,
one
in
a
regulatory
region
of
the
mCry3A
gene
and
two
in
the
pmi
coding
sequence.
The
mCry3A
and
pmi
genes
are
closely
linked.

Classification:
Acceptable
461556­
03
Characterization
of
modified
Cry3A
protein
produced
in
event
MIR604­
derived
maize
(
corn)
and
comparison
with
modified
Cry3A
protein
expressed
in
recombinant
Escherichia
coli
This
study
evaluated
the
similarity
of
modified
Cry3A
(
mCry3A)
insecticidal
protein
expressed
in
corn
event
MIR604
and
mCry3A
protein
expressed
in
a
recombinant
E.
coli
test
system.
The
mCry3A
protein
derived
from
corn
event
MIR604
and
recombinant
E.
coli
had
the
same
approximate
molecular
weight
(
ca.
67,700
Da.)
based
on
mass
spectral
analysis
(
from
MRID
461556­
06).
The
mCry3A
protein
from
both
sources
was
immunologically
cross­
reactive
with
the
same
anti­
mCry3A
antibody.
Both
proteins
produced
comparable
toxicities
toward
Western
Corn
Rootworm
larvae,
based
on
LC50
values.
There
was
no
evidence
of
post­
translational
glycosylation
of
mCry3A
protein
from
either
source.
It
was
concluded
that
the
mCry3A
proteins
from
corn
event
MIR604
and
from
recombinant
E.
coli
were
substantially
the
same.

Classification:
Acceptable
­
11
­
461556­
04
Quantification
of
modified
Cry3A
and
pmi
proteins
in
transgenic
maize
(
corn)
tissues,
whole
plants,
and
silage
derived
from
transformation
event
MIR604
The
plant
extracts
(
including
leaves,
roots,
kernels,
silk,
pollen,
silage,
and
whole
plants)
from
inbred
and
hybrid
corn
lines
derived
from
MIR604
field
plants
were
quantitatively
analyzed
for
mCry3A
by
ELISA.
The
magnitude
of
expression
for
tissue
types
was
as
follows
in
descending
order:
leaves,
roots,
silage,
and
kernels.
All
control
tissues
were
negative
for
the
expression
of
mCry3A.
The
mean
extraction
efficiency
for
mCry3A
over
all
tissues
(
except
for
pollen
or
silk
tissue,
where
levels
were
too
low
to
be
determined)
was
76.6%.
Low,
but
quantifiable,
levels
of
PMI
protein
were
found
in
most
of
the
Event
MIR604­
derived
plant
tissues
analyzed
including
pollen.
The
mCry3A
and
PMI
proteins
were
stably
expressed
in
four
backcross
generations
in
leaf
tissue
analyzed
at
anthesis
stage.
Mean
levels
across
all
backcross
generations
were
ca.
11.8
­
15.5
µ
g/
g
dry
weight
and
1.1
­
1.3
µ
g/
g
dry
weight
for
mCry3A
and
PMI
proteins,
respectively.

Classification:
Acceptable
461556­
05
Characterization
of
modified
Cry3A
test
substance
mCry3A­
0102
and
certificate
of
analysis
This
study
characterized
test
material
MCRY3A­
0102,
a
microbially
produced
protein
preparation
containing
a
modified
Cry3A
(
mCry3A)
protein.
The
purity
of
mCry3A
in
the
test
material
was
shown
to
be
ca.
90.3%
by
weight
using
SDS­
PAGE
analysis.
Western
blot
analysis
of
the
test
material
showed
a
single
immunoreactive
band
corresponding
to
the
predicted
molecular
weight
of
ca.
67,700
Da.
The
test
material
was
insecticidally
active
and
had
a
144­
hour
LC50
of
1.4
µ
g/
mL
diet
(
95%
confidence
interval:
0.7
­
2.2
µ
g/
mL)
against
Western
corn
rootworm
(
WCRW)
larvae.
Two
forms
of
mCry3A
were
found
in
the
test
material,
designated
mCry3A­
SF
and
mCry3A­
LF,
respectively,
and
were
both
insecticidally
active
against
WCRW.
On
this
basis,
and
taking
into
account
the
high
degree
of
structural
homology
(
97.4%
amino
acid
identity),
the
two
forms
of
mCry3A
in
test
material
MCRY3A­
0102
were
considered
to
be
equivalent.
The
test
material
was
re­
analyzed
ca.
9
months
after
its
initial
characterization
and
found
to
be
substantially
stable
when
stored
at
­
20
°
C.

Classification:
Acceptable
461556­
06
Further
Characterization
of
Modified
Cry3A
Test
Substance
MCRY3A­
0102
The
test
material,
MCRY3A­
0102,
containing
mCry3A
protein
was
shown,
by
SDS­
PAGE
and
MALDI
TOF
mass
spectrometry,
to
contain
two
closely
related
components
in
a
ratio
of
ca.
2:
3.
The
lesser
of
the
two
components,
with
the
lower
molecular
weight,
corresponded
to
the
intended
mCry3A
protein
with
598
amino
acids.
The
other
component
contained
the
same
598
amino
acids
as
the
first
component
but
also
contained
an
additional
16
amino
acids
at
the
N 
terminal
end
of
the
protein.
The
identity
of
the
two
components
was
conclusively
determined
by
peptide
mapping
using
tandem
(
MS/
MS)
mass
spectrometry
and
by
MALDI
TOF
mass
spectrometry
of
the
intact
proteins.
The
molecular
weight
data
showed
the
two
proteins
to
have
masses
of
67,519
and
69,138
Da.

Classification:
Acceptable
­
12
­
461556­
07
In
vitro
digestibility
of
modified
Cry3A
protein
(
mCry3A­
0102
and
IAPMIR604­
0103)
under
simulated
mammalian
gastric
conditions
The
susceptibility
of
mCry3A
protein
to
proteolytic
degradation
was
evaluated
in
simulated
gastric
fluid
(
SGF)
containing
pepsin.
Modified
Cry3A
protein
from
transgenic
corn
and
recombinant
E.
coli
(
test
material
MCRY3A­
0102)
was
readily
degraded
in
SGF.
The
data
support
a
conclusion
that
mCry3A
protein
expressed
in
transgenic
plants
will
be
readily
digested
as
conventional
dietary
protein
under
typical
mammalian
gastric
conditions.

Classification:
Acceptable
461556­
08
Effect
of
temperature
on
the
stability
of
modified
Cry3A
protein
(
mCry3A­
0102)
At
95
°
C
mCry3A
protein
was
completely
inactivated
against
WCRM.
At
4
°
C,
25
°
C,
and
37
°
C
there
was
little
or
no
effect
on
mCry3A
bioactivity,
while
at
65
°
C
there
was
some
reduction
in
the
bioactivity.

Classification:
Acceptable
461556­
09
Analysis
for
the
presence
of
modified
Cry3A
protein
in
wet
and
dry
milled
fractions,
corn
oil
and
corn
chips
from
corn
(
maize)
event
MIR604
Among
the
wet­
milled
fractions,
the
medium
fiber
(
0.46
µ
g
mCry3A/
g),
fine
fiber
(
0.26
µ
g
mCry3A/
g),
and
gluten
meal
(
0.24
µ
g
mCry3A/
g)
fractions
yielded
quantifiable
amounts
of
mCry3A.
Among
the
dry­
milled
fractions
the
highest
concentrations
were
found
in
the
flaking
grits
(
2.12
µ
g
mCry3A/
g),
the
corn
hulls
(
1.42
µ
g
mCry3A/
g),
and
the
coarse
grit
(
0.92
µ
g
mCry3A/
g)
fractions.
Levels
of
mCry3A
found
in
the
other
dry­
milled
fractions,
including
fine
grits,
corn
meal,
corn
cone
and
corn
flour,
were
between
0.32
and
0.69
µ
g
mCry3A/
g.
Although
the
concentration
of
mCry3A
protein
measured
in
the
flour
used
to
prepare
the
corn
chips
was
0.32
µ
g
mCry3A
/
g,
no
mCry3A
protein
was
detected
in
the
corn
chips.
Similarly,
mCry3A
protein
was
not
detectable
in
oil,
whereas
the
starting
material,
flaking
grits,
contained
2.12
µ
g
mCry3A/
g.

Classification:
Acceptable
461556­
10
Acute
oral
toxicity
study
of
modified
Cry3A
protein
(
mCry3A­
0102)
in
the
mouse
MCRY3A­
0102
was
not
acutely
toxic
to
mice.
There
was
no
evidence
of
toxicity
at
2,632
mg
MCry3A­
0102/
kg
body
weight,
representing
ca.
2,377
mg
mCry3A
protein/
kg
body
weight.
The
estimated
LD50
value
for
pure
mCry3A
protein
in
male
and
female
mice
was
>
2,377
mg/
kg
body
weight,
the
single
dose
used.

Classification:
Acceptable
461556­
11
Modified
Cry3A
protein
as
expressed
in
transgenic
maize
event
MIR604:
assessment
of
amino
acid
homology
with
known
toxins
The
National
Center
for
Biotechnology
Information
(
NCBI)
GenBank
Database
(
NCBI,
2003)
containing
all
publicly
available
protein
sequences
was
queried
for
proteins
with
amino
acid
sequences
having
significant
homology
to
mCry3A
protein
and
that
were
toxins.
The
query
found
no
significant
amino
acid
homology
between
any
protein
toxin
and
the
mCry3A
protein.

Classification:
Acceptable
­
13
­
461556­
12
Modified
Cry3A
protein
as
expressed
in
transgenic
maize
event
MIR604:
assessment
of
amino
acid
homology
with
known
allergens
No
significant
similarity
was
found
between
any
of
the
mCry3A
80­
amino
acid
peptides
and
any
entries
in
the
SBI
Allergen
Database.
Also,
there
were
no
alignments
of
eight
or
more
contiguous
amino
acids
between
the
mCry3A
protein
and
any
of
the
proteins
in
the
allergen
database.
Overall,
the
mCry3A
protein
showed
no
significant
amino
acid
homology
to
any
known
or
putative
allergenic
protein.

Classification:
Acceptable
465974­
01
Analytical
Method
for
the
Detection
of
the
Plant­
Incorporated
Protectant
Modified
Cry3A
Protein
in
Event
MIR604
Corn
Grain
and
Independent,
Third­
Party
Validation
of
Said
Method
An
ELISA
method
to
quantify
modified
Cry3A
(
mCry3A)
protein
in
corn
tissues
was
validated
by
an
independent
third­
party
laboratory
(
EnvironLogix,
Inc.)
using
grain
from
Event
MIR604,
which
contains
mCry3A,
and
25
other
commercially­
available
transgenic
and
non­
transgenic
corns
which
do
not
contain
mCry3A.
The
average
mCry3A
LOD
for
three
different
lots
of
reagents
tested
was
5.5
±
4.6
ppb,
which
established
the
sensitivity
of
the
ELISA
assay
at
approximately
25%.
In
a
ground
grain
assay,
the
mean
mCry3A
protein
detected
was
159.1
±
70.2
ng/
g
fresh
weight
with
a
coefficient
of
variation
(%
CV)
of
44.1%.
However,
the
data
submitted
showed:
less
than
optimal
LOD;
low
sensitivity;
high
standard
deviations;
the
%
CV
exceeding
the
recommended
value
(
20%);
anomalous
frequency
of
false
positives
in
the
non­
MIR604
corn
(
both
transgenic
and
conventional)
as
well
as
false
negatives
in
the
MIR604
corn;
and
unexpected
cross
reactivity
with
other
Cry3Bb1
samples.

Classification:
Supplemental
461556­
13
Phosphomannose
Isomerase
as
expressed
in
transgenic
maize
event
MIR604:
assessment
of
amino
acid
homology
with
known
toxins
Two
nucleotide
changes
were
discovered
in
the
pmi
gene
sequence
inserted
in
corn
Event
MIR604.
This
resulted
in
two
changes
in
the
PMI
protein;
valine­
61
was
replaced
by
alanine,
and
glutamine­
210
was
replaced
by
histidine.
These
substitutions
have
not
resulted
in
any
apparent
functional
change
in
the
PMI
protein.
The
NCBI
GenBank
Database
containing
all
publicly
available
protein
sequences
was
queried
for
proteins
with
amino
acid
sequences
having
significant
homology
to
this
modified
PMI
protein
that
were
toxins.
The
query
found
no
significant
amino
acid
homology
between
any
protein
toxin
and
the
PMI
protein
expressed
in
corn
Event
MIR604.

Classification:
Acceptable
464252­
01
Phosphomannose
Isomerase
as
expressed
in
transgenic
maize
event
MIR604:
assessment
of
amino
acid
homology
with
known
allergens
No
significant
similarity
was
found
between
any
of
the
PMI
80­
amino
acid
peptides
and
any
entries
in
the
SBI
Allergen
Database.
However,
in
the
eight
or
more
contiguous
amino
acids
homology
search,
there
was
an
alignment
between
the
PMI
protein
and
a
recently
identified
allergen,
 ­
parvalbumin
from
Rana
species
CH2001
(
a
frog
of
Indonesian
origin).
However,
a
serum
screening
concluded
that
there
is
no
cross­
reactivity
between
PMI
and
serum
IgE
(
obtained
from
an
allergic
individual
who
displayed
food­
induce
anaphylaxis
from
 ­
parvalbumin).
Bovine
serum
albumin
was
also
tested
as
an
internal
check.
The
observed
low
degree
of
sequence
identity
between
MIR604
PMI
and
 ­
parvalbumin
is
not
biologically
relevant.

Classification:
Acceptable
­
14
­
465974­
01
Independent
Laboratory
Validation
of
Monoclonal­
based
ELISA
method
for
the
Detection
of
mCry3A
in
Event
MIR604
corn
grain
A
monoclonal
based
enzyme­
linked
immunosorbent
assay
(
ELISA)
method
was
developed
by
Syngenta
to
detect
modified
Cry3A
(
mCry3A)
protein
expressed
in
MIR604
seed
and
leaf
tissues.
An
independent,
third
party
laboratory
(
EnvironLogix,
Inc.)
utilized
the
assay
methodology,
Event
MIR604
seed,
negative
isoline
seed,
and
three
lots
of
mCry3A
monoclonal
antibody
for
validation
of
assay
protocol,
sensitivity
and
cross­
reactivity,
according
to
the
USDA
GIPSA
directive.
The
assay
sensitivity
was
estimated
at
1
positive
Event
MIR604
kernel
in
999
non­
MIR604
kernels
(
0.1%),
which
was
based
on
a
minimum
of
120
ground
seed
samples
(
with
a
LOD
of
0.33
ppb
for
seed
material).
The
average
quantification
was
2.07
ppb
(
ng/
g
fresh
weight
corn).
No
cross
reactivity
with
other
commercial,
conventional
and
transgenic
corn
was
detected
with
the
exception
of
products
expressing
the
Cry3Bb1
protein.
Moreover,
extraction
efficiency
was
determined
at
59%
for
ground
seed
and
75%
for
leaf
tissue.

CLASSIFICATION:
ACCEPTABLE­
The
monoclonal
assay
antibody­
based
commercial
ELISA
detection
assay
(
tested
by
EnviroLogix,
Inc.)
satisfies
the
EPA
Residue
Chemistry
Guidelines
OPPTS
860.1340(
c)(
6)
Residue
Analytical
Methods
and
PR
Notice
96­
1.
However,
the
EPA's
Analytical
Method
Laboratory
located
in
Fort
Meade
(
Maryland)
will
have
to
independently
validate
Syngenta's
ELISA
protocol
for
accuracy,
precision,
and
sensitivity.

REFERENCES
Chen,
E.
&
Stacy,
C.
(
2003)
Modified
Cry3A
toxins
and
nucleic
acid
sequences
coding
therefore.
WO
Patent
No.
03/
018810.

Food
and
Agriculture
Organization
of
the
United
Nations
and
World
Health
Organization.
(
2003).
Foods
Derived
from
Biotechnology.
Codex
Alimentarius.
Sec.
4,
No.
38,
pg.
16.

Negrotto,
et
al.
(
2000)
The
use
of
phosphomannose­
isomerase
as
a
selectable
marker
to
recover
transgenic
maize
plants
(
Zea
mays
L.
via
Agrobacterium
transformation.
Plant
Cell
Reports,
19:
798­
803.

Sekar
et
al.
(
1987)
Molecular
cloning
and
characterization
of
the
insecticidal
crystal
protein
gene
of
Bacillus
thuringiensis
var.
tenebrionis.
Proc.
Natl.
Acad.
Sci.
USA
8:
7036­
7040.

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