UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
OFFICE
OF
PREVENTION
PESTICIDES
AND
TOXIC
SUBSTANCES
FEB
09
2006
MEMORANDUM
SUBJECT
Environmental
Fate
and
Risk
Assessment
for
mCry3A
Protein
in
Syngenta's
MIR604
field
corn
(
MRIDs
462656­
01,
462656­
13,
and
462656­
14).

TO
Michael
Mendelsohn
Senior
Regulatory
Action
Leader
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

FROM
Tessa
Milofsky,
M.
S.
Agronomist
signed
2­
9­
06
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

THROUGH
Zigfridas
Vaituzis,
Ph.
D,
Microbiologist
signed
2­
9­
06
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

Recommendations
It
is
recommended
that
additional
studies
should
be
conducted
to
evaluate
plant­
produced
mCry3A
protein
degradation,
accumulation,
and
persistence
in
a
variety
of
soil
types,
including
those
high
in
clay
and
humic
acids,
into
which
all
non­
harvested
corn
plant
material
is
incorporated.

Background
Syngenta,
Ltd.
is
requesting
a
full
and
unrestricted
FIFRA
Section
3
registration
for
commercialization
of
MIR604
corn
rootworm
protected
field
corn,
a
new
end­
use
product
containing
the
mCry3A
protein
and
the
genetic
material
necessary
for
its
production
in
corn,
which
includes
the
PMI
inert
marker.
This
memo
includes
summaries
and
DERs
for
the
Environmental
Safety
Assessment,
Environmental
Fate
Assessment,
and
Laboratory
Soil
Degradation
studies
submitted
in
support
of
the
MIR604
registration.
Reviews
of
the
non­
target
studies
included
in
this
data
package
are
covered
in
a
separate
memo.

Study:
Environmental
Safety
Assessment
(
summary
document)
of
Modified
Cry3A
Protein
and
Event
MIR604
to
Non­
Target
Organisms
MRID
No.:
462656­
01
Classification:
Supplemental
Study
Summary:
Summary
information
is
presented
in
support
of
the
Section
3
registration
of
MIR604
rootworm
protected
field
corn,
which
contains
the
mCry3A
protein.
The
safety
assessment
document
provides
summaries
of
the
non­
target
laboratory
studies
that
were
used
to
evaluate
the
potential
adverse
effects
of
mCry3A
on
non­
target
organisms.
According
to
expected
environmental
concentration
(
EEC)
calculations
performed
and
justified
by
Syngenta,
non­
target
test
species
were
exposed
to
11.2
to
2,600
times
the
EEC
or
daily
dietary
dose
of
mCry3A
without
significant
adverse
effects.
BPPD
Review:
The
reviewer
generally
agrees
with
summary
information
presented
by
the
study
authors.
Justifications/
explanations
provided
for
EEC
calculations
seem
reasonable.
It
is
noted,
however,
that
many
of
the
assumptions
used
to
calculate
margins
of
exposure
for
test
species
were
not
submitted
to
EPA
for
2
review
and
therefore,
could
not
be
verified
by
the
reviewer.
It
is
further
noted
that
the
potency
of
cornderived
protein
was
found
to
be
twice
the
potency
of
bacteria­
derived
MCRY3A­
0102
(
MRID
461556­
03).
Despite
the
lower
relative
toxicity
of
the
bacteria­
derived
protein,
the
reviewer
agrees
that,
based
on
study
results
and
EEC
calculations
presented
by
the
registrant,
it
is
unlikely
that
exposure
to
plant­
produced
mCry3A
protein
will
result
in
significant
adverse
effects
to
non­
target
organisms.

Study:
Environmental
Fate
Assessment
of
Modified
Cry3A
Protein
in
Event
MIR604
Corn
MRID
No.:
462656­
13
Classification:
Supplemental
Study
Summary:
MIR604
corn
plants
have
been
shown
to
express
mCry3A
protein
in
leaves,
kernels,
roots,
and
silks,
but
the
protein
was
not
detected
in
corn
pollen.
Corn
leaf
assays
were
used
to
verify
that
mCry3A
expression
is
stable
over
multiple
generations
and
a
soil
degradation
study
showed
that
MCRY3A­
0102
degrades
readily,
with
a
DT50
of
7.6
days
in
silty
clay
loam
soil.
Due
to
corn's
lack
of
invasive
characteristics
and
the
low
probability
that
the
mcry3A
gene
from
Event
MIR604
would
transfer
to
a
wild
relative
of
corn,
it
is
unlikely
that
mCry3A
will
spread
beyond
cultivated
sites
and
persist
in
weedy
populations.
It
is
also
unlikely
that
genes
present
in
MIR604
corn
would
be
subject
to
horizontal
gene
transfer
at
a
frequency
that
exceeds
the
rate
of
transfer
in
other
plants.
In
the
unlikely
event
that
mcry3A
is
stably
integrated
and
expressed
in
a
soil
microorganism,
no
harmful
effects
are
expected.
Laboratory­
based
non­
target
studies
indicate
that
expected
environmental
exposure
to
mCry3A
protein
will
not
result
in
unreasonable
adverse
effects
to
beneficial
organisms.
The
submission
also
notes
the
low
likelihood
that
aquatic
organisms
will
be
exposed
to
mCry3A,
since
it
is
unlikely
that
the
protein
will
enter
watercourses
via
soil
particle
movement,
pollen
dispersal,
or
seed
spillage.
BPPD
Review:
The
reviewer
agrees
with
the
above
conclusions.

Study:
Laboratory
Soil
Degradation
of
Modified
Cry3A
Protein
(
MCRY3A­
0102)
MRID
No.:
462656­
14
Classification:
Supplemental
Study
Summary:
Degradation
of
MCRY3A­
0102
(
microbe­
produced
mCry3A
protein)
in
silty
clay
loam
soil
was
evaluated
in
a
laboratory
study,
where
treated
soil
samples
were
maintained
under
conditions
that
mimicked
the
field
environment.
MCRY3A­
0102
activity
in
soil
was
evaluated
with
a
Colorado
potato
beetle
(
CPB)
(
Leptinotarsa
decemlineata)
larval
bioassay.
The
test
protein
was
incorporated
into
the
test
soil
at
a
nominal
rate
of
230
µ
g
MCRY3A­
0102/
g
of
dry
soil.
Soil
samples
were
collected
at
days
0,
1,
3,
7,
12,
and
30
and
frozen
until
needed
for
CPB
diet
formulation.
CPB
larvae
were
maintained
on
each
soil/
diet
mixture
for
72
hrs,
after
which
mortality
was
determined.
Larval
mortality
was
48­
53%
when
fed
diets
containing
soil
from
days
0,
1,
3,
and
7;
mortality
declined
to
9%
when
fed
diet
containing
soil
from
day
30.
Based
on
these
results,
a
simple
first­
order
kinetic
model
determined
that
the
DT50
for
mCry3A
in
this
silty
clay
loam
soil
is
7.6
days.
BPPD
Review:
These
results
indicate
that
microbe­
produced
MCRY3A­
0102
insecticidal
protein
degrades
rapidly
in
silt
loam
soil.
However,
silt
loam
soil
is
just
one
of
many
soil
classes
used
for
corn
production
in
the
United
States.
A
more
useful
study
would
evaluate
protein
degradation,
accumulation,
and
persistence
in
a
range
of
soil
types,
including
those
with
high
clay
and
humic
acid
content,
due
to
their
known
binding
affinity
for
proteins.

It
is
also
noted
that
this
study
utilized
field
soil
spiked
with
microbe­
derived
insecticidal
protein.
This
approach
is
useful
because
dose
responses
can
be
easily
quantified.
However,
the
degradation
and
accumulation
of
Cry
proteins
found
within
decaying
plant
tissue
may
behave
differently
than
proteins
in
artificially
spiked
soil
and
MCRY3A­
0102
was
shown
to
be
half
as
potent
as
plant­
produced
protein
(
MRID
461556­
03).
Consequently,
aside
from
showing
that
MCRY3A­
0102
does
degrade
in
soil,
the
studies
are
incomplete
and
the
long
term
relevance
of
these
study
results
is
unclear.
3
To
account
for
the
above
concerns,
it
is
recommended
that
additional
studies
should
be
conducted
to
evaluate
plant­
produced
mCry3A
protein
degradation,
accumulation,
and
persistence
in
a
variety
of
soil
types,
including
those
high
in
clay
and
humic
acids,
into
which
all
non­
harvested
corn
plant
material
is
incorporated.
Sampling
should
be
conducted
each
year
for
three
years
in
a
field
sown
with
continuous
MIR604
corn.
Soil
should
be
monitored
for
a
minimum
of
one
growing
season
after
harvest
and
continued
until
the
mCry3A
protein
can
no
longer
be
detected.
It
is
recommended
that
these
studies
should
be
included
as
a
condition
of
registration.
4
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis,
M.
S.
EPA
Secondary
Reviewer:
Tessa
Milofsky,
M.
S.

STUDY
TYPE:
Nonguideline
MRID
NO:
46265601
DP
BARCODE:
DP303605
TEST
MATERIAL:
mCry3A
Protein
in
Event
MIR604
Corn
STUDY
NO:
Not
provided
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
N/
A
TITLE
OF
REPORT:
Environmental
Safety
Assessment
of
Modified
Cry3A
Protein
and
Event
MIR604
Corn
to
Non­
Target
Organisms
AUTHOR:
Raybould,
A.

STUDY
COMPLETED:
April
27,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
is
a
compilation
and
is
not
subject
to
GLP
standards.

STUDY
SUMMARY:
Summary
information
is
presented
in
support
of
the
Section
3
registration
of
MIR604
rootworm
protected
field
corn,
which
contains
the
mCry3A
protein.
The
safety
assessment
document
provides
summaries
of
the
non­
target
laboratory
studies
that
were
used
to
evaluate
the
potential
adverse
effects
of
mCry3A
on
non­
target
organisms.
According
to
expected
environmental
concentration
(
EEC)
calculations
performed
and
justified
by
Syngenta,
non­
target
test
species
were
exposed
to
11.2
to
2,600
times
the
EEC
or
daily
dietary
dose
of
mCry3A
without
significant
adverse
effects.

CLASSIFICATION:
Supplemental
Introduction
5
Laboratory
studies
were
conducted
using
a
range
of
non­
target
organisms
to
evaluate
the
environmental
safety
of
modified
Cry3A
(
mCry3A)
protein
(
Table
1).
To
ensure
an
adequate
"
safety
margin,"
the
studies
were
designed
to
expose
non­
target
arthropods
to
at
least
10
times
the
estimated
environmental
concentration
(
EEC)
of
mCry3A,
which
was
calculated
to
be
50
µ
g
mCry3A/
g
based
on
preliminary
studies
indicating
that
the
mCry3A
concentration
in
leaves
of
MIR604
hybrid
corn
was
5
µ
g/
g
fresh
weight.
Other
plant
tissues
contained
less
than
this
value,
and
because
non­
target
arthropods
tend
not
to
eat
corn
leaf
tissue,
this
exposure
estimate
was
considered
to
be
a
worst­
case
exposure
scenario.
To
achieve
this
safety
margin
(
10
x
EEC),
most
of
the
test
species
were
exposed
using
a
preparation
of
microbe­
produced
mCry3A
(
test
substance
MCRY3A­
0102,
90.3%
w/
w
mCry3A)
in
artificial
diets,
rather
than
using
plant
material
containing
mCry3A
(
lower
dose
test
substance).
The
mCry3A
in
MCRY3A­
0102
was
shown
to
be
chemically
and
biologically
equivalent
to
mCry3A
produced
in
Event
MIR604
corn.

Further
evaluation
of
corn
leaf
tissue
indicated
that
mCry3A
expression
over
four
time
points
ranged
from
3.8
µ
g
mCry3A/
g
(
anthesis)
to
7.8
µ
g
mCry3A/
g
at
seed
maturity,
with
an
average
over
all
time
periods
of
6.6
µ
g
mCry3A/
g
fresh
weight.
Based
on
these
findings,
the
50
µ
g
mCry3A/
g
exposure
level
is
less
than
10
x
the
exposure
level
in
leaves.
However,
Syngenta
argues
that
"
mCry3A
concentration
in
corn
leaf
tissue
is
not
a
realistic
EEC,
because
(
by
definition)
non­
target
organisms
do
not
consume
significant
quantities
of
crop
plant
tissues".

The
EEC
for
earthworms
was
based
on
the
concentration
of
mCry3A
protein
present
in
senescent
corn
leaf
tissue,
while
the
exposure
level
used
for
the
bird
study
was
calculated
from
mCry3A
concentrations
present
in
corn
grain.
The
fish
study
was
the
only
non­
target
organism
study
that
did
not
use
microbe­
produced
MCRY3A­
0102
as
the
test
substance.
In
this
case,
corn
grain
was
incorporated
in
fish
feed.

TABLE
1.
Ecotoxicolgy
studies
using
exposure
to
mCry3A
Test
species
Exposure
route
Minimum
Margin
of
Exposure
Dosing
Endpoints
Bobwhite
quail
(
Colinus
virginianus)
Capsule
1400
X
DDD1
Single
oral
14
d
mortality,
body
wt,
feed
consumption
Rainbow
trout
(
Oncorhyncus
mykiss)
Feed
formulated
with
50%
MIR604
grain
37.0
X
EEC2
Daily
28
d
mortality
and
growth
Flower
bug
(
Orius
insidiosus)
Protein
in
artificial
diet
10.6
X
EEC
Daily
Pre­
imaginal
mortality
Ladybird
beetle
(
Coccinella
septempunctata)
Aphids
dipped
in
solution
of
protein
12.3
X
EEC
Daily
until
pupation
and
after
adult
emergence
Pre­
imaginal
mortality
and
development,
adult
mortality
Rove
beetle
(
Aleochara
bilineata)
Protein
in
artificial
diet
15.6
X
EEC
Daily
for
first
35
days
of
test
Fecundity
Carabid
beetle
Protein
11.2
X
EEC
Daily
until
Pre­
imaginal
mortality
6
(
Poecilus
cupreus)
injected
into
blowfly
pupae
pupation
Honeybee
(
Apis
mellifera)
Protein
in
sugar
solution
36
X
EEC
Daily
for
first
5
days
of
test
Brood
development,
adult
and
larval
mortality
Earthworm
(
Eisenia
foetida)
Protein
in
artificial
soil
46
X
EEC
Single
14
d
mortality,
body
wt
Mouse3
Protein
in
methylcellulos
e
solution
2600
X
DDD
Single
oral
14
d
mortality,
body
wt,
organ
wt,
feed
consumption
1DDD
=
Daily
dietary
dose
2EEC
=
Estimated
environmental
exposure
3Mammalian
toxicity
study
Results
of
Toxicity
Tests
Bobwhite
quail
(
MRID
46155616)

Five
male
and
five
female
young
adult
northern
bobwhite
quail
(
Colinus
virginianus)
were
administered
a
single
oral
dose
of
722
mg
MCRY3A­
0102/
kg
body
wt
(
nominally
equivalent
to
approximately
microbe­
produced
652
mg
mCry3A
protein/
kg
body
wt)
and
observed
for
14
days.
There
were
no
treatment­
related
adverse
clinical
signs
or
mortality.
Body
weight
and
feed
consumption
of
the
test
birds
were
comparable
to
those
of
controls.
The
acute
oral
nominal
LD50
was
greater
than
722
mg
MCRY3A­
0102/
kg
body
wt
(
microbe­
produced
652
mg
mCry3A
protein/
kg
body
wt),
and
the
nominal
NOEL
was
equal
to
722
mg
MCRY3A­
0102/
kg
body
wt
(
microbe­
produced
652
mg
mCry3A
protein/
kg
body
wt).

The
margin
of
exposure
for
seed­
eating
birds
was
calculated
using
the
formula
DDD
=
(
FIR/
bw)
x
C,
where
DDD
is
daily
dietary
dose,
FIR
is
food
intake
rate,
bw
is
body
weight,
and
C
is
the
mCry3A
concentration
in
the
food.
Based
on
a
FIR/
bw
ratio
of
0.11
to
0.35
for
seed­
eating
birds
and
an
average
mCry3A
concentration
of
1.3
µ
g
mCry3A/
g
fresh
weight
in
kernels
of
MIR604
hybrids,
the
daily
dietary
dose
for
seed­
eating
birds
ranges
from
0.14
to
0.46
mg
mCry3A/
kg
body
weight.
The
652
mg
MCRY3A­
0102/
kg
dose
used
in
the
quail
study
would
therefore
provide
1417
to
4657
times
the
estimated
daily
dietary
dose
of
mCry3A
for
seed­
eating
birds
that
ate
a
diet
of
100%
fresh
kernels
from
MIR604
plants.

Rainbow
trout
(
MRID
46155617)

In
a
28­
day
flow­
through
acute
toxicity
study,
40
juvenile
rainbow
trout
(
Onchorhynchus
mykiss)
were
fed
twice
daily
with
a
formulated
fish
feed
containing
50%
by
weight
Event
MIR604
grain,
which
contains
mCry3A
protein.
Transient
discoloration,
sounding,
and
surfacing
were
seen
in
one
to
three
fish
in
the
test
group
after
day
15,
and
one
fish
was
found
dead
on
day
21.
No
significant
differences
were
detected
in
the
weight
of
the
control
or
test
fish
at
0,
14,
or
28
days,
and
no
significant
difference
in
length
was
seen
at
14
or
28
days.
The
actual
concentration
of
mCry3A
in
the
grain
used
to
prepare
the
fish
feed
was
subsequently
determined
to
be
0.30
µ
g/
g
fresh
weight,
and
the
concentration
of
mCry3A
in
the
formulated
feed
was
0.09
±
0.005
µ
g/
g
fresh
weight
(
MRID
46265602).

The
margin
of
exposure
via
the
feed
prepared
in
the
study
was
calculated
to
be
37
times
the
EEC,
based
on
the
assumptions
that
feed
would
not
likely
be
formulated
using
100%
grain
from
MIR604
hybrids
and
that
the
adoption
rate
of
MIR604
hybrids
would
be
no
more
than
about
5%
of
field
corn
grown
in
the
U.
S.
7
Insidious
Flower
Bug
(
MRID
46265609)

Predatory
bug
(
Orius
insidiosus)
nymphs
were
provided
artificial
meat­
based
diet
containing
MCRY3A­
0102
at
a
rate
nominally
equivalent
to
50
µ
g
microbe­
produced
mCry3A/
g
diet
for
21
days.
A
negative
control
group
received
artificial
diet
mixed
with
deionized
water
only,
and
a
positive
control
group
received
diet
containing
teflubenzuron
(
0.01
mg
a.
i./
g
of
diet).
At
the
end
of
the
test,
there
was
no
statistically
significant
difference
in
the
percent
mortality
of
the
MCRY3A­
0102
and
negative
control
groups.
Mortality
in
the
positive
control
group
was
appropriate.
The
nominal
LC50
was
>
50
µ
g
microbe­
produced
mCry3A/
g
diet,
and
the
nominal
NOEC
was
50
µ
g
mCry3A/
g
diet.
Subsequent
analysis
of
the
treated
diet
recovered
95.6%
of
the
mCry3A,
indicating
mCry3A
was
present
at
the
intended
concentration
(
50
µ
g/
g
diet),
and
a
CPB
larvae
assay
showed
the
mCry3A
was
intact
in
the
diet
for
at
least
4
days
(
MRID
46265610).

Potential
routes
of
field
exposure
of
Orius
to
mCry3A
include
ingestion
of
corn
silks
and
spider
mites
(
Tetranychus
urticae)
that
feed
on
MIR604
hybrids.
Assuming
a
concentration
of
1.7
µ
g
mCry3A/
g
fresh
weight
of
MIR604
corn
silks,
the
exposure
for
Orius
in
the
above
study
(
50
µ
g/
g
diet)
was
calculated
to
be
29.4
times
the
EEC.
Assuming
a
concentration
of
4.7
µ
g
mCry3A/
g
fresh
weight
of
spider
mites,
the
exposure
was
10.6
times
the
EEC.

Ladybird
beetle
(
MRID
46265603)

Four­
day­
old
ladybird
beetle
larvae
(
Coccinella
septempunctata)
were
fed
daily
with
live
pea
aphids
(
Acyrthosiphon
pisum)
that
had
been
immersed
for
30
seconds
in
diluted
wetting
solution
containing
approximately
50
µ
g
of
MCRY3A­
0102
protein/
ml
solution.
Negative
control
larvae
received
aphids
dipped
in
wetting
solution
only,
and
positive
control
larvae
received
aphids
dipped
in
teflubenzuron.
The
larvae
were
allowed
to
pupate
(
approximately
9
to
10
days
from
study
initiation
to
pupation),
and
adult
beetles
that
emerged
were
fed
treated
aphids
for
14
days.
There
were
no
statistically
significant
differences
in
pre­
imaginal
or
adult
survival
between
the
negative
control
and
mCry3A
treatments;
all
larvae
in
the
positive
control
treatment
died
in
the
pre­
imaginal
stage.
There
was
no
significant
difference
in
the
mean
number
of
days
for
pupae
to
form
in
the
negative
control
and
mCry3A
treatments,
but
the
mean
number
of
days
to
adult
emergence
was
significantly
lower
in
the
mCry3A
group.
A
subsequent
study
(
MRID
46265604)
determined
that
the
actual
mCry3A
concentration
in
the
treated
aphids
was
9
µ
g
MCRY3A­
0102/
g
of
aphid
(
9
ng
MCRY3A­
0102/
aphid).

Potential
routes
of
field
exposure
of
Coccinella
to
mCry3A
include
aphids
and
spider
mites
feeding
on
MIR604
corn
hybrids.
Based
on
the
aphids
containing
0.026
µ
g
mCry3A/
g
fresh
weight
and
the
mites
containing
4.7
µ
g
mCry3A/
g
fresh
weight,
the
EEC
for
ladybird
beetle
larvae
consuming
a
diet
of
85%
aphids
and
15%
spider
mites
would
be
0.73
µ
g
mCry3A/
g
fresh
weight
of
diet.
The
exposure
in
the
above
ladybird
beetle
toxicity
test
(
9
µ
g
mCry3A/
g
of
aphid)
was
therefore
12.3
times
the
EEC.

Rove
beetle
(
MRID
46265607)

Adult
rove
beetles
(
Aleochara
bilineata)
were
provided
beef
paste
diet
containing
MCRY3A­
0102
at
a
rate
nominally
equivalent
to
50
µ
g
microbe­
produced
mCry3A/
g
of
diet
for
35
days.
Negative
control
beetles
were
fed
diet
mixed
with
deionized
water
only,
and
positive
control
beetles
were
fed
diet
mixed
with
teflubenzuron
(
0.01
mg
a.
i./
g
of
diet).
To
assess
the
effects
of
treatment
on
fecundity,
approximately
500
onion
fly
(
Delia
antiqua)
pupae,
which
are
parasitized
by
rove
beetles,
were
placed
in
the
beetle
habitat
on
days
14,
21,
and
28
of
the
study.
During
days
35
to
76,
emergence
of
adult
rove
beetles
from
the
onion
fly
pupae
was
monitored.
There
was
no
statistically
significant
difference
in
day
35
mortality
among
any
of
the
rove
beetle
groups,
and
no
statistically
significant
difference
in
the
mean
number
of
progeny
produced
by
the
negative
control
and
MCRY3A­
0102
groups
from
days
35
to
76.
Reproduction
in
the
positive
control
group
was
significantly
reduced.
Subsequent
analysis
(
MRID
46265608)
of
the
treated
diet
recovered
91.7%
of
the
mCry3A,
indicating
the
test
material
was
present
at
the
intended
concentration
(
50
µ
g/
g
diet).
8
Rove
beetles
(
Staphylinidae)
generally
feed
on
other
invertebrates,
although
some
species
feed
on
decaying
plant
material
or
parasitize
other
insects.
A
worst­
case
EEC
was
calculated
using
an
MIR604
root
concentration
of
3.7
µ
g
mCry3A/
g
fresh
weight.
If
soil
invertebrates
incorporated
the
root
mCry3A
at
1.4
times
less
than
the
root
concentration,
the
soil
invertebrates
would
average
2.6
µ
g
mCry3A/
g
fresh
weight.
The
rove
beetle
exposure
in
the
above
study
(
50
µ
g/
g
diet)
was
then
19.2
times
the
EEC.
If
rove
beetles
consumed
decaying
plant
material
containing
3.2
µ
g
mCry3A/
g
fresh
weight
(
based
on
15­
day
silage),
the
margin
of
exposure
in
the
above
study
would
be
15.6
times
the
EEC.

Ground­
dwelling
beetle
(
MRID
42665605)

Ground­
dwelling
beetle
(
Poecilus
cupreus)
larvae
were
fed
daily
with
blowfly
(
Calliphora
vomitoria)
pupae
that
had
been
injected
with
MCRY3A­
0102
at
a
rate
nominally
equivalent
to
50
µ
g
microbeproduced
mCry3A
protein/
g
fly
pupa.
A
negative
control
of
fly
pupae
injected
with
deionized
water
only,
and
a
positive
control
of
fly
pupae
injected
with
teflubenzuron
(
0.664
ng
a.
i./
g
of
fly
pupa)
were
also
used
in
the
test.
When
the
P.
cupreus
larvae
pupated,
feeding
stopped
and
adult
emergence
was
monitored.
There
was
no
statistically
significant
difference
in
the
percent
pre­
imaginal
mortality
or
mean
weight
of
emerged
P.
cupreus
adults
between
the
MCRY3A­
0102
and
negative
control
groups.
Treatment
with
the
positive
control
produced
100%
pre­
imaginal
mortality.
A
subsequent
study
(
MRID
46465606)
found
the
mean
concentration
of
microbe­
produced
mCry3A
in
the
fly
pupae
used
in
the
test
was
12
µ
g
mCry3A/
g
pupa.

The
likely
route
of
field
exposure
of
non­
target
Carabids
to
mCry3A
would
be
prey
that
had
ingested
tissue
of
MIR604
hybrids,
e.
g.,
black
cutworm
(
Agrotis
ipsilon)
larvae.
Assuming
the
cutworm
larvae
contained
0.15
µ
g
mCry3A/
g
fresh
weight
(
based
on
studies
with
Cry1Ab),
the
beetle
exposure
in
the
above
study
(
12
µ
g/
g
pupa)
was
80
times
the
EEC.
In
a
worst
case
exposure
based
on
the
cutworm
larvae
containing
1.07
µ
g
mCry3A/
g
fresh
weight
(
using
a
spider
mite
mCry3A
concentration
1.4
times
lower
than
that
of
MIR604
tissue),
the
exposure
in
the
beetle
study
would
be
11.2
times
the
EEC.

Honeybee
(
MRID
46155618)

Honeybees
(
Apis
mellifera)
were
exposed
via
in­
hive
feeders
to
a
nominal
concentration
of
50
µ
g
MCRY3A­
0102/
g
of
sucrose
solution,
0.375
mg
of
diflubenzuron
insect
growth
regulator/
mL
of
sucrose
solution,
or
50%
w/
v
sucrose
solution
alone
for
5
days
and
monitored
for
21
days.
There
were
no
statistically
significant
differences
in
egg
cell
mortality,
larval
cell
mortality,
or
pre­
and
post­
test
hive
condition
between
the
test
and
sucrose
solution­
only
groups.
The
diflubenzuron
positive
control
produced
100%
mortality
in
both
egg
and
larval
cells,
and
significantly
reduced
post­
treatment
hive
condition.
Adult
bees
were
generally
not
affected
by
any
of
the
treatments.

Honeybees
are
not
likely
to
be
exposed
to
mCry3A
via
MIR604,
since
corn
does
not
produce
nectar
and
detectable
amounts
of
mCry3A
are
not
found
in
the
pollen.
The
honeybee
was
used
to
represent
the
order
Hymenoptera,
which
contains
wasps
that
are
parasites
of
corn
pests,
primarily
Lepidoptera.
There
is
currently
no
validated
test
to
expose
parasitic
Hymenoptera
larvae
to
high
doses
of
protein,
so
honeybee
larvae
were
used.
Assuming
the
average
concentration
of
mCry3A
in
Lepidoptera
larvae
is
1.4
µ
g/
g
fresh
weight
(
based
on
studies
with
Cry1Ab),
the
margin
of
exposure
for
honeybee
larvae
in
the
above
study
was
35.7
times
the
EEC.

Earthworm
(
MRID
46265611)

Groups
of
10
earthworms
(
Eisenia
fetida)
in
artificial
soil
were
exposed
to
MCRY3A­
0102
equivalent
to
a
nominal
concentration
of
250
µ
g
microbe­
produced
mCry3A/
g
moistened
soil
for
14
days.
Controls
were
exposed
to
the
same
soil
without
MCRY3A­
0102.
The
LC50
for
a
positive
control
(
2­
chloroacetamide)
group
in
the
same
soil
was
also
determined.
At
test
end,
the
test
material
group
9
worms
had
a
mortality
rate
of
5%
and
a
mean
weight
loss
of
5.8%.
Negative
control
worms
had
a
mortality
rate
of
0%
and
a
mean
weight
loss
of
11.4%.
The
LC50
for
the
positive
control
worms
was
18.0
mg/
kg
dry
soil.
Subsequent
extraction
of
mCry3A
from
treated
soil
samples
collected
on
days
0,
3,
7,
and
14
of
the
study
recovered
18.1%,
14.8%,
6.3%,
and
7.6%
of
the
nominal
concentration,
respectively
(
MRID
46265612).
The
low
recovery
rate
at
day
0
was
attributed
to
low
extractability
from
the
soil,
because
a
CPB
larval
bioassay
showed
that
the
test
material
was
active
for
at
least
5
days,
and
a
Western
blot
assay
showed
intact
mCry3A
was
present
at
14
days,
although
some
degradation
did
occur.

The
most
likely
route
of
field
exposure
of
earthworms
to
mCry3A
would
be
ingestion
of
senescent
plant
material
incorporated
into
the
soil.
Assuming
that
earthworms
ate
100%
senescing
MIR604
leaves
(
conservative
scenario)
containing
5.5
µ
g
mCry3A/
g
fresh
weight,
the
250
µ
g
mCry3A/
g
moistened
soil
in
the
above
study
would
be
46
times
the
EEC.

Other
toxicity
tests
No
ecotoxicity
studies
were
carried
out
using
wild
mammals,
but
an
acute
oral
toxicity
study
with
mice
was
conducted.
Mice
(
5/
sex)
received
a
single
oral
dose
of
MCRY3A­
0102
representing
approximately
2377
mg
mCry3A/
kg
body
weight.
After
14
days,
there
were
no
signs
of
toxicity,
and
all
endpoints
were
comparable
between
the
treated
and
control
groups.
The
LD50
for
males
and
females
was
>
2377
mg
MCRY3A­
0102/
kg
body
weight.

Rodents
such
as
squirrels,
mice,
voles,
and
woodchucks,
and
larger
mammals
such
as
raccoons
and
deer
ingest
corn
seed
or
ripening
ears.
The
DDD
of
mCry3A
for
mammals
can
be
calculated
using
the
formula
described
above
for
birds.
The
FIR/
bw
ratios
for
the
harvest
mouse
(
Micromys
minutus)
and
the
wood
mouse
(
Apodemus
sylvaticus)
consuming
cereal
seeds
have
been
estimated
to
be
0.33
and
0.28,
respectively.
Based
on
MIR604
hybrid
kernels
averaging
1.3
µ
g
mCry3A/
g
fresh
weight,
and
assuming
100%
of
the
diet
is
kernels,
the
DDD
for
rodents
was
estimated
to
be
0.43
mg
mCry3A/
kg
body
weight.
The
dose
used
in
the
acute
oral
toxicity
study
with
mice
discussed
above
would
be
over
5500
times
the
DDD.

Animals
grazing
on
corn
ears
may
consume
both
leaf
material
and
kernels.
Assuming
that
the
ears
contain
10.1
µ
g
mCry3A/
g
fresh
weight
(
a
worst­
case
exposure,
since
MIR604
hybrid
leaves
average
10.1
µ
g
mCry3A/
g
fresh
weight,
while
the
kernels,
which
would
compose
most
of
the
ear,
average
only
1.6
µ
g
mCry3A/
g
fresh
weight)
and
the
FIR/
bw
ratio
for
deer
is
0.09,
the
DDD
for
large
mammals
eating
a
100%
diet
of
MIR604
corn
ears
is
0.91
mg
mCry3A/
kg
body
weight.
The
dose
used
in
the
acute
oral
toxicity
study
with
mice
discussed
above
would
be
2600
times
the
large
mammal
DDD.

Potential
for
exposure
of
non­
target
Coleoptera
The
registrant
states
that
mCry3A
activity
is
expected
to
be
limited
to
beetles,
particularly
some
members
of
the
Chrysomelidae
(
leaf
beetles,
flea
beetles,
rootworms),
Curculionidae
(
weevils
and
snout
beetles),
and
Tenebrionidae
(
darkling
beetles)
families.
The
Chrysomelidae
and
Curculionidae
are
herbivores
and
could
potentially
be
exposed
to
MIR604
pollen.
However,
since
mCry3A
has
not
been
detected
in
MIR604
pollen,
exposure
of
these
beetles
is
unlikely.
Tenebrionidae
larvae
and
adults
are
plant
material
scavengers,
and
some
mealworm
species
are
pests
of
stored
grain.
Darkling
beetles
are
not
recorded
as
a
pest
of
corn,
but
may
be
exposed
to
mCry3A
by
feeding
on
senescent
MIR604
tissue
in
cornfields.
The
LD50
of
a
native
Cry3A
to
yellow
mealworm
(
Tenebrio
molitor),
a
representative
Tenebrionid,
has
been
found
to
be
11.4
µ
g
Cry3A/
g
larvae.
Assuming
that
the
activity
of
mCry3A
to
T.
molitor
is
roughly
equal
to
that
of
native
Cry3A,
and
the
mCry3A
concentration
in
senescing
MIR604
hybrid
leaves
is
5.5
µ
g/
g
fresh
weight,
a
larva
would
need
to
ingest
about
2.1
g
of
senescing
leaf
tissue
to
receive
the
median
lethal
dose.
This
represents
about
3500
times
the
body
weight
of
a
first
instar
larva
and
about
15
times
the
body
weight
of
a
final
instar.
Since
Tenebrionidae
10
are
omnivorous
and
mCry3A
rapidly
degrades
in
soil,
darkling
beetles
are
unlikely
to
be
exposed
to
sufficient
mCry3A
to
be
significantly
impacted.

Endangered
species
considerations
The
registrant
states
that
no
harmful
effects
of
mCry3A,
native
Cry3A,
or
other
Cry3
proteins
have
been
shown
in
taxa
outside
Coleoptera.
The
activity
of
mCry3A
is
expected
to
be
limited
to
certain
species
of
the
Chrysomelidae,
Curculionidae,
and
Tenebrionidae
families,
and
there
are
currently
no
endangered
or
threatened
species
in
these
families.
Additionally,
there
are
no
known
endangered
or
threatened
beetle
species
in
habitats
where
corn
is
grown.
Therefore,
no
endangered
or
threatened
beetles
are
expected
to
be
harmed
by
mCry3A
pollen
expressed
in
MIR604
hybrids.

The
lack
of
detectable
expression
of
mCry3A
in
MIR604
pollen,
the
lack
of
weediness
in
corn,
the
absence
of
sexually
compatible
wild
relatives
of
corn
in
the
U.
S.,
and
the
low
probability
of
significant
mCry3A
dispersal
in
soil
indicates
that
endangered
or
threatened
species
would
only
be
exposed
to
mCry3A
by
eating
MIR604
plants
or
organisms
that
had
fed
on
MIR604
tissue.
The
Agency
has
reviewed
the
likelihood
of
endangered
bird
and
bat
species
exposure
to
Cry3Bb1,
a
coleopteran­
active
protein
expressed
in
Event
MON863,
concluding
that
endangered
birds
and
bats
rarely
forage
in
agricultural
fields
and
that
insectivorous
species
tend
to
take
flying
insects
rather
than
larvae
(
the
life
stage
most
likely
to
contain
Cry
protein).
Similarly,
cultivation
of
MIR604
is
unlikely
to
have
any
harmful
effects
on
endangered
or
threatened
species.
11
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis,
M.
S.
EPA
Secondary
Reviewer:
Tessa
Milofsky,
M.
S.

STUDY
TYPE:
Nonguideline
MRID
NO:
46265613
DP
BARCODE:
DP303605
TEST
MATERIAL:
mCry3A
Protein
in
Event
MIR604
Corn
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
N/
A
TITLE
OF
REPORT:
Environmental
Fate
Assessment
of
Modified
Cry3A
Protein
in
Event
MIR604
Corn
AUTHOR:
Raybould,
A.

STUDY
COMPLETED:
April
27,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
is
a
compilation
and
is
not
subject
to
GLP
standards.

STUDY
SUMMARY:
MIR604
corn
plants
have
been
shown
to
express
mCry3A
protein
in
leaves,
kernels,
roots,
and
silks,
but
the
protein
was
not
detected
in
corn
pollen.
Corn
leaf
assays
were
used
to
verify
that
mCry3A
expression
is
stable
over
multiple
generations
and
a
soil
degradation
study
showed
that
mCry3A
degrades
readily,
with
a
DT50
of
7.6
days
in
silty
clay
loam
soil.
Due
to
corn's
lack
of
invasive
characteristics
and
the
low
probability
that
the
mCry3A
gene
from
Event
MIR604
would
transfer
to
a
wild
relative
of
corn,
it
is
unlikely
that
mCry3A
will
spread
beyond
cultivated
sites
and
persist
in
weedy
populations.
It
is
also
unlikely
that
genes
present
in
MIR604
corn
would
be
subject
to
horizontal
gene
transfer
at
a
frequency
that
exceeds
the
rate
of
transfer
in
other
plants.
In
the
unlikely
event
that
mcry3A
is
stably
integrated
and
expressed
in
a
soil
microorganism,
no
harmful
effects
are
expected.
Laboratory­
based
non­
target
studies
indicate
that
expected
environmental
exposure
to
mCry3A
protein
will
not
result
in
unreasonable
adverse
effects
to
beneficial
organisms.
The
submission
also
notes
the
low
likelihood
that
aquatic
organisms
will
be
exposed
to
mCry3A,
since
it
is
unlikely
that
the
protein
will
enter
watercourses
via
soil
particle
movement,
pollen
dispersal,
or
seed
spillage.
12
CLASSIFICATION:
Supplemental
Introduction
The
cry3A
gene
from
Bacillus
thuringiensis
subsp.
tenebrionsis
was
synthetically
recreated
to
optimize
for
expression
in
corn,
and
changed
so
that
the
encoded
modified
Cry3A
(
mCry3A)
protein
has
enhanced
activity
against
the
western
corn
rootworm
(
Diabrotica
virgifera
virgifera)
and
the
northern
corn
rootworm
(
D.
longicornis
barberi).
Corn
plants
transformed
with
mCry3A,
such
as
Event
MIR604,
display
resistance
to
these
pests.

mCry3A
Levels
in
MIR604
Hybrids
In
MIR604
corn
plants
sampled
at
four
growth
stages
(
whorl
stage,
6
weeks
after
planting;
anthesis,
10­
11
weeks
after
planting;
seed
maturity,
18­
20
weeks
after
planting;
and
senescence,
about
24
weeks
after
planting),
mCry3A
was
found
in
the
leaves,
kernels,
roots,
and
silks
at
each
growth
stage,
but
not
in
pollen
(
limit
of
detection
=
0.07
µ
g/
g
fresh
wt).
The
expression
profile
appears
to
be
stable
over
multiple
generations.
In
four
successive
backcrosses
under
greenhouse
conditions,
leaf
tissue
sampled
at
anthesis
showed
that
the
concentration
of
mCry3A
in
each
generation
was
similar,
with
no
trend
toward
increased
or
decreased
expression.

Degradation
in
Soil
Evidence
indicates
that
Cry
proteins
undergo
enzymatic
degradation
by
soil
proteases.
Laboratory
soil
degradation
studies
on
lepidopteran­
active
proteins
(
Cry1Ab,
Cry1Ac,
Cry1F)
indicate
they
are
rapidly
degraded,
with
a
DT50
(
time
for
the
Cry
protein
concentration
or
bioactivity
to
fall
to
half
its
initial
value)
of
2
to
22
days
for
Cry1A
proteins
and
a
DT50
of
just
over
3
days
for
Cry1F.
The
DT50
for
Cry3Bb1
protein,
which
is
active
against
certain
Coleoptera,
is
between
less
than
1
day
and
up
to
9
days.
In
a
laboratory
degradation
test
using
mCry3A
incorporated
into
a
silty
clay
loam
soil
(
MRID
46265614),
a
Colorado
potato
beetle
bioassay
determined
the
DT50
of
MCRY3A­
0102
protein
was
7.6
days.

Spread
and
Persistence
of
the
mCry3A
gene
The
probability
of
the
mcry3A
gene
from
MIR604
transferring
to
wild
relatives
of
corn
is
very
low.
Corn
will
hybridize
with
a
group
of
taxa
collectively
known
as
teosinte,
which
are
native
to
Central
America.
Teosinte
species
have
co­
existed
with
cultivated
corn
for
several
thousand
years,
but
have
remained
genetically
distinct
from
cultivated
varieties
despite
occasional
introgression.
While
teosinte
species
are
grown
in
the
U.
S.
in
botanical
gardens,
fertilization
of
these
plants
by
MIR604
pollen
is
unlikely.

Species
of
the
genus
Tripsacum,
three
of
which
occur
in
the
U.
S.,
are
considered
to
be
close
relatives
of
corn.
However,
hybrids
between
Tripsacum
and
corn
are
difficult
to
obtain
and
are
often
sterile.
T.
dactyloides
is
widespread,
but
does
not
hybridize
readily
with
corn,
and
the
probability
of
backcross
or
F2
progeny
of
Tripsacum
x
Zea
hybrids
being
produced
in
the
field
is
negligible.
In
the
environmental
assessment
section
of
the
BRAD
for
Bt­
plant
incorporated
protectants
(
2001),
the
Agency
concluded
that
the
chance
of
natural
introgression
of
genes
from
corn
to
Tripsacum
was
extremely
remote.
13
In
its
reassessment
of
the
environmental
safety
of
Bt
plant­
incorporated
protectants,
the
Agency
reviewed
information
concerning
the
theoretical
risks
of
horizontal
gene
transfer
(
HGT),
and
found
no
evidence
for
HGT
under
field
conditions.
Non­
agency
reviews
found
very
few
examples
of
HGT,
and
these
cases
involved
artificially
high
sequence
homology
between
the
transgene
and
the
potential
recipient.
Data
comparing
full
genomic
sequences
of
various
prokaryotes
and
eukaryotes
have
identified
putative
HGT
events,
although
the
data
are
subject
to
interpretation.
There
is
no
reason
to
suppose
that
corn
derived
from
Event
MIR604
is
likely
to
transfer
genes
by
HGT
at
a
higher
rate
than
any
other
plant.

The
likelihood
of
exposure
to
mCry3A
via
microorganisms
expressing
the
mcry3A
gene
from
MIR604
is
minimal.
Should
a
cry
gene
be
transferred
from
a
transgenic
plant
to
a
microorganism,
there
should
be
no
significant
hazards.
B.
thuringiensis
is
common
in
soil,
and
cry
genes
have
been
available
for
HGT
to
other
species
for
long
periods.
No
harmful
effects
appear
to
have
resulted
from
the
prolonged
exposure.
Soil
microorganisms
have
not
previously
been
exposed
to
the
mcry3A
gene
contained
in
MIR604.
The
DNA
sequence
of
mcry3A
was
substantially
altered
from
native
cry3A
to
optimize
codon
use
for
expression
in
plants,
so
the
mcry3A
gene
likely
has
lower
homology
to
potential
recombination
sites
in
soil
microorganisms
than
the
native
cry3A
gene
does.
Should
mcry3A
be
integrated
into
a
plasmid
or
chromosome
of
a
bacterium,
mCry3A
protein
is
unlikely
to
be
produced
because
the
maize
metallothionen
promoter
linked
to
mcry3A
in
MIR604
is
unlikely
to
function
in
bacteria
and
codon
use
in
mcry3A
is
optimized
for
expression
in
corn,
not
bacteria.
Laboratory
tests
of
the
effects
of
mCry3A
protein
on
a
variety
of
non­
target
organisms
have
revealed
no
adverse
effects.
In
the
unlikely
event
that
mcry3A
is
stably
integrated
and
expressed
in
a
soil
microorganism,
no
harmful
effects
are
expected.

Weediness
Corn
has
lost
the
ability
to
survive
outside
cultivation,
and
is
unlikely
to
form
self­
sustaining
populations
as
a
weed
since
a)
it
is
easily
controlled
with
herbicides,
b)
seed
dispersal
is
limited,
c)
the
seeds
lack
dormancy
and
are
exposed
to
winter
conditions,
d)
it
requires
disturbed
ground
to
germinate,
and
e)
it
is
not
competitive
with
perennial
vegetation.
The
lack
of
invasiveness
of
corn
in
non­
agricultural
habitats
makes
it
unlikely
that
mCry3A
will
spread
from
cultivated
sites
and
persist
as
weedy
populations
of
MIR604.

Environmental
Exposure
and
Fate
Ecologically
significant
exposure
to
mCry3A
outside
cultivation
is
unlikely
to
occur
through
contact
with
pollen,
since
mCry3A
protein
has
not
been
detected
in
MIR604
hybrid
pollen.
Due
to
its
rapid
degradation
in
soil,
mCry3A
is
not
likely
to
spread
from
cultivation
to
surface
or
ground
water.
Exposure
to
mCry3A
during
cultivation
would
be
limited
to
contact
with
MIR604
tissues,
and
possibly
short­
term
exposure
to
exuded
proteins
in
soil.
After
harvest,
mCry3A
may
remain
in
MIR604
plant
tissue
until
the
tissue
degrades.
The
mCry3A
is
expected
to
degrade
rapidly
and
not
move
offsite.

Aquatic
organisms
are
not
likely
to
be
exposed
to
mCry3A
since
it
is
unlikely
that
mCry3A
will
enter
watercourses
via
soil
particle
movement,
pollen
dispersal,
or
seed
spillage.
Other
than
humans
or
animals
that
eat
corn
grain,
forage,
or
silage,
the
only
organisms
likely
to
be
exposed
to
mCry3A
from
cultivation
of
MIR604
are
pests
and
non­
target
organisms
that
feed
on
corn
tissue,
predators
and
parisitoids
of
these
animals,
and
soil
organisms.
Exposure
of
non­
target
organisms
feeding
on
the
above­
ground
parts
of
corn
will
be
limited
to
the
period
of
cultivation.
14
Soil
organisms
may
be
exposed
to
mCry3A
during
cultivation
and
afterward
by
material
incorporated
into
the
soil
following
harvest.
Due
to
the
rapid
degradation
of
mCry3A
in
soil,
the
post­
harvest
exposure
of
soil
organisms
to
significant
amounts
of
mCry3A
would
be
limited
to
a
few
weeks.
A
second
exposure
of
non­
target
organisms
could
arise
if
MIR604
volunteers
occur.
This
exposure
would
be
much
lower.
15
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis,
M.
S.
EPA
Secondary
Reviewer:
Tessa
Milofsky,
M.
S.

STUDY
TYPE:
Nonguideline
MRID
NO:
46265614
DP
BARCODE:
DP303605
TEST
MATERIAL:
MCRY3A­
0102
STUDY
NO:
SSB­
015004
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Syngenta
Biotechnology,
Inc.,
Regulatory
Science
Laboratory,
Research
Triangle
Park,
NC
27709­
2257
TITLE
OF
REPORT:
Laboratory
Soil
Degradation
of
Modified
Cry3A
Protein
(
MCRY3A­
0102)

AUTHORS:
Kramer,
C.
and
R.
Joseph
STUDY
COMPLETED:
April
27,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
GLP
compliant.

STUDY
SUMMARY:
Degradation
of
MCRY3A­
0102
(
microbe
produced
mCry3A
protein)
in
silty
clay
loam
soil
was
evaluated
in
a
laboratory
study,
where
treated
soil
samples
were
maintained
under
conditions
that
mimicked
the
field
environment.
MCRY3A­
0102
activity
in
soil
was
evaluated
with
a
Colorado
potato
beetle
(
CPB)
(
Leptinotarsa
decemlineata)
larval
bioassay.
The
test
protein
was
incorporated
into
the
test
soil
at
a
nominal
rate
of
230
µ
g
microbe­
produced
mCry3A/
g
of
dry
soil.
Soil
samples
were
collected
at
days
0,
1,
3,
7,
12,
and
30
and
frozen
until
needed
for
CPB
diet
formulation.
CPB
larvae
were
maintained
on
each
soil/
diet
mixture
for
72
hrs,
after
which
mortality
was
determined.
Larval
mortality
was
48­
53%
when
fed
diets
containing
soil
from
days
0,
1,
3,
and
7;
mortality
declined
to
9%
when
fed
diet
containing
soil
from
day
30.
Based
on
these
results,
a
simple
first­
order
kinetic
model
determined
that
the
DT50
for
mCry3A
in
this
silty
clay
loam
soil
is
7.6
days.

CLASSIFICATION:
Supplemental
16
Test
Material
The
test
substance,
MCRY3A­
0102,
E.
coli­
produced
mCry3A
protein,
Batch
Number
not
provided,
was
supplied
by
the
sponsor
with
a
reported
purity
of
90.3%
w/
w.
No
expiration
date
was
reported.
The
test
substance
was
stored
at
­
20
°
C
prior
to
use.

Test
Methods
The
test
soil
was
a
silty
clay
loam
collected
from
a
corn­
growing
region
of
Iowa.
Characterization
of
the
soil
is
provided
in
Appendix
A
of
MRID
462656­
14.
The
soil
was
passed
through
a
2
mm
sieve
and
acclimated
at
25
±
1
°
C
at
a
constant
moisture
level
for
10
days
prior
to
the
test.
One
day
prior
to
the
test,
50
g
of
equivalent
dry
weight
soil
(~
59
g)
at
18.25%
moisture
level
was
added
to
250­
mL
glass
incubation
flasks.

At
test
start,
MCRY3A­
0102
was
prepared
in
deionized
water
at
a
concentration
of
2.3
mg
microbe­
produced
mCry3A/
mL
and
applied
in
a
volume
of
5
mL
to
each
of
24
flasks
of
soil
for
a
dose
corresponding
to
230
µ
g
microbe­
produced
mCry3A/
g
of
soil
on
a
dry
weight
basis.
One
control
flask
and
6
biomass
determination
flasks
were
prepared
using
soil
dosed
with
5
mL
of
deionized
water
only.
The
flasks
were
capped,
shaken,
and
weighed
to
determine
moisture
content.
Flasks
were
maintained
at
25
±
1
°
C
and
moisture
levels
were
adjusted
throughout
the
test
to
75
±
12%
of
field
moisture
capacity
at
1/
3
bar.
Samples
were
collected
from
the
dosed
flasks
after
0,
1,
3,
7,
12,
and
30
days
of
incubation
and
frozen
prior
to
their
use
in
a
degradation
bioassay
using
Colorado
potato
beetle
(
CPB)
(
Leptinotarsa
decemlineata)
larvae.
Microbial
activity
of
soil
in
the
biomass
determination
flasks
was
measured
on
days
0
and
42
using
a
Micro­
Oxymax
analyzer
(
procedure
described
in
Appendix
A
of
MRID
462656­
14).

For
the
bioassay,
soil
collected
at
each
incubation
time
point
was
incorporated
into
a
stock
CPB
diet
at
a
concentration
of
10%
w/
w,
and
the
resulting
suspension
was
poured
into
Petri
dishes.
A
negative
control
dosed
with
sterile
water
only
was
prepared
in
the
same
manner,
and
a
positive
control
containing
no
soil
was
prepared
by
adding
MCRY3A­
0102
test
solution
to
CPB
diet
to
give
a
concentration
of
23
µ
g
microbe­
produced
mCry3A/
g
of
diet
(
equivalent
to
that
in
the
day
0
soil
samples).
Ten
freshly­
hatched
CPB
larvae
(
from
eggs
obtained
from
New
Jersey
Dept
of
Agriculture,
Trenton)
were
added
to
each
dish
of
diet,
covered,
and
maintained
under
ambient
laboratory
conditions.
The
test
material
and
negative
control
treatments
were
replicated
12
times
(
120
larvae
total
each)
and
the
positive
control
plates
were
replicated
4
times
(
40
larvae
total).
Larval
mortality
was
assessed
at
72
hours.

Results
Summary
Results
of
the
larval
bioassay
are
given
in
Table
1.
Mean
CPB
larvae
mortality
in
MCRY3A­
0102­
treated
soil
was
48­
54%
during
the
first
week,
then
declined
rapidly
to
9%
on
day
30.
The
loss
rate
of
mCry3A
activity
was
estimated
by
plotting
the
mean
percent
mortality
against
days
of
incubation
and
fitting
simple
first
order
kinetics
to
the
data
using
ModelManager
(
Cherwell
Scientific
Publishing,
Oxford,
UK).
The
DT50
value
(
time
for
50%
of
initial
bioactivity
to
dissipate)
for
degradation
of
MCRY3A­
0102
in
this
soil
was
estimated
to
be
7.6
days.
Biomass
determinations
of
soil
at
study
start
and
end
showed
that
microbial
activity
was
maintained
during
the
study.
17
TABLE
1.
MCRY3A­
0102
activity
in
treated
soil
as
measured
by
Colorado
potato
beetle
mortality
(%)

Mean
percent
mortality
MCRY3A­
0102
Negative
control
Positive
control
Day
0
Day
1
Day
3
Day
7
Day
12
Day
30
18
53
53
54
48
51
11
9
Data
from
p.
15,
MRID
46265614
Study
Authors'
Conclusions
The
study
authors
concluded
that
mCry3A
was
rapidly
degraded
in
soil,
with
a
first­
order
DT50
of
7.6
days.
