Document ID: EPA-HQ-OPP-2006-0035-0043
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2006-03-20T05:00Z

Introduction
&
Background
­
MIR604
Modified
Cry3A
Protein
Bt
Corn
Mike
Mendelsohn
Senior
Regulatory
Specialist
Environmental
Protection
Agency
Office
of
Pesticide
Programs
2
Background

Native
Cry3A
is
currently
found
in
several
microbial
insecticide
products
and
as
a
plantincorporated
protectant
(
PIP)
in
Bacillus
thuringiensis
(
Bt)
potato.

Syngenta
Seeds,
Inc.
synthetically
recreated
a
cry3A
gene
from
Bt
subsp.
tenebrionis
to
optimize
for
expression
in
maize
(
corn)
with
enhanced
activity
against
WCRM
and
NCRM.

Result
 
Event
MIR604
corn
plants
with
resistance
to
these
pests.
3
Regulatory
Status

Syngenta
Seeds,
Inc.
seeks
to
commercialize
the
plant­
incorporated
protectant
(
PIP)
Bt
Modified
Cry3A
and
the
genetic
material
necessary
for
its
production
in
corn
plants
derived
from
Event
MIR604.

Experimental
Usage
Permit
(
EUP)
issued
for
field
testing
prior
to
commercialization.

Temporary
tolerance
exemption
issued
and
extended.
4
Current
mCry3A
Approvals

Experimental
Use
Permit,
EPA
EUP
No.
67979­

EUP­
4

Allows
Testing
of
6,894
Acres
of
Corn

Temporary
Tolerance
Exemption,
40
CFR
Part
174.456

Permits
the
use
of
the
corn
food
commodities
when
treated
in
accordance
with
the
provisions
of
the
experimental
use
permit
67979­

EUP­
4
5
Current
mCry3A
Applications

Full
Commercial
Use
Registration
EPA
File
Symbol
67979­
L
Would
Allow
MIR604
to
Be
Sold
To
and
Planted
By
Farmers
6
Current
MIR604
Applications

Permanent
Tolerance
Exemption
Pesticide
Petition
Number
PP
4F6838
Would
Allow
mCry3A
Residues
in
Corn
Food
Commodities
with
No
Numerical
Limit
7
MIR604
Risk
Assessments

Human
Health
Risk
Assessment
Used
to
Determine
Whether:

Reasonable
Certainty
of
No
Harm,
A
Finding
Necessary
for
Tolerance
Exemption
No
Unreasonable
Adverse
Effects,
A
Finding
Necessary
to
Support
Registration
8
MIR604
Risk
Assessments

Ecological
Risk
Assessment
Used
to
Determine
Whether
No
Unreasonable
Adverse
Effects,
A
Finding
Necessary
to
Support
Registration
9
Environmental
Effects
Assessment
for
MIR604,
Modified
Cry3A
Corn
Tessa
S.
Milofsky,
M.
S.
Agronomist
and
Mika
J.
Hunter,
Biologist
U.
S.
Environmental
Protection
Agency
Office
of
Pesticide
Programs
11
Outline

Hazard
Assessment
Process

Risk
Assessment
Process

Summary
of
Event
MIR604
Environmental
Assessment

Introduction
to
SAP
Question
12
Environmental
Hazard
Assessment
Process

Assessment
includes:

 
Effect
on
wildlife
 
Fate
in
environment
(
e.
g.
soil
fate,
HGT,
gene
flow)

 
Effect
on
endangered
species

Assessment
based
on:

 
Data
submitted
to
the
Agency
 
FIFRA
SAP
recommendations
 
Consultations
with
scientific
experts
 
Public
comments
13
Environmental
Hazard
Assessment
Process

Tiered
testing
system
 
Assess
toxicity
to
representative
nontarget
organisms
 
Tier
I
single
species
testing

Useful
for
short­
term
hazard
assessment

Maximum
Hazard
Dose
(
MHD)
approach
 
MHD
 
10
x
Expected
Environmental
Concentration
(
EEC)

Mortality
as
end
point

Negative
results
provide
high
degree
of
confidence
that
no
unreasonable
adverse
effects
are
likely
to
occur
 
Higher
tier
testing
(
Tier
II­
IV)

Useful
for
assessment
of
long­
term
effects

May
require
multiple
year
field
testing
14
Environmental
Hazard
Assessment
Process

SAP
guidance
for
tiered
testing
of
Bt
PIPs:

 
1996
­
MHD
approach
recommended
for
naturally
occurring
and
altered
active
ingredients
and
toxins
 
1999
 
MHD
approach
appropriate
for
assessment
of
protein
PIPs
 
2000
 
NAS
and
SAP
recommended
that
direct
field
testing
should
be
included
as
component
of
hazard
assessment
 
2002
 
agreed
that
both
Tier
I
and
higher
tier
(
e.
g.
field
testing)
studies
should
be
used
for
hazard
assessment,
with
emphasis
on
Tier
I
testing
of
invertebrates
exposed
to
the
crop
in
field
environment
15
Environmental
Risk
Assessment
Process
Risk
=
Hazard
x
Exposure
16
Environmental
Risk
Assessment
Process

LC
50
/
LD
50
>
MHD
=
"
no
adverse
effect"

assessment

LC
50
/
LD
50
<
MHD
=
lower
dose
testing
necessary
to
establish:

 
Definitive
LD
50
 
Lowest
Observable
Effect
Concentration
(
LOEC)

EEC
<
LOEC
=
"
no
adverse
effect"
assessment
17
Nontarget
Wildlife
Toxicity
Testing

Testing
complies
with
Tier
I
microbial
toxin
requirements
(
Test
Guideline
Series
885)

Toxicity
evaluated
for:

 
Mammals
 
Birds
 
Fish
 
Terrestrial
and
aquatic
invertebrates
 
Earthworms
 
Plants
18
Nontarget
Wildlife
Toxicity
Testing:

Test
Substance

Pesticide:
Modified
Cry3A
protein
(
mCry3A)

 
Synthetic
cry3A
gene
derived
from
Bacillus
thuringiensis
tenebrionis,
optimized
for
expression
in
corn,
with
enhanced
activity
against
WCRM
and
NCRM

Source
of
test
substance:

 
Plant
material
(
e.
g.
corn
grain)

 
Bacteria­
produced
purified
protein
(
MCRY3A­
0102)

2000
SAP
guidance:

 
Plant
material
preferred
 
Bacteria­
derived
protein
also
valid
where:

Test
animals
do
not
consume
plant
tissue

Large
amounts
of
Cry
protein
are
needed
for
MHD
testing

Plant
material
toxic
(
e.
g.
gossypol
in
cotton)
19
Nontarget
Wildlife
Toxicity
Testing:

Mammalian
Wildlife

Study
type:
Wild
Mammal
Testing,
Tier
1

Test
animal:
Mouse
(
Mus
musculus);
10
mice

Test
material/
dosage:
2,377
mg
MCRY3A­
0102/
kg
body
weight

Control
treatment:
Negative

Test
duration:
Single
oral
dose;
14­
day
observation
period

Results:
No
significance
in
clinical
signs,
body
weight,

microscopic
or
macroscopic
findings
(
necropsy)
between
MCRY3A
and
negative
control.

Hazard
assessment:
No
hazard
to
mammalian
wildlife
is
anticipated.
biology.
plosjournals.
org
biology.
plosjournals.
org
biology.
plosjournals.
org
20
Nontarget
Wildlife
Toxicity
Testing:

Avian
Wildlife

Study
type:
Avian
Acute
Oral
Toxicity
Test,
Tier
1

Test
animal:
Young
adult
Northern
Bobwhite
quail
(
Colinus
virginianus);
20
birds

Test
material/
dosage:
Single
dose
of
722
mg
MCRY3A­

0102/
kg
body
weight

Control
treatment:
Negative

Test
duration:
Single
dose;
14­
day
observation
period

Results:
No
significant
difference
in
body
weight
and
feed
consumption
was
seen
between
MCRY3A
and
negative
control
groups.
Acute
oral
LD50
>
722
mg
MCRY3A­

0102/
kg
body
weight.

Hazard
assessment:
No
avian
hazard
expected
from
incidental
dietary
exposure.
Six­
week
broiler
study
required
to
address
effects
of
long­
term
exposure.
www.
fnal.
gov
21
Nontarget
Wildlife
Toxicity
Testing:

Avian
Wildlife

Study
type:
Avian
Oral,
Tier
1

Test
animal:
Broiler
chickens
(
Gallus
domesticus);
300
birds

Test
material/
dosage:
Diet
contained
57.5
­
67.5%
Event
MIR604
corn
grain

Control
treatment:
Negative

Test
duration:
49­
day
feeding
study

Results:
No
significant
difference
in
carcass
weight
and
mortality
was
seen
between
mCry3A
and
negative
control.

Hazard
assessment:
No
hazard
to
avian
species
is
anticipated
from
continuous
dietary
exposure.
www.
hi.
is
22
Nontarget
Wildlife
Toxicity
Testing:

Aquatic
Wildlife

Study
type:
Freshwater
Fish
Testing,
Tier
1

Test
animal:
Juvenile
Rainbow
Trout
(
Onchorhynchus
mykiss);
40
fish

Test
material/
dosage:
Diet
contained
50%
Event
MIR604
corn
grain

Control
treatments:
Negative

Test
duration:
28­
day
feeding
study

Results:
No
significant
differences
in
fish
weight,
length,
or
mortality
were
seen
among
treatment
groups.

Hazard
assessment:
No
hazard
to
fish
species
is
anticipated
from
continuous
dietary
exposure.
www.
cnr.
vt.
edu
23
Nontarget
Wildlife
Toxicity
Testing:

Aquatic
Wildlife

Study
type:

 
Estuarine
and
Marine
Animal
Testing,
Tier
1
 
Aquatic
Invertebrate
Acute
Toxicity
Test,
Tier
1

Waiver
justification:

 
Low
potential
for
aquatic
exposure
to
corn

Hazard
assessment:
No
hazard
to
aquatic
animals
is
expected
from
incidental
exposure.
24
Nontarget
Wildlife
Toxicity
Testing:

Terrestrial
Invertebrates

Study
type:
Nontarget
Insect
Testing,
Tier
1

Test
animal:
Lady
beetle
(
Coccinella
septempunctata);
120
larvae

Test
material/
dosage:
9
µ
g
MCRY3A­
0102/
g
aphid

Control
treatments:
Negative,
positive
(
teflubenzuron)

Test
duration:
Feeding
period
from
study
initiation
to
14
days
following
adult
emergence
(
no
feeding
during
pupation)

Results:
No
significant
difference
in
pre­
imaginal
or
adult
survival
between
MCRY3A
and
negative
control;
100%
pre­
imaginal
mortality
among
larvae
in
positive
control.
No
significant
difference
in
rate
of
pupal
development
between
MCRY3A
and
negative
control;
time
to
adult
emergence
significantly
shorter
for
MCRY3A
treatment,
compared
to
negative
control.

Hazard
assessment:
No
hazard
to
lady
beetles
is
anticipated
at
field
exposure
levels.
insektenfotos.
de
www.
gardensafari.
net
25
Nontarget
Wildlife
Toxicity
Testing:

Terrestrial
Invertebrates

Study
type:
Nontarget
Insect
Testing,
Tier
1

Test
animal:
Carabid
beetle
(
Poecilus
cupreus);
120
larvae

Test
material/
dosage:
12
µ
g
MCRY3A­
0102/
g
pupa

Control
treatments:
Negative,
positive
(
teflubenzuron)

Test
duration:
24
to
48
hour­
old
larvae
fed
daily
until
pupation;

32
day
observation
period
(
until
adult
emergence).

Results:
No
significant
difference
in
the
percent
of
pre­
imaginal
mortality
or
mean
weight
of
emerged
adults
between
MCRY3A
and
negative
control
groups;
all
larvae
in
positive
control
died
in
pre­
imaginal
stage.

Hazard
assessment:
No
hazard
to
Carabid
beetles
is
anticipated
at
field
exposure
levels.
www.
nobodyhere.
com
26
Nontarget
Wildlife
Toxicity
Testing:

Terrestrial
Invertebrates

Study
type:
Nontarget
Insect
Testing,
Tier
1

Test
animal:
Rove
beetle
(
Aleochara
bilineata);
240
beetles

Test
material/
dosage:
50
µ
g
MCRY3A­
0102/
g
meat

Control
treatments:
Negative,
positive
(
teflubenzuron)

Test
duration:
35­
day
feeding
period;
76­
day
observation
period

Results:
No
significant
difference
in
number
of
F1
progeny
or
in
reproductive
capacity
of
beetles
was
seen
between
MCRY3A
and
negative
control.
Mortality
was
similar
among
all
treatments.

Hazard
assessment:
No
hazard
to
rove
beetles
is
anticipated
at
field
exposure
levels.
www.
parasitoides.
univ­
rennes1.
fr
27
Nontarget
Wildlife
Toxicity
Testing:

Terrestrial
Invertebrates

Study
type:
Nontarget
Insect
Testing,
Tier
1

Test
animal:
Insidious
flower
bug
(
Orius
insidiosus);
120
bugs

Test
material/
dosage:
47.8
µ
g
MCRY3A­
0102/
g
diet

Control
treatments:
Negative,
positive
(
teflubenzuron)

Test
duration:
21­
day
feeding
and
observation
period

Results:
No
significant
difference
in
mortality
seen
between
mCry3A
and
negative
control;
mortality
significantly
higher
for
positive
control.
Development
time
similar
among
all
treatments.

Hazard
assessment:
No
hazard
to
insidious
flower
bugs
is
anticipated
at
field
exposure
levels.
www.
mda.
state.
mn.
us
28
Nontarget
Wildlife
Toxicity
Testing:

Terrestrial
Invertebrates

Study
type:
Honey
Bee
Testing,
Tier
1

Test
animal:
Honey
bee
eggs
and
larvae
(
Apis
mellifera);

2,400
eggs

Test
material/
dosage:
50
µ
g
MCRY3A­
0102/
g
sucrose
solution

Control
treatments:
Negative,
positive
(
diflubenzuron)

Test
duration:
5­
day
feeding
period;
21­
day
observation
period

Results:
No
significant
difference
in
brood
mortality
(
eggs
and
larvae)
between
MCRY3A
and
negative
control;
mortality
was
100%
among
egg
cells
in
positive
control.
Adults
not
affected
by
any
treatment.

Hazard
assessment:
No
hazard
to
honey
bee
eggs
or
larvae
is
anticipated
at
field
exposure
levels.
www.
beekeeping.
com
www.
genome.
gov
29
Nontarget
Wildlife
Toxicity
Testing:

Terrestrial
Invertebrates

Study
type:
Earthworm
Subchronic
Toxicity
Study

Test
animal:
Earthworm
(
Eisenia
fetida);
80
worms

Test
material/
dosage:
370
µ
g
MCRY3A­
0102/
g
soil

Control
treatments:
Negative,
positive
(
2­
chloroacetamide)

Test
duration:
14­
day
exposure
period

Results:
No
significant
difference
in
weight
loss
or
mortality
was
seen
between
MCRY3A
and
negative
control
groups;

mortality
was
100%
in
the
positive
control
treatment.
LC
50
>
370
µ
g
MCRY3A­
0102/
g
soil.

Hazard
assessment:
No
hazard
to
earthworms
is
anticipated
at
field
exposure
levels.
www.
amystewart.
com
30
Nontarget
Wildlife
Toxicity
Testing:

Wild
Plants

Study
type:
Nontarget
Plant
Studies,
Tier
1

Waiver
justification:
mCry3A
is
nontoxic
to
plants

Hazard
assessment:
No
hazard
to
plants
is
expected
from
exposure
to
Event
MIR604
corn
31
Nontarget
Wildlife
Toxicity
Testing:

Environmental
Fate

Background:
Soil
Fate
 
October
2000
SAP
concluded
that
earthworms
and
springtails
(
Collembola)
are
appropriate
indicator
species
for
Cry
protein
testing
 
Soil
organisms
may
be
subject
to
long­
term
exposure
to
Cry
proteins
 
Data
received
by
EPA
indicate
that
Cry
proteins
degrade
rapidly
in
field
soils
 
Significant
accumulation/
persistence
in
soil,
resulting
in
adverse
effects
to
nontarget
soil
organisms,
is
not
anticipated
 
Post­
registration
confirmatory
data
of
soil
persistence
are
recommended
32
Nontarget
Wildlife
Toxicity
Testing:

Environmental
Fate

Study
type:
Soil
Fate

Test
soil:
Silty
clay
loam;
24
flasks
Test
animal:
Colorado
potato
beetle
(
Leptinotarsa
decemlineata);

160
larvae

Test
material/
dosage:
230
µ
g
MCRY3A­
0102/
g
dry
weight
soil

Control
treatments:
Negative,
positive
(
pure
MCRY3A­
0102)

Test
duration:
0,1,3,7,12,
and
30­
day
incubation
periods

Results:
Mean
CPB
larval
mortality
was
48­
54%
or
9%
when
fed
MCRY3A
soil
incubated
0­
7
days
or
30
days,
respectively.
DT50
for
degradation
of
MCRY3A
estimated
to
be
7.6
days.

Hazard
assessment:
mCry3A
protein
appears
to
degrade
rapidly
in
soil
and
is
not
expected
to
adversely
affect
soil
organisms
at
field
exposure
levels.
Three­
year
field
study
recommended
to
address
effects
of
long­
term
exposure.
33
Nontarget
Wildlife
Toxicity
Testing:

Environmental
Fate

Background:
Horizontal
gene
transfer
(
HGT)

 
HGT
=
transfer
of
Bt
genes
from
transgenic
plants
to
soil
inhabiting
bacteria
(
source
of
Bt
genes)

 
The
mCry3A
gene
is
not
expressed
by
a
bacterial
promoter
in
Event
MIR604
corn,
so
if
transferred
to
bacteria
the
gene
would
not
be
expressed
 
Experiments
conducted
to
assess
the
likelihood
of
HGT
have
been
unable
to
detect
gene
transfer
under
typical
field
conditions
 
HGT
has
only
been
detected
under
laboratory
conditions
designed
to
favor
DNA
transfer
 
The
Agency
has
concluded
that
there
is
no
significant
risk
of
HGT
with
Event
MIR604
corn
34
Nontarget
Wildlife
Toxicity
Testing:

Environmental
Fate

Background:
Gene
flow
 
In
2001,
EPA
determined
that
gene
capture
and
expression
of
Bt
Cry
toxins
by
wild
or
weedy
relatives
of
corn
was
unlikely
to
occur
in
the
U.
S.,

its
possessions,
or
territories.
35
Nontarget
Wildlife
Toxicity
Testing:

Endangered
Species
Considerations

Hazard
Assessment:
Comparison
of
the
county­
level
distribution
of
endangered
coleopteran
species
relative
to
corn
producing
counties
in
the
U.
S.
indicate
that
there
will
be
no
overlap
with
corn
cultivation
and
endangered
coleopteran
breeding
habitats.

Risk
Assessment:
Non­
target
data
confirm
that
Event
MIR604
corn
will
have
no
adverse
effect
on
endangered
and/
or
threatened
species
listed
by
the
U.
S.
Fish
and
Wildlife
Service,

including
mammals,
birds
or
terrestrial
and
aquatic
plants
and
invertebrate
species.

USFWS
Consultation:
No
consultation
is
required
under
the
Endangered
Species
Act
due
to
no
"
may
affect"
finding.
36
Conclusions

This
assessment
finds
no
hazard
to
the
environment
from
cultivation
of
Event
MIR604
corn
for
a
time­
limited
registration
period

Supplemental
long
term
effects
studies
would
provide
added
weight
to
support
the
Agency's
conclusions
37
Additional
Data

Statistically
valid
invertebrate
field
study

Long
range
soil
persistence
field
study
on
large
plots,
that
include
a
variety
of
high
clay
soil
types,
that
have
been
planted
with
Event
MIR604
corn
for
at
least
three
consecutive
years
38
SAP
Question

The
Panel
is
requested
to
comment
on
the
Agency's
analysis
of
the
currently
available
data
on
the
potential
impacts
of
Event
MIR604
corn
on
nontarget
species
39
Overview
of
EPA's
Product
Characterization
and
Human
Health
Assessment
of
Modified
Cry3A
Annabel
Fellman
Environmental
Protection
Agency
Office
of
Pesticide
Programs
41
Outline

Background
of
product

EPA
process
for
reviewing
product
characterization
for
plant­
incorporated
protectants
(
PIPs)

Product
Characterization
of
mCry3A

EPA
process
for
reviewing
potential
toxicity
and
allergenicity
for
PIPs

Human
Health
Assessment
of
mCry3A
42
Background

Native
Cry3A
is
currently
found
in
several
microbial
insecticide
products
and
as
a
plantincorporated
protectant
(
PIP)
in
Bacillus
thuringiensis
(
Bt)
potato.

Syngenta
Seeds,
Inc.
synthetically
recreated
a
cry3A
gene
from
Bt
subsp.
tenebrionis
to
optimize
for
expression
in
maize
(
corn)
with
enhanced
activity
against
WCRM
and
NCRM.

Result
 
Event
MIR604
corn
plants
with
resistance
to
these
pests.
43
How
EPA
Characterizes
PIPs
Molecular
Characterization
Test
Substance
Equivalence
Protein
Expression
Levels
Analytical
Methods
Product
Characterization
44
Comparison
of
Native
Cry3A
to
mCry3A

Insect
gut
proteases
function
in
aiding
the
insect
in
obtaining
needed
amino
acids
from
dietary
protein.

Serine
proteases
can
be
dominant
in
the
coleopteran
gut
lumen.

Cathepsin­
G 
a
chymotrypsin­
like
serine
protease.

The
mCry3A
was
designed
to
have
significantly
greater
toxicity
to
WCRM
by
inserting
a
site
that
could
be
recognized
by
this
protease.
45
Comparison
of
Native
Cry3A
to
mCry3A
(
cont.)

mCry3A
 
Its
N­
terminus
corresponds
to
methionine­
48
of
the
native
protein
and
a
Cathepsin­
G
recognition
site
introduced
into
AA
sequence
Modified
Cry3A

598
AA

MW
ca.
67
kDa
Native
Cry3A

644
AA

MW
ca.
73
kDa
46
Comparison
of
Native
Cry3A
to
mCry3A
(
cont.)

Mode
of
action
appears
to
be
similar
to
other
Bt
Cry
toxins

To
be
insecticidal,
most
known
 ­
endotoxins
must:

°
be
ingested
by
the
insect
and
solubilized
in
the
gut,

°
be
activated
by
specific
proteolytic
cleavages,

°
bind
to
specific
receptors
on
surface
of
the
insect
midgut
°
form
ion­
channels.
47
Comparison
of
Native
Cry3A
to
mCry3A
(
cont.)

Comparison
of
Insecticidal
Properties

Solubilization
 
Both
readily
soluble
in
pH>
10
and
sparingly
soluble
at
neutral
pH

Proteolytic
Processing
 
Rapid
and
complete
processing
of
the
67
to
55
kDa.

 
In
coleopteran
gut
lumen,
the
smaller
49
kDa
of
the
2
processed
forms
is
soluble
at
neutral
pH
and
retains
insecticidal
activity.

 
Addition
of
Capthepsin­
G
recognition
site
permits
a
more
facilitated
and
rapid
processing
to
the
stable
form
48
Comparison
of
Native
Cry3A
to
mCry3A
(
cont.)

Receptor
binding
 
Persistence
of
binding
of
the
55
kDa
form
of
mCry3A
protein
to
first
instar
WCRW
membranes

Membrane
pore
forming
properties
 
mCry3A
is
able
to
form
discrete
ion
channels
in
artificial
membrane
systems.

 
The
mCry3A
protein
has
enhanced
toxicity
to
NCRW
and
WCRW.
49
Molecular
Characterization
of
mCry3A

Transformation
was
conducted
using
immature
maize
embryos
derived
from
a
proprietary
Zea
mays
line,
via
Agrobacterium­
mediated
transformation.

Event
MIR604
maize
also
contains
the
pmi
gene,

which
was
introduced
along
with
the
mCry3A
protein
via
the
same
pZM26
transformation
vector.

The
pmi
gene
represents
the
manA
gene
from
Escherichia
coli
and
encodes
the
enzyme
phosphomannose
isomerase
(
PMI).
50
Plasmid
Map
(
pZM26)
51
Molecular
Characterization
(
cont.)

T­
DNA
insert
(
via
the
pZM26
plasmid)
in
Event
MIR604
was
analyzed
via
Southern
blot
analyses:

 
single
copies
of
mcry3A
and
the
pmi
genes
were
present
and
closely
linked
in
MIR604.

DNA
sequencing
revealed
a
44
and
43
bp
truncation
at
RB
and
LB
points
of
the
T­
DNA
insert,

respectively,
during
the
transformation
process.

Expected
Mendelian
inheritance
ratio
of
positive
and
negative
plants
for
the
traits
(
3:
1).
52
Test
substance
Equivalence

Production
of
bacterial­
derived
mCry3A
protein
as
test
material
(
MCRY3A­
0102).

Tests
were
conducted
in
support
of
utilizing
the
mCry3A
protein
expressed
in
recombinant
E.
coli
as
a
surrogate
for
maizeexpressed
mCry3A
protein.

Equivalence
has
been
confirmed.
53
Test
substance
Equivalence
(
cont.)

Molecular
weight
determined
via
SDS­
PAGE.

Lowered
MW
than
predicted.

2nd
significant
band
of
MW
of
55.4
kDa
in
plant­
derived
sample
54
Test
substance
Equivalence
(
cont.)

Western
Blot

Molecular
weight
(
67.7
kDa)
was
also
determined
by
Western
Blot

Immunologically
cross­
reactive
to
same
anti­
Cry3A
antibody
55
Test
Substance
Equivalence
(
cont.)

Integrity
and
Purity
of
mCry3A
Protein
in
Sample
MCRY3A­
0102

Densitometric
analyses­

Purity
of
90.3%

Western
Blot
shows
single
immunoreactive
band
with
predicted
MW
of
~
67.7
kDa
Initial
analysis
Re­
analysis­

9
months
later
56
Test
Substance
Equivalence
(
cont.)

Comparable
toxicities
to
WCRM
larvae

No
post­
translational
glycosylation
of
mCry3A
protein
from
either
source
 
mCry3A
proteins
from
corn
event
MIR604
and
from
recombinant
E.
coli
are
equivalent.
57
Equivalence
for
mCry3A
components
in
microbially­
derived
test
material

Examination
of
the
nucleotide
sequence
of
the
cry1Ac
promoter
utilized
for
expression
of
the
mCry3A
protein
in
the
MCRY3A­
0102
test
substance.

An
"
ATG"
initiation
codon
sequence
was
identified
within
the
cry1Ac
promoter
indicating
the
presence
of
an
additional
putative
open
reading
frame
(
ORF)

short
form
mCry3A
long
form
mCry3A
Cry1Ac
promoter
ATG
ATG
58
Equivalence
for
mCry3A
components
in
microbially­
derived
test
material
(
cont.)

ORF
would
be
inframe
with
the
mcry3A
gene
and
would
result
in
an
additional
16
amino
acids
on
the
N­
terminus
of
the
intended
mCry3A
protein.
cry1Ac
promoter
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

121
TGCATTTTTT
CATAAGATGA
GTCATATGTT
TTAAATTGTA
GTAATGAAAA
ACAGTATTAT
ACGTAAAAAA
GTATTCTACT
CAGTATACAA
AATTTAACAT
CATTACTTTT
TGTCATAATA
long
form
mcry3A
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

cry1Ac
promoter
short
form
mcry3A
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~

M
N
W
Y
L
N
K
R
D
G
G
L
N
G
S
T
M
T
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

181
ATCATAATGA
ATTGGTATCT
TAATAAAAGA
GATGGAGGTT
TAAACGGATC
CACCATGACG
TAGTATTACT
TAACCATAGA
ATTATTTTCT
CTACCTCCAA
ATTTGCCTAG
GTGGTACTGC
long
form
mcry3A
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

short
form
mcry3A
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

A
D
N
N
T
E
A
L
D
S
S
T
T
K
D
V
I
Q
K
G
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

241
GCCGACAACA
ACACCGAGGC
CCTGGACAGC
AGCACCACCA
AGGACGTGAT
CCAGAAGGGC
CGGCTGTTGT
TGTGGCTCCG
GGACCTGTCG
TCGTGGTGGT
TCCTGCACTA
GGTCTTCCCG
59
Equivalence
for
mCry3A
components
in
microbially­
derived
test
material
(
cont.)

Identity
of
both
components
was
analyzed
by:

 
Peptide
mapping
using
tandem
(
MS/
MS)
mass
spectrometry;
and
 
MALDI
TOF
mass
spectrometry
of
the
intact
proteins.
60
148
98
64
50
36
22
1
2
3
4
1
2
3
4
A
B
Equivalence
for
mCry3A
components
in
microbially­
derived
test
material
(
cont.)

SDS­
PAGE
(
figure
A)
and
Western
(
figure
B)
gels
indicated
single
intense
bands:

 
69.5
kDa 
mCry3A­
LF
 
67.7
kDa 
mCry3A­
SF

Both
forms
were
also
insecticidally
active
against
WCRW

High
degree
of
structural
homology
 
Both
forms
of
mCry3A
are
considered
equivalent
61
Protein
Expression
Levels

Plant
extracts
from
inbred
and
hybrid
corn
lines
derived
from
Event
MIR604
field
plants
were
quantitatively
analyzed
for
mCry3A
by
ELISA.

Magnitude
of
expression
for
tissue
types
was
as
follows
in
descending
order:

leaves
 
roots
 
silage
 
kernels

All
control
tissues
were
negative.

Mean
extraction
efficiency
for
mCry3A
 
76.6%.
62
Protein
Expression
Levels
(
cont.)

Levels
of
mCry3A
present
were
also
determined
via
ELISA
in
standard
wet­
milled
and
dry­
milled
processing
fractions,
corn
oil,
and
corn
chips
from
grain
derived
from
transgenic
corn
Event
MIR604.

 
Residues
of
the
mCry3A
protein,
and/
or
breakdown
products
thereof,
while
present
in
corn
grain
and
other
tissues
of
Event
MIR604­

derived
corn
are
in
the
ppm
range,
and
is
expected
to
have
little
to
no
human
dietary
exposure.
63
Analytical
Methods

Validation
of
ELISA
method
by
independent
third
party
laboratory

Assay
sensitivity
was
1
positive
Event
MIR604
kernel
in
999
non­
MIR604
kernels
(
0.1%).

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.
64
Product
Characterization
Question
#
1
Previously
submitted
studies
for
Event
MIR604
demonstrated
the
equivalence
of
the
plant­
and
bacterialproduced
test
substances.

However,
two
forms
of
mCry3A
(
designated
as
mCry3A­
sf
and
mCry3A­
lf)
were
found
in
the
bacterial­
produced
test
material
(
MCRY3A­
0102).

 
Please
comment
on
the
Agency's
conclusion
for
the
purpose
of
the
Agency's
risk
assessment;
and
that
the
equivalence
is
confirmed
for
the
two
forms
of
the
bacterial­
produced
mCry3A
test
material.
65
How
EPA
Assesses
Human
Health
Effects
for
PIPs
Human
Health
Assessment
Acute
Oral
Toxicity
Biochemical
Properties
Amino
Acid
Sequence
Homology
66
Human
Health
Assessment

Section
408(
c)(
2)(
A)(
i)
of
the
FFDCA
allows
EPA
to
establish
an
exemption
from
the
requirement
for
a
tolerance
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."
67
Toxicity
and
Allergenicity

Vast
majority
of
proteins
pose
no
risks
to
humans.

Of
the
thousands
of
proteins
in
food,
only
a
tiny
fraction
exhibit
toxicity
by
the
oral
route
or
elicit
allergic
reactions

Toxicity
and
allergenicity
are
unusual
properties
among
proteins
68
Toxicity
Determination
(
cont.)

No
evidence
of
toxicity
after
a
single
oral
dose.

Acute
oral
LD50
of
MCRY3A­
0102
in
mice
was
>
2632
mg/
kg
body
weight.

No
significant
amino
acid
homology
between
any
protein
toxin
and
the
mCry3A
protein.

Inactivated
against
WCRM,
when
heated
to
95
°
C
for
30
minutes;

mCry3A
is
not
glycoslylated;
and

Present
in
low
levels
in
corn
tissue.
69
Toxicity
Determination
(
cont.)

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.

 
mCry3A
protein
is
not
considered
toxic
to
humans.
70
Allergenicity

EPA
uses
"
Weight
of
the
evidence
approach"

Following
factors
are
considered:

 
source
of
the
trait;

 
amino
acid
sequence
similarity
with
known
allergens;
and
 
biochemical
properties
of
the
protein
(
eg.­
in
vitro
digestibility
in
simulated
gastric
fluid
(
SGF),
and
glycosylation).
71
Source
of
Trait

Source 
Bacillus
thuringiensis

In
general,
proteins
are:

 
Composed
of
amino
acids
 
Upon
digestion,
degrade
into
small
peptides
and
amino
acids
 
Provide
essential
nutrients
72
In
vitro
Digestibility

Figures
A
and
B
show
Coomassie
blue­
stained
SDSPAGE
gels
of
digests
of
microbially­
and
plant­
derived
mCry3,
respectively.

No
intact
mCry3A
(
MW
~
67.7
KDa)
was
detected
in
either
mCry3A
protein
after
only
2
min.
of
digestion.

A
B
73
In
vitro
Digestibility
(
cont.)

1
2
3
4
5
6
7
8
9
10
11
210
105
78
55
45
34
17
16
7
4
mCry3A
mCry3A
breakdown
product
mol
wt
[
kDa]

No
intact
mCry3A
(
MW
~
67.7
KDa)
was
detected
in
either
mCry3A
protein
after
only
2
min.
of
digestion.

 
mCry3A
will
be
as
readily
digested
as
a
conventional
dietary
protein
in
a
typical
mammalian
gastric
environment.
74
Amino
Acid
Homology
for
mCry3A

No
significant
similarity
was
found
between
any
of
the
mCry3A
80­
amino
acid
peptides
and
any
entries
in
the
SBI
Allergen
Database.

No
alignments
of
8
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.
75
Conclusion

Lack
of
toxicity
of
the
mCry3A
protein
following
acute
oral
high­
dose
exposure
to
mice

No
post­
translational
glycosylation

Rapid
degradation
of
mCry3A
in
SGF
fluid

Lack
of
significant
AA
sequence
homology
of
the
mCry3A
protein
to
toxins
or
allergens.

 
Little
to
no
human
dietary
exposure
to
mCry3A
protein
is
expected
to
occur
via
transformed
corn.
76
Conclusion
(
cont.)

 
Reasonable
certainty
that
no
harm
will
result
from
aggregate
exposure
to
the
U.
S.

population
to
mCry3A
protein
and
the
genetic
material
necessary
for
its
production.

 
No
adverse
effects
on
human
health
from
the
proposed
use
of
mCry3A
protein
expressed
in
corn.
77
Human
Health
Assessment
Question
#
2
Previously
submitted
studies
demonstrated
the
lack
of
toxicity
of
the
mCry3A
protein.
Moreover,
little
to
no
human
dietary
exposure
to
mCry3A
protein
is
expected
to
occur
via
transformed
corn.
Therefore,
dietary
exposure
to
mCry3A
is
not
anticipated
to
pose
any
dietary
risk
to
the
U.
S.
population.

Please
comment
on
the
Agency's
conclusions
regarding
the
lack
of
mammalian
toxicity
and
allergenicity
of
mCry3A.
78
Acknowledgements
John
Kough
and
Annabel
Fellman
Janet
Andersen
Phil
Hutton
and
Dennis
Szuhay
Mike
Mendelsohn
Tessa
Milofsky
Zig
Vaituzis
and
Mika
Hunter
79
SAP
Panel
Questions
Tessa
Milofsky
and
Annabel
Fellman
Environmental
Protection
Agency
Office
of
Pesticide
Programs
81
Environmental
Effects
Assessment
SAP
Question
#
1
The
weight
of
evidence
from
the
reviewed
data
indicates
that
there
will
not
be
a
hazard
to
wildlife
from
the
commercialization
of
Event
MIR604
corn.
Although
the
mCry3A
protein
expressed
by
Event
MIR604
corn
is
known
to
affect
only
coleopteran
insect
species,
EPA
assessed
the
potential
risks,
to
a
wide
variety
of
non­
target
organisms
(
i.
e.

mammals,
birds,
fish,
invertebrates
and
plants),
that
could
potentially
result
from
exposure
to
this
Bt
protein.
The
emphasis
of
this
nontarget
risk
assessment,
however,
was
on
invertebrate
species
that
dominate
corn
agroecosystems.
The
Agency
also
evaluated
a
soil
fate
study
that
was
intended
to
provide
information
on
the
persistence
and
rate
of
degradation
of
mCry3A
protein
in
the
soil
environment.
After
reviewing
data
submitted
in
support
of
the
Event
MIR604
Bt
corn
registration,
EPA
concluded
that
aquatic
and
terrestrial
wildlife,

including
soil
organisms,
were
not
likely
to
be
adversely
affected
and
that
this
Bt
corn
product
is
not
likely
to
threaten
the
long­
term
survival
of
any
non­
target
wildlife
populations.
82
Environmental
Effects
Assessment
SAP
Question
#
1
(
cont.)

The
Panel
is
requested
to
comment
on
the
Agency's
analysis
of
the
currently
available
data
on
the
potential
impacts
of
Event
MIR604
corn
on
nontarget
species.
83
Product
Characterization
SAP
Question
#
2

Previously
submitted
studies
for
Event
MIR604
demonstrated
the
equivalence
of
the
plant­
and
bacterial­
produced
test
substances
by
showing
similar
molecular
weights,
purity
of
90.3%,
a
lack
of
post­
translational
glycosylation
of
mCry3A
from
either
source,
and
comparable
toxicities
toward
western
corn
rootworm
(
WCRW).

However,
two
forms
of
mCry3A
(
designated
as
mCry3A­
sf
and
mCry3A­
lf)
were
found
in
the
bacterial­
produced
test
material
(
MCRY3A­
0102).
The
molecular
weights
of
the
short
and
long
form
of
mCry3A
were
67.5
kDa
and
69.1
kDa,
respectively,

determined
via
SDS­
PAGE
and
MALDI­
TOF
Mass
spectrometry.
The
lesser
of
the
two
components,
with
the
lower
molecular
weight,
corresponded
to
the
intended
mCry3A
protein
with
598
amino
acids.
84
Product
Characterization
SAP
Question
#
2
(
cont.)

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.
Both
mCry3A
forms
were
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.

Please
comment
on
the
Agency's
conclusion
that
the
mCry3A
proteins
from
corn
event
MIR604
and
from
recombinant
E.
coli
are
substantially
the
same
for
the
purpose
of
the
Agency's
risk
assessment;
and
that
the
equivalence
is
confirmed
for
the
two
forms
of
the
bacterial­
produced
mCry3A
test
material
(
MCRY3A­

0102).
85
Human
Health
Assessment
SAP
Question
#
3

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.

Moreover,
little
to
no
human
dietary
exposure
to
mCry3A
protein
is
expected
to
occur
via
transformed
corn.
Therefore,

dietary
exposure
to
mCry3A
is
not
anticipated
to
pose
any
dietary
risk
to
the
U.
S.
population.

Please
comment
on
the
Agency's
conclusions
regarding
the
lack
of
mammalian
toxicity
and
allergenicity
of
mCry3A.