1
MEMORANDUM
SUBJECT:
Review
of
Non­
Target
Insect
Studies,
Select
Non­
target
Organism
Studies,
and
Test
Substance
Analysis
Studies
Submitted
for
Registration
of
Event
MIR604
Which
Contains
the
Modified
Cry3A
Protein
(
mCry3A)
in
Maize.
DP
#
303605
MRID
Numbers:
46265602
through
46265612,
46265615
and
46155618
FROM:
Mika
J.
Hunter,
Biologist
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

THROUGH:
Tessa
Milofsky,
M.
S.,
Agronomist
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

Zigfridas
Vaituzis,
Ph.
D,
Microbiologist
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

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

Background
Syngenta
Seeds,
Inc.
submitted
data
to
the
EPA
in
support
of
registration
of
a
new
genetically
modified
maize,
Event
MIR604.
MIR604
maize
contains
a
modified
Cry3A
protein
(
mCry3A)
that
targets
the
western
corn
rootworm,
Diabrotica
virgifera.
This
memorandum
contains
reviews
of
Syngenta's
submitted
non­
target
insect
studies
for
ladybird
beetle
(
Coccinella
septempunctata),
carabid
beetle
(
Poecilus
cupreus),
rove
beetle
(
Aleochara
bilineata),
insidious
UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
OFFICE
OF
PREVENTION,
PESTICIDES
AND
TOXIC
SUBSTANCES
2
flower
bug
(
Orius
insidiosus),
and
honeybee
(
Apis
mellifera).
In
addition,
this
document
also
includes
reviews
of
the
earthworm
toxicity
study
(
Eisenia
fetida),
analysis
of
soil
used
in
the
earthworm
study,
fish
feed
analysis
study,
and
broiler
chicken
study
(
Gallus
domesticus).
Data
evaluation
records
are
attached
at
the
end
of
this
document.

This
document
also
includes
discussion
of
information
provided
in
the
submission
titled
"
Environmental
Safety
Assessment
of
Modified
Cry3A
Protein
and
Event
MIR604
Corn
to
Non­
Target
Organisms"
(
MRID
46265601).
A
complete
review
and
DER
for
this
study
is
included
in
the
Ecological
Risk
Assessment
(
Milofsky,
2006).
Only
information
pertinent
to
this
document
was
included.

Conclusions
and
Recommendations
The
submitted
studies
were
all
scientifically
sound
and
are
classified
as
acceptable.
However,
several
studies
contained
deviations
from
the
OPPTS
testing
guidelines.
Such
deviations
included,
but
are
not
limited
to,
length
of
observation
period,
control
group
mortality,
and
actual
dose
of
test
substance
ingested
by
test
organisms.
Specific
aspects
of
particular
studies
that
were
significant
to
note
are
described
in
detail
below.
It
was
confirmed
for
each
species,
except
for
honeybee,
that
bioactive
mCry3A
(
MCRY3A­
0102)
was
present
in
the
ladybird
beetle,
carabid,
rove
beetle,
insidious
flower
bug,
and
earthworm
test
diets.
Event
MIR604
maize
does
not
express
mCry3A
in
pollen.
Overall,
the
submitted
studies
show
that
direct
dietary
exposure
to
mCry3A
protein
at
levels
encountered
in
the
field,
because
not
all
tests
were
done
at
10X
the
concentration
in
maize
leaves,
results
in
no
significant
adverse
effects
on
ladybird
beetles,
carabid
beetles,
rove
beetles,
insidious
flower
bugs,
honeybees,
earthworms,
and
chickens.

Ladybird
Beetle
Testing
(
Coccinellidae:
Coccinella
septempunctata)

MRID
46265603
A
Tier
I
non­
target
insect
study
on
ladybird
beetles
was
submitted
based
on
OPPTS
Guideline
885.4340.
The
study
looked
at
both
larvae
and
adult
ladybird
beetles.
In
addition,
developmental
time
from
larvae
to
adult
was
also
reported.
In
the
study,
administered
between
September
27,
2003
and
October
26,
2003,
4­
day­
old
ladybird
beetle
larvae
were
fed
daily
with
live
pea
aphids
(
Acyrthosiphon
pisum).
Three
treatments
were
established
(
test
treatment,
positive
and
negative
control)
and
each
treatment
was
replicated
40
times.
Each
replication
contained
one
ladybird
beetle.
The
test
substance
used
was
MCRY3A­
0102,
a
purified
preparation
of
microbially
expressed
mCry3A.
The
test
substance
was
formulated
so
that
a
solution
was
created
that
consisted
of
50
µ
g
MCRY3A­
0102/
mL
Agral
solution
(
5.5
mg
MCRY3A­
0102
in
100
mL
of
0.004
v/
v
Agral
90
solution).
This
concentration
is
approximately
10X
the
amount
of
mCry3A
protein
expressed
in
transgenic
maize
leaves.
Aphids
fed
to
the
negative
control
group
were
immersed
in
the
Agral
90
solution
only.
Aphids
fed
to
the
positive
control
group
were
immersed
in
a
solution
of
0.5
mL
of
Nemolt
(
teflubenzuron)/
L
of
0.004%
v/
v
Agral
90.
Agral
90
is
a
non­
ionic
surfactant.
Aphids
were
immersed
in
the
appropriate
solution
for
approximately
30
seconds
and
allowed
to
dry
for
30
minutes.
Freshly­
treated
live
aphids
were
provided
daily
until
larvae
pupated.
The
number
of
aphids
fed
to
the
larvae
increased
with
3
age
of
larvae
(
table
provided
in
Date
Evaluation
Record).
No
other
food
or
water
source
was
provided.
The
test
was
conducted
in
a
room
maintained
at
21­
27
°
C,
35­
92%
relative
humidity,
and
16
hours
light.
After
adult
beetles
emerged,
they
were
moved
into
clean
dishes
(
1
male
and
1
female
per
dish,
where
numbers
allowed).
Adult
beetles
were
fed
approximately
50
treated
pea
aphids
3
times
a
week
for
14
days.
The
condition
of
beetle
larvae
was
assessed
daily,
while
the
condition
of
the
adult
beetles
was
assessed
at
each
feeding.
In
addition,
it
should
be
noted
that
the
study
was
compliant
with
OECD
and
UK
Good
Laboratory
Practice
(
GLP)
regulations.

There
were
no
statistically
significant
differences
in
pre­
imaginal
or
adult
survival
between
the
negative
control
and
MCRY3A­
0102
treatments.
All
larvae
in
the
positive
control
died
in
the
pre­
imaginal
stage.
Results
for
individual
treatments
were
compared
to
the
control
using
a
Chisquare
test.

There
was
no
significant
difference
in
the
mean
number
of
days
for
pupae
to
form
in
the
negative
control
and
MCRY3A­
0102
treatments.
However,
the
mean
number
of
days
from
larvae
to
adult
emergence
was
significantly
lower
in
the
MCRY3A­
0102
treatment
(
no
P
value
provided
in
study
report).
Mean
developmental
time
was
compared
between
the
test
treatment
and
control
treatment
using
t­
tests
for
unmatched
pairs.

An
additional
study
(
MRID
46265604)
was
conducted
to
analyze
the
concentration
of
mCry3A
that
was
present
on
the
surface
of
immersed
aphids.
An
ELISA
analysis
showed
that
treated
aphids
contained
9
µ
g
MCRY3A­
0102/
g
of
aphid
(
9
ng
mCry3A/
aphid).
This
concentration
is
1.8X
greater
than
the
concentration
of
mCry3A
expressed
in
maize
leaves.
The
diet
analysis
study
also
showed,
through
Western
blot
analysis,
that
intact
proteins
were
present
on
the
aphids.

Discussion:
The
diet
analysis
study
(
46265604)
showed
that
the
actual
amount
of
MCRY3A­
0102
protein
consumed
by
larvae
and
adult
ladybird
beetles
was
below
the
targeted
concentration
of
50
µ
g/
g
aphid.
The
actual
concentration
of
protein
ingested
was
9
µ
g
MCRY3A­
0102/
g
aphid.
Although
this
study
did
not
dose
the
ladybird
beetles
at
the
targeted
concentration
of
10X
the
concentration
expressed
in
maize
leaves,
the
study
shows
that
this
ingestion
of
MCRY3A­
0102
protein
does
not
result
in
negative
effects
to
ladybird
beetles.
The
decrease
in
development
time
from
larvae
to
adult
does
not
indicate
a
substantial
negative
adverse
effect.
Because
each
larva
was
considered
a
separate
replicate,
the
statistical
analyses
might
have
been
exaggerated.
In
the
results
section
of
the
study,
the
author
reports
that
statistical
significance
was
observed
with
a
P
value
<
0.05.
The
author
does
not
indicate
how
close
to
0.05
the
value
was
which
would
have
provided
a
better
indication
of
significance.
In
addition,
the
mean
numbers
of
days
to
pupae
formation
was
not
statistically
different.
However,
it
would
have
been
beneficial
to
report
if
there
were
significant
differences
in
development
from
pupae
to
adult.

A
discussion
of
the
margin
of
exposure
provided
by
the
registrant
(
MRID
46265601)
presents
information
regarding
the
estimated
concentration
of
Cry1Ab
protein
in
ladybird
beetle
prey.
Researchers
estimate
that
aphids
contain
at
most
a
concentration
of
Cry1Ab
that
is
250
times
less
than
the
corn
on
which
they
are
feeding.
In
addition,
the
concentration
of
Cry1Ab
in
mites
has
been
shown
to
be
1.4
times
lower
than
the
corn
they
were
feeding
on.
Additionally,
the
author
4
states
that
a
worst
case
exposure
scenario
for
ladybird
beetles
would
be
a
diet
consisting
of
15%
mites
and
85%
aphids.
The
author
expects
that
the
concentration
of
mCry3A
that
ladybird
beetle
prey
contains
is
substantially
lower
than
10X
the
concentration
expressed
in
maize
leaves.
BPPD
agrees
with
the
rationale
the
registrant
submitted.

It
is
important
to
take
into
consideration
the
likely
route
of
exposure,
as
well
as
the
likely
concentration
of
exposure
that
the
non­
target
insects
will
be
subject
to.
However,
it
is
important
that
the
study
sponsor
and
study
author
include
explanations
of
anticipated
and
appropriate
dose
concentrations
within
the
study
submission.
In
the
submitted
ladybird
beetle
study,
the
sponsor
does
not
include
an
explanation
why
they
were
not
dissatisfied
that
the
ladybird
beetles
were
not
dosed
with
the
intended
concentration
of
MCRY3A­
0102
(
50
µ
g).
It
is
recommended
that
if
the
sponsor
thought
that
it
was
not
appropriate
to
dose
the
ladybird
beetles
at
10X
the
concentration
expressed
in
the
maize
leaves,
the
sponsor
should
have
provided
that
information
along
with
the
submitted
study.
Therefore,
whether
adverse
effects
would
have
been
seen
at
10X
concentrations
has
not
been
determined.

Conclusion:
This
study
is
considered
acceptable.
Although
the
test
was
not
conducted
at
the
targeted
10X
concentrations,
the
results
do
show
that
no
hazard
to
ladybird
beetles
is
expected
at
mCry3A
concentrations
that
could
be
encountered
in
the
field.

Ground
Beetle
Testing
(
Carabidae:
Poecilus
cupreus)

MRID
46265605
A
Tier
I
non­
target
insect
study
on
carabid
beetle
larvae
was
submitted
based
on
OPPTS
guideline
885.4340.
The
submitted
study
was
OECD
and
UK
GLP
compliant.
The
study
was
conducted
between
November
21,
2003
and
January
8,
2004.
During
the
study,
larvae
of
Poecilus
cupreus
were
fed
daily
with
blowfly
pupae
(
Calliphora
vomitoria)
that
had
been
injected
with
MCRY3A­
0102
solution.
The
MCRY3A­
0102
was
a
sample
of
modified
Cry3A
protein
(
mCry3A)
that
was
purified
from
cells
of
recombinant
E.
coli.
The
solution
that
was
injected
into
the
blowfly
pupa
contained
50
µ
g
MCRY3A­
0102/
g
of
pupa.
The
negative
control
carabid
larvae
were
fed
blowfly
pupa
that
were
injected
with
only
deionized
water.
A
positive
control
included
blowfly
pupa
that
were
injected
with
teflubenzuron.
After
being
injected,
fly
pupae
were
frozen
to
preserve
for
later
use.
Each
treatment
was
replicated
40
times,
and
each
replicate
contained
one
carabid
larva.
24
to
48
hour­
old
beetle
larvae
were
put
in
glass
tubes
filled
with
25
g
(
dry
weight)
of
sandy
soil
maintained
at
approximately
35%
of
its
water
holding
capacity.
One
fly
larva
was
placed
in
each
container,
and
capped
with
a
ventilated
stopper.
Tubes
were
maintained
at
19
º
to
22
º
(
64%­
90%
relative
humidity)
and
were
placed
upright
in
plastic
boxes
with
ventilated
lids
in
constant
darkness.
The
fly
pupae
were
replaced
daily
with
freshly
defrosted
pupae
until
the
beetle
began
pupation.
The
beetle
larvae
were
assessed
3
times
per
week
for
the
first
two
weeks
of
the
study,
and
two
times
per
week
thereafter
until
pupation.
Starting
at
day
32,
the
test
containers
were
checked
daily
for
emerging
adult
beetles.
The
emerged
adults
were
weighed
and
sexed,
and
overall
mortality
and
final
adult
weights
were
analyzed
statistically.
5
In
another
study
(
46265606),
the
diet
of
the
carabid
beetles
was
analyzed
and
tested
for
activity
of
MCRY3A­
0102.
The
study
determined
that
the
actual
concentration
of
MCRY3A­
0102
was
12
µ
g/
g
fly
pupae.
This
amount
is
less
than
10X
the
expressed
concentration
in
maize
leaves
(
50
µ
g).
The
study
also
showed,
through
Western
blot
analysis,
that
active
MCRY3A­
0102
was
likely
present
in
the
fly
pupa.

Discussion:
Although
carabid
beetle
larvae
were
not
dosed
at
10X
the
concentration
expressed
in
maize
leaves,
the
study
was
scientifically
sound
and
the
beetle
larvae
were
dosed
at
a
level
greater
than
the
concentration
expressed
in
maize
leaves.

The
registrant
submitted
supporting
information
regarding
EEC's
in
a
separate
document
(
MRID
46265601)
that
is
important
to
note
in
this
review.
In
the
document
the
registrant
presents
rationale
for
their
expected
maximum
EEC.
The
author
states
that
the
main
route
of
exposure
of
non­
target
carabids
to
mCry3A
will
be
through
eating
prey
that
has
eaten
tissue
of
MIR604
hybrids.
One
common
prey
organism
for
carabid
beetles
is
larvae
of
the
black
cutworm.
The
author
states
that
black
cutworm
larvae
contain
a
concentration
of
Cry1Ab
that
is
10
times
less
than
the
concentration
expressed
in
the
plant.
The
registrant
further
discusses
EECs
and
routes
of
exposure,
stating
that
an
extreme
worst­
case
scenario
is
to
assume
that
the
concentration
of
MCRY3A­
0102
in
the
cutworm
larvae
is
1.4
times
lower
than
in
the
MIR604
hybrids.
This
conversion
factor
is
derived
from
the
concentration
of
Cry1Ab
in
spider
mites
relative
to
their
diet.
Therefore,
the
registrant
concludes
that
the
maximum
concentration
that
carabid
beetles
could
be
exposed
to
is
much
less
than
10X
the
concentration
in
maize
leaves.
BPPD
agrees
with
the
rationale
the
registrant
provided.

Conclusion:
This
study
is
considered
acceptable.
This
study
shows
that
at
a
concentration
of
12
µ
g/
g
fly
pupae,
no
significant
adverse
effects
were
observed
with
regards
to
carabid
beetle
mortality
or
adult
weight.
There
was
no
significant
difference
in
the
percent
of
pre­
imaginal
mortality
or
mean
weight
of
emerged
adults
between
the
MCRY3A­
0102
treatment
and
the
negative
control
group.
The
positive
treatment
resulted
in
100%
pre­
imaginal
mortality
Rove
Beetle
Testing
(
Staphylindae:
Aleochara
bilineata)

MRID
46265607
A
Tier
I
non­
target
insect
study
using
rove
beetles
was
submitted
based
on
OPPTS
Guideline
885.4340.
The
76­
day
study
looked
at
effects
of
mCry3A
protein
on
adult
beetle
mortality
and
their
larval
offspring.
The
study
was
conducted
between
July
2,
2003
and
October
1,
2003
and
was
OECD
and
UK
GLP
compliant.
The
rove
beetles
were
obtained
in
advance
of
the
bioassay
as
parasitized
pupae
of
the
onion
fly,
Delia
antique.
After
emergence,
adult
beetles
were
placed
in
a
dark
cold
room
(
0­
8
°
C)
for
six
days
to
delay
their
physiological
development.
Three
days
before
the
study
commenced,
beetles
were
removed
from
the
cold
room
and
maintained
at
23­
25
°
C
and
64­
79%
relative
humidity.
For
the
remainder
of
the
study
beetles
were
maintained
at
6
this
temperature
and
humidity
(
with
minor
fluctuations)
with
a
16
hour
photoperiod.
Beetles
were
physiologically
4­
days
old
at
the
start
of
the
study.
For
the
first
7
days
of
the
study,
beetles
(
10
female/
10
male)
were
kept
in
round,
plastic
pots
(
9
cm
in
diameter
and
5
cm
deep).
From
day
7
to
day
35,
test
arenas
comprised
polystyrene
boxes
(
17
cm
x
11.1
cm
x
6
cm
deep).
The
boxes
were
filled
with
at
least
4
cm
of
quartz
sand
that
was
maintained
at
a
constant
wet
weight.
Beetles
were
provided
one
of
three
treatments
of
cooked
minced
beef.
The
mCry3A
test
substance
was
derived
from
E.
coli
producing
mCry3A
(
MCRY3A­
0102).
The
MCRY3A­
0102
treatment
beetles
were
given
approximately
0.2
g
of
treated
minced
beef.
The
beef
for
the
MCRY3A­
0102
test
treatment
was
mixed
with
a
solution
containing
MCRY3A­
0102
that
resulted
in
a
concentration
of
50
µ
g
mCry3A
protein/
g
meat.
The
MCRY3A­
0102
beef
was
frozen
to
maintain
freshness
and
defrosted
as
needed.
Negative
control
beetles
were
fed
cooked
minced
meat
that
had
been
mixed
with
deionozed
water
(
90
g
meat/
10
mL
deionized
water).
Positive
control
beetles
were
fed
cooked
minced
beef
that
was
mixed
with
teflubenzuron
at
a
rate
of
0.01
mg
a.
i.
/
g
diet.
Food
was
replaced
daily
in
each
treatment.
Each
treatment
of
20
beetles
was
replicated
four
times.
To
assess
the
fecundity
of
the
beetles,
host
fly
pupae
(
D.
antique)
were
incorporated
beneath
the
surface
of
the
sand
in
each
test
box
on
day
14,
21,
and
28.
On
each
occasion,
approximately
500
fly
pupae
were
placed
in
each
box.
The
boxes
were
uncovered
on
day
35
and
any
live
beetles
were
removed.
The
sand
was
allowed
to
dry
for
approximately
one
week,
after
which
the
fly
pupae
were
separated
from
the
sand
and
placed
in
plastic
pots
according
to
treatment.
The
base
of
each
pot
acted
as
a
sieve
through
which
emerging
beetles
could
crawl.

The
condition
of
the
beetles
was
assessed
on
days
1,
7
and
35
as
being
alive,
moribund,
dead,
or
missing.
For
the
statistical
analyses,
any
moribund
insects
were
grouped
with
the
dead
and
missing
beetles
for
the
calculation
of
percent
mortality.
The
number
of
F1
beetles
that
developed
from
the
onion
fly
pupae
was
recorded
every
1­
4
days
up
to
day
76.
The
test
ended
when
the
mean
number
of
beetles
emerging
per
replicate
fell
to
below
2
per
day
in
the
control
treatment.

The
control
group
resulted
in
34%
mortality
of
the
parental
generation
on
day
35,
compared
to
31%
in
the
MCRY3A­
0102
treatment
and
35%
in
the
positive
control
treatment.
When
corrected
for
the
control
mortality,
the
percentage
mortalities
were
0%
and
2%
in
the
test
treatment
and
positive
control,
respectively.
These
results
did
not
differ
significantly
from
the
control
treatment.
The
author
expected
the
low
mortality
of
the
positive
control
group.
Teflubenzuron
is
a
chitin
synthesis
inhibitor
that
is
known
to
affect
the
fertility
of
adult
Coleoptera.
The
study
author
notes
that
extended
laboratory
tests
with
A.
bilineata
about
20­
40%
of
the
introduced
adult
beetles
are
not
recovered
alive
in
the
negative
control
treatment
after
28
days.
In
the
reproduction
assessment,
the
mean
number
of
F1
progeny
in
the
control
was
647
compared
with
663
beetles
in
the
MCRY3A­
0102
treatment.
These
results
did
not
differ
significantly.
In
the
positive
control
treatment,
only
3
beetles
emerged.

An
additional
study
(
MRID
46265608)
analyzed
the
diet
fed
to
the
rove
beetles
and
tested
MCRY3A­
0102
bioactivity
using
Western
Blot
Analysis
as
well
as
a
bioassay
using
Colorado
Potato
Beetle
(
CPB).
The
study
found
that
91.7%
of
the
nominal
50
µ
g
MCRY3A­
0102/
g
diet
was
recovered
from
the
test
diet.
In
addition,
the
Western
Blot
Analysis
found
that
the
diet
contained
bioactive
protein.
The
activity
was
confirmed
in
the
CPB
bioassay,
resulting
in
57%
7
and
77%
mortality
after
96
hours
for
the
CPB
test
diets
prepared
with
10%
and
20%
rove
beetle
test
diet.

Discussion:
The
beetles
were
dosed
daily
at
a
concentration
that
exceeds
the
concentration
expressed
in
maize
leaves.
The
approximate
concentration
of
MCRY3A­
0102
protein
in
the
beetle
diet
was
45.85
µ
g/
g
diet.
However,
this
study
does
not
draw
any
conclusions
regarding
the
fitness
of
the
test
beetle's
offspring
or
other
factors
associated
with
the
second
generation.
In
addition,
the
study
author
states
that
long­
term
bioassays
using
this
particular
species
of
rove
beetle
usually
results
in
high
numbers
of
control
beetle
mortality.
In
the
submitted
study,
34%
mortality
was
observed
in
the
negative
control.
Current
OPPTS
guidelines
state
that
control
treatment
mortality
should
not
exceed
20%.
Although
it
is
unlikely
that
the
high
mortality
of
the
control
group
impacted
the
ability
to
observe
effects
in
the
test
group,
it
is
noted
in
this
review.

The
registrant
submitted
supporting
information
regarding
EEC's
in
a
separate
document
(
MRID
46265601)
that
is
important
to
note
in
this
review.
In
the
document
the
registrant
presents
rationale
for
their
expected
maximum
EEC.
They
state
that
a
worst­
case
EEC
would
be
based
on
consumption
of
soil
invertebrates
that
have
been
exposed
to
mCry3A
via
protein
expression
in
the
maize
roots.
The
author
states
that
the
concentration
of
mCry3A
in
maize
roots
is
equivalent
to
3.7
µ
g/
g
fresh
weight.
The
author
states
that
the
insect
with
the
highest
concentration
of
Cry1Ab
is
the
spider
mite,
which
generally
contains
a
concentration
that
is
1.4
times
less
than
the
amount
expressed
in
the
plant.
Therefore,
the
registrant
concludes
that
the
actual
concentration
ingested
by
rove
beetles
would
be
much
less
than
10X
the
concentration
in
maize
leaves.
BPPD
agrees
with
the
rationale
that
the
registrant
submitted.

Conclusion:
This
study
is
classified
as
acceptable.
The
dose
of
45.85
µ
g/
g
diet
did
not
result
in
an
increase
in
beetle
mortality
or
negatively
impact
their
ability
to
produce
offspring.

Predatory
Hemiptera
(
Orius
insidiosus)

MRID
46265609
A
Tier
I
non­
target
insect
study
on
insidious
flower
bugs
(
O.
insidiosis)
was
submitted
based
on
OPPTS
Guideline
885.4340.
The
study
was
carried
out
between
July
22,
2003
and
August
13,
2003
and
was
compliant
with
OECD
and
UK
GLP
guideline
requirements.
The
aim
of
the
study
was
to
determine
under
laboratory
conditions
whether
mCry3Aprotein
fed
to
O.
insidiosis
would
result
in
adverse
effects.
Again,
the
protein
test
substance
was
purified
from
cells
of
E.
coli.
The
mCry3A
protein
was
administered
to
nymphal
O.
insidiosis
during
their
development
for
21
days.
A
diet
consisting
of
beef,
liver,
yeast,
honey,
egg,
sugar,
water,
and
Nipagin
(
preservative)
was
fed
to
the
insects.
The
diet
was
properly
heated
so
that
raw
ingredients
were
cooked.
The
MCRY3A­
0102
test
diet
was
formulated
to
contain
a
concentration
of
50
µ
g/
g
of
diet.
The
control
diet
was
formulated
by
mixing
20
mL
deionized
water
with
180
g
diet.
The
positive
control
contained
teflubenzuron
at
a
concentration
of
0.01
mg
a.
i.
/
g
diet.
Approximately
0.2
g
of
diet
was
placed
into
a
small
plastic
cup
and
a
short
length
of
parafilm
was
stretched
over
the
8
cup.
The
insects
were
capable
of
piercing
the
parafilm
to
reach
the
diet.
Diet
cups
were
replaced
daily
and
additional
diet
cups
were
frozen
until
needed.
Insects
were
contained
in
clear,
plastic
pots
(
35
mm
deep
x
35
mm
internal
diameter
at
base
x
30
mm
internal
diameter
at
top).
The
sides
of
the
containers
were
treated
with
a
1.5
cm
deep
strip
of
Fluon
®
suspension
around
the
top
sidewalls
that
created
a
frictionless
surface
to
deter
insects
from
crawling
up
the
sides.
The
study
was
conducted
in
a
controlled
environment
room
maintained
at
25­
26
°
C
and
61­
87%
relative
humidity
with
16
hours
of
light.
The
condition
of
the
nymphs
was
assessed
each
day
until
they
became
adults,
or
until
21
days
after
test
initiation.
Insects
were
assessed
as
being
alive,
moribund,
dead,
or
missing/
squashed.
It
was
assumed
that
any
bugs
noted
as
missing
that
were
not
found
in
subsequent
assessments
had
escaped
from
their
test
containers.
Such
missing
bugs,
or
any
that
were
accidentally
squashed
or
injured
during
the
study,
were
excluded
from
any
data
analysis.
The
percentage
of
pre­
imaginal
mortality
of
insects
was
calculated
for
each
treatment
before
and
after
correction
for
control
mortality.

Mortality
in
the
control
treatment
was
23%
while
the
mortality
of
the
MCRY3A­
0102
treatment
was
18%.
The
positive
control
treatment
resulted
in
98%
mortality.
The
study
author
did
not
calculate
the
average
pre­
imaginal
development
time.
The
average
development
time
in
the
MCRY3A­
0102
and
negative
control
treatment
was
calculated
as
part
of
this
review
and
was
found
to
not
be
significantly
different
(
P
>
0.5).

A
non­
guideline
study
(
MRID
46265610)
analyzed
the
diet
that
was
used
in
the
Orius
study.
The
study
used
ELISA
following
extraction
of
MCRY3A­
0102
from
the
test
diet
and
recovered
95.6%
of
the
nominal
concentration
put
into
the
diet
(
50
µ
g
/
g
diet).
Therefore,
the
test
diet
contained
a
concentration
of
47.8
µ
g
(
MCRY3A­
0102)/
g
diet.
Western
blot
analysis
indicated
that
the
mCry3A
protein
did
not
degrade
when
the
Orius
test
diet
was
prepared.
In
addition,
a
bioassay
was
conducted
that
exposed
Colorado
potato
beetles
(
CPB)
to
a
diet
containing
mCry3A
and
the
Orius
test
diet.
The
bioassay
resulted
in
83%
and
90%
mortality
of
CPB
larvae
following
exposure
to
diet
containing
10%
and
20%
Orius
diet,
respectively.

Discussion:
EPA
guidelines
state
that
control
treatment
mortality
should
not
exceed
20%.
In
this
study,
control
treatment
mortality
was
23%,
and
treatment
mortality
was
18%.
Although
it
is
unlikely
that
the
high
mortality
of
the
control
group
impacted
the
ability
to
observe
effects
in
the
test
group,
it
should
be
noted.

The
registrant
submitted
supporting
information
regarding
EEC's
in
a
separate
document
(
MRID
46265601)
that
is
important
to
note.
In
the
document
the
registrant
presents
rationale
for
their
expected
maximum
EEC.
They
state
that
a
worst­
case
EEC
would
be
based
on
the
consumption
of
only
spider
mites,
which
have
been
shown
to
accumulate
the
most
Cry1Ab
protein
compared
to
other
tested
insects
(
1.4X
less
than
the
concentration
in
plants).
Therefore,
the
registrant
concludes
that
the
actual
EEC
that
Orius
would
be
exposed
to
is
much
less
than
the
concentration
expressed
in
maize
plants.
BPPD
agrees
with
this
rationale.
9
Conclusion:
This
study
is
acceptable.
This
study
shows
that
when
Orius
are
fed
a
test
diet
containing
47.8
µ
g
MCRY3A­
0102/
g
diet
during
their
development
from
nymphs
to
adults
no
negative
adverse
effects
are
observed
Honey
Bee
Testing
(
Hymenoptera:
Apis
mellifera)

MRID
46155618
A
semi­
field
study
was
conducted
to
fulfill
OPPTS
guideline
885.4380.
The
aim
of
the
study
was
to
determine
whether
mCry3A
protein
has
harmful
effects
on
brood
development
and
adult
worker
bee
survival
in
hives
of
the
honeybee,
Apis
mellifera
(
Hymenoptera:
Apidae).
The
study
was
carried
out
between
July
15
and
August
11,
2003
in
a
private
garden
in
the
United
Kingdom.
The
honeybees
were
exposed
to
microbially
produced
mCry3A
protein
via
in­
hive
feeders.
The
MCRY3A­
0102
protein
test
substance
was
purified
from
cells
of
recombinant
E.
coli.
The
feeders
were
filled
daily
for
five
days
with
one
of
three
sucrose
solutions.
The
MCRY3A­
0102
protein
was
suspended
in
200
mL
sucrose
solution
(
at
a
concentration
of
50
µ
g
MCRY3A­
0102
protein/
g
sucrose
solution).
This
concentration
was
approximately
equal
to
10X
the
concentration
of
mCry3A
protein
expressed
in
maize
leaves.
A
negative
control
treatment
contained
only
sucrose,
and
a
positive
control
treatment
contained
a
solution
of
sucrose
and
an
insect
growth
regulator
(
Dimilin
Flo).
The
sucrose
solution
was
completely
consumed
each
day.
Each
treatment
was
replicated
4
times.
In
each
hive,
100
eggs
and
100
1­
3
day
old
larvae
were
located
and
recorded
by
marking
a
transparent
acetate
sheet
over
the
hive
frames
and
tracing
the
cells.
Therefore,
each
replicate
followed
the
development
of
200
bees.
To
assess
brood
success,
the
relative
proportions
of
brood
and
food
(
pollen
and
nectar)
on
the
frames
within
each
hive
were
recorded
before
the
treatments
were
administered.
At
the
end
of
the
study,
this
assessment
was
repeated.
To
assess
adult
bee
mortality,
dead
bee
traps
were
placed
at
the
opening
of
each
hive.
After
the
five
day
feeding
period,
bee
development
and
brood
success
was
monitored
for
21
days.
Egg
cells
were
assessed
at
days
4,
6,
12,
19,
and
21.
Larval
cells
were
assessed
at
days
4,
6,
12,
and
19.
The
dead
bee
traps
were
assessed
at
the
start
of
the
study
and
every
1
to
3
days
thereafter.
The
hives
were
assessed
for
general
condition
on
days
0
and
22.
This
study
was
UK
and
OECD
GLP
compliant.

Egg
cell
mortality
in
the
negative
control,
MCRY3A­
0102
treatment,
and
positive
control
was
28.5%,
27.3%,
and
100%,
respectively.
Larval
cell
mortality
was
6.0%,
6.8%,
and
100%,
respectively.
Mortality
was
not
significantly
different
between
the
negative
control
and
MCRY3A­
0102
treatment.
In
addition,
there
was
no
significant
difference
in
pre­
and
post­
test
hive
condition
between
negative
control
and
MCRY3A­
0102
treatments.
The
number
of
dead
adult
bees
found
during
the
test
was
too
low
for
statistical
comparison
among
treatments
(
36
in
negative
control,
27
in
mCry3A
treatment,
and
29
in
positive
control).

Discussion:
Certain
aspects
of
the
study
were
inconsistent
with
OPPTS
guideline
885.4380.
However,
these
inconsistencies
likely
do
not
affect
the
study's
results
but
are
noted
in
this
review.
OPPTS
10
guideline
885.4380
states
that
the
control
and
treated
bees
should
be
observed
for
at
least
30
days
after
dosing.
In
the
submitted
study,
bees
were
only
observed
for
21
days.
However,
the
21
days
was
sufficient
to
observe
bee
development.
In
addition,
the
bees
were
only
dosed
for
five
days.
The
study
author
states
that
the
study
site
did
not
have
any
areas
of
flowering
crops
within
foraging
distance;
however,
no
distance
was
given.
Honey
bees
are
known
to
travel
far
distances
in
search
of
food.
Although
it
is
unlikely
that
this
absence
impacted
the
results,
a
numerical
distance
should
have
been
included.
Along
those
lines,
the
distance
between
hives
was
not
stated
in
the
study
report
or
indicated
on
the
submitted
map
of
the
test
area.
Further,
the
negative
control
group
resulted
in
more
than
20%
mortality
(
28.5%).
OPPTS
guidelines
state
that
for
a
study
to
be
valid,
control
mortality
should
not
exceed
20%.
The
negative
control
mortality
was
similar
to
the
treatment
mortality.
The
study
did
not
analyze
the
diet
that
was
used,
but
it
was
observed
that
the
sucrose
solutions
were
completed
consumed
each
day.

Conclusion:
This
study
is
classified
as
acceptable.
The
results
of
the
study
showed
that
there
were
no
adverse
effects
on
honeybee
development
as
a
result
of
exposure
to
MCRY3A­
0102
protein.

Characterization
of
Fish
Feed
Test
Substance
MRID
46265602
This
study
was
submitted
to
supplement
the
28­
day
fish
toxicity
study
(
MRID
46155617)
that
was
submitted
to
support
the
experimental
use
permit
for
MIR604.
The
fish
toxicity
study
was
reviewed
in
a
memo
titled
Ecological
risk
assessment
for
67979­
EUP­
U,
from
Syngenta,
Inc.
for
corn
containing
Bacillus
thuringiensis
Cry3A
protein
for
control
of
corn
root
worm
(
Diabrotica
spp.),
dated
February
15,
2005
(
Tomimatsu,
2005).

In
the
current
study,
the
corn
and
feed
pellets
that
were
fed
to
the
fish
were
analyzed
to
determine
the
actual
amount
of
mCry3A
present.
This
study
was
US
EPA
GLP
compliant.
The
grain
that
was
used
to
produce
the
fish
feed
was
derived
from
two
field
maize
hybrids.
One
hybrid
was
transgenic,
expressing
the
mCry3A
protein.
The
other
hybrid
was
non­
transgenic,
and
was
a
similar
hybrid
to
the
transgenic
maize.
Both
hybrids
were
grown
concurrently
in
Bloomington,
IL
according
to
local
agronomic
practices.
After
harvest,
the
grain
was
shipped
to
Syngenta
and
was
ground
to
a
powder
suitable
for
making
fish
feed
pellets.
A
sample
of
transgenic
and
non­
transgenic
powder
was
saved
for
mCry3A
quantification.
The
powder
was
sent
to
Zeigler
Brothers
Inc.
where
it
was
formulated
into
feed
pellets.
The
feed
pellets
were
formulated
to
contain
50%
maize
powder.
The
remaining
50%
was
formulated
with
several
fish
feed
items,
including
soybean
meal,
fish
meal,
vitamins
and
minerals.
Samples
of
transgenic
and
non­
transgenic
feed
pellets
were
saved
for
further
analysis.
The
mCry3A
protein
was
extracted
from
the
feed
pellets
and
powder
using
standard
operating
procedures
and
quantified
using
ELISA.
For
the
fish
feed,
three
separate,
random
samples
of
the
retained
pellets
were
analyzed,
to
check
for
homogeneity.

No
mCry3A
protein
was
detected
in
the
control
maize
powder,
or
in
the
control
non­
transgenic
fish
feed.
The
measured
mCry3A
concentration
in
the
transgenic
maize
powder
was
0.3
µ
g
11
mCry3A/
g
fresh
weight.
The
mean
concentration
in
the
fish
feed
was
0.09
µ
g
mCry3A/
g
fresh
weight.
The
mCry3A
was
uniformly
distributed
in
the
feed
pellets
at
a
concentration
of
0.09
±
005
µ
g
mCry3A/
g
fresh
weight.

Discussion/
Conclusion:
This
study
is
acceptable.
The
submitted
study
adequately
characterizes
the
amount
of
mCry3A
present
in
the
fish
feed
used
in
the
28­
day
fish
toxicity
study
Earthworm
Toxicity
(
Eisenia
fetida)

MRID
46265611
A
laboratory
study
was
conducted
to
determine
the
effect
on
earthworms
when
exposed
to
MCRY3A­
0102
in
the
soil
for
14
days.
The
study
took
place
between
October
14,
2003
and
November
5,
2003
and
was
OECD
and
EPA
GLP
compliant.
Earthworms
may
be
exposed
to
mCry3A
protein
when
they
forage
around
the
roots
of
the
transgenic
maize.
To
expose
the
earthworms
to
mCry3A,
an
artificial
soil
substrate
was
used
in
which
a
concentration
of
MCRY3A­
0102
was
added.
The
MCRY3A­
0102
protein
was
purified
from
cells
of
recombinant
E.
coli.
The
artificial
soil
was
composed
of
10%
peat,
20%
clay,
and
70%
sand
with
1.5
g
CaCo3
added
to
adjust
the
pH
to
6.0
±
0.5.
During
the
test
the
soil
was
maintained
at
about
50%
water
holding
capacity.
To
formulate
the
test
soil,
a
solution
of
MCRY3A­
0102
in
deionized
water
was
added
to
the
soil
at
a
concentration
of
370
µ
g/
g
dry
soil.
This
is
equivalent
to
334
µ
g
MCRY3A­
0102
protein/
g
dry
weight
soil.
This
concentration
is
approximately
10X
the
maximum
concentration
of
mCry3A
expressed
in
maize
roots.
A
positive
control
treatment
contained
2­
choroacetamide
at
several
concentrations
(
10,
20,
30,
40,
or
50
mg/
kg
of
dry
soil)
to
determine
a
dose
response.
The
negative
control
treatment
contained
deionized
water.
The
test
chambers
for
this
study
were
1­
liter
glass
jars
with
hinged
glass
lids.
Each
jar
contained
10
adult
earthworms.
The
test
material
and
negative
control
treatments
were
replicated
four
times,
while
the
positive
control
treatments
were
replicated
three
times.
The
test
was
conducted
in
a
controlled
environment
room
maintained
at
18­
20
º
C
and
57­
89%
relative
humidity
with
constant
overhead
light.
Before
being
placed
in
the
glass
containers,
worms
were
placed
in
a
large
container
of
artificial
soil
to
acclimate
to
the
conditions
of
the
study.
Only
adult
worms,
with
clitellum,
and
weighing
between
300
and
600
mg
were
selected
for
the
study.
On
day
0
of
the
study
worms
were
removed
from
the
artificial
soil,
washed
and
dried,
and
weighed
on
an
analytical
balance.
Within
30
minutes
of
treatment
incorporation
into
the
soil,
worms
were
placed
on
the
surface
of
the
soil
in
the
test
containers
and
the
lids
were
closed.
Worm
mortality
was
assessed
on
days
7
and
14.
Worms
were
recorded
as
being
alive,
dead,
or
missing.
It
was
assumed
that
any
worms
that
were
not
found
were
dead.
Worms
classified
as
`
dead'
did
not
respond
to
mechanical
stimulus.
On
day
14,
living
worms
were
washed
and
dried
before
being
re­
weighed.

Earthworm
mortality
and
mean
weight
loss
were
comparable
between
the
MCRY3A­
0102
and
negative
control
treatments.
The
positive
control
resulted
in
100%
mortality
on
day
7
for
all
groups
receiving
greater
than
or
equal
to
30
mg
2­
chloroacetamide/
kg
soil.
The
LC50
for
the
toxic
reference
treatment
was
calculated
to
be
18
g/
kg
dry
weight
of
artificial
soil.
In
a
12
supporting
study
(
MRID
46265612),
the
artificial
soil
was
analyzed
for
presence
and
activity
of
MCRY3A­
0102.
Soil
samples
were
collected
on
days
0,
3,
7,
and
14.
Extraction
of
MCRY3A­
0102
from
treated
soil
samples
were18.1%,
14.8%,
6.3%,
and
7.6%
of
the
nominal
concentration,
respectively.
The
study
author
comments
that
the
declining
recovery
of
the
protein
indicates
degradation
of
the
protein.
In
addition,
the
author
comments
that
the
actual
concentration
of
MCRY3A­
0102
in
the
test
soil
is
likely
higher
than
detected,
and
the
difficulty
in
extracting
the
protein
from
the
soil
is
caused
by
the
physico­
chemical
properties
of
the
soil
matrix.
In
the
study,
Western
blot
analysis
detected
a
band
of
immunoreactive
material
corresponding
to
the
predicted
molecular
weight
of
MCRY3A­
0102.
To
further
confirm
activity
of
the
protein,
a
bioassay
was
conducted
using
Colorado
potato
beetle
(
CPB)
larvae.
The
samples
of
mCry3A
treated
soil
showed
bioactivity
at
all
sampling
times,
resulting
in
CPB
larvae
mortality
of
60%
and
83%
after
120
hours
for
the
CPB
diets
prepared
with
5%
and
10%
w/
w
treated
soil.

Discussion:
The
mortality
of
the
positive
control
group
indicates
that
the
earthworms
were
ingesting
soil
during
the
study
and
the
study
was
an
adequate
model
to
assess
earthworm
toxicity.
It
should
be
noted
that
the
actual
concentration
of
MCRY3A­
0102
in
the
artificial
soil
was
less
than
the
anticipated
concentration.
However,
throughout
the
study
the
extracted
concentration
of
MCRY3A­
0102
was
greater
than
(
days
0­
3)
or
near
the
concentration
(
days
7­
14)
expressed
in
maize
roots.
The
study
author
comments
that
the
investigators
were
unable
to
effectively
extract
and
measure
the
MCRY3A­
0102
from
the
soil
due
to
physico­
chemical
soil
properties.
The
study
author
does
not
elaborate
on
this
conclusion,
or
describe
properties
of
the
artificial
soil
that
would
lead
to
a
reduction
in
extraction.
In
addition,
EPA
guideline
850.6200
recommends
that
earthworm
toxicity
studies
are
conducted
over
28
days.

In
addition,
the
registrant
provided
supporting
information
regarding
EEC's
in
a
separate
document
(
MRID
46265601)
that
is
important
to
note
in
this
review.
In
the
document
the
registrant
presents
rationale
for
their
expected
maximum
EEC.
The
most
likely
route
of
exposure
for
earthworms
would
be
through
ingestion
of
senescent
plant
materials
that
are
incorporated
into
the
soil.
The
registrant
states
that
the
average
concentration
of
mCry3A
in
senescent
roots
of
MIR604
plants
is
5.5
µ
g/
g
fresh
weight.
Therefore,
the
registrant
states
that
the
maximum
EEC
that
earthworms
could
be
exposed
to
is
much
less
than
the
concentration
of
mCry3A
in
the
artificial
soil.
BPPD
agrees
with
this
rationale.

Conclusion:
This
study
is
classified
as
acceptable.
The
results
show
that
no
hazard
to
earthworms
is
expected
at
mCry3A
concentrations
that
could
be
encountered
in
the
field.

Broiler
Chicken
Toxicity
Study
(
Gallus
domesticus)

MRID
46265615
A
long­
term
feeding
study
using
broiler
chickens
was
conducted
to
determine
the
toxicity
of
MIR604
maize
grain
to
chickens.
The
submitted
study
was
not
USEPA
GLP
compliant,
but
was
13
conducted
according
to
accepted
scientific
methods.
Chickens
were
fed
formulated
diets
containing
either
transgenic
MIR604
corn
with
the
mCry3A
protein,
a
MIR604
isoline
(
nontransgenic
or
a
non­
transgenic
commercial
corn
grown
in
North
Carolina
in
2003
(
NC
2003).
The
concentration
of
mCry3A
in
the
transgenic
diets
was
reported
to
be
0.04
µ
g/
g
dry
weight,
0.06
µ
g/
g
dry
weight,
and
0.08
µ
g/
g
dry
weight
of
the
starter,
grower,
and
finisher
diets,
respectively.
The
test
diets
were
formulated
to
be
similar
in
nutrient
composition,
so
that
available
nutrition
did
not
influence
the
test
results.
Following
the
standardization
of
the
different
corn
types
to
the
same
nutrient
content,
three
standard
base
diets
(
starter,
grower,
and
finisher)
were
formulated
to
contain
57.5%,
63.0%,
and
67.5%
of
the
standardized
corn.
The
chickens
were
fed
for
49
days.
Broiler
chicks
were
hatched
from
eggs
produced
and
incubated
at
the
North
Carolina
State
University.
The
parent
stock
was
a
commercial
strain
of
Ross
344
males
and
feather­
sexable
Ross
308
females.
Chicks
were
maintained
on
coccidostat
but
were
not
given
any
vaccinations.
Chicks
were
sexed
and
placed
into
pens
that
measured
4
x
12
x
8
feet
high.
Each
pen
contained
25
birds
of
the
same
sex.
Each
treatment
group
contained
12
cages
of
birds,
6
cages
of
males
and
6
cages
of
females.
The
pens
were
housed
in
a
curtain­
sided
poultry
house.
The
chickens
were
provided
with
continuous
access
to
feed
and
water.
The
birds
were
given
access
to
2.5
lbs
of
starter
diet
per
bird
at
the
start
of
the
study.
The
grower
diet
was
provided
at
day
16,
and
the
finisher
diet
was
introduced
at
day
31.
Access
to
feed
was
discontinued
on
the
50th
day
of
the
experiment.
Total
pen
weights
were
collected
at
hatch
(
1
day),
16
days,
31
days,
and
49
days
of
age.
At
the
later
three
times,
feed
consumption
per
pen
was
determined
in
order
to
calculate
feed
conversion
ratios
and
adjusted
feed
conversion
ratios.
At
51
days,
two
birds
from
each
pen
were
sacrificed
and
processed
in
order
to
determine
carcass
meat
yield.
The
remaining
birds
that
had
been
fed
the
transgenic
corn
containing
feed
were
killed
and
buried
in
a
secure
landfill
along
with
the
remaining
unused
feed
and
corn.
Body
weight,
feed
conversion,
and
survival
data
were
analyzed
in
order
to
determine
statistical
differences
between
corn
diets
and
sex.
Statistical
analyses
were
performed
using
the
General
Linear
Model
(
GLM)
procedure
of
SAS
with
sex
and
corn
source
as
independent
variables
in
a
two­
way
analysis
of
variance
within
a
randomized
complete
block
design.
Carcass
data
on
a
gross
and
adjusted
to
body
weight
basis
were
analyzed
for
effects
due
to
corn
source
within
sex
using
a
one­
way
analysis
of
variance
as
effects
due
to
sex
are
well
known.

The
report
of
the
quantification
and
characterization
of
mCry3A
in
the
test
diets
was
not
submitted
to
EPA.
The
study
author
does
report
that
the
amount
of
mCry3A
in
the
MIR604
transgenic
corn
grain
was
determined
through
ELISA
to
be
0.23
±
0.035
µ
g/
g
dry
weight.
In
addition,
the
amount
of
mCry3A
protein
in
the
three
diets
as
determined
by
ELISA
were
as
follows:
a)
starter:
0.04
±
0.005
µ
g/
g
dry
weight;
b)
grower:
0.06
±
0.005
µ
g/
g
dry
weight;
c)
finisher:
0.08
±
0.013
µ
g/
g
dry
weight.

The
results
showed
that
at
16
days
of
age
sex
effects
became
evident
with
the
males
weighing
significantly
more
than
the
females.
These
effects
were
also
observed
at
35
and
49
days
of
age.
There
was
statistically
significant
body
weight
difference
due
to
corn
source
at
31
and
49
days
of
age
in
the
NC
2003
treatment,
where
lower
body
weights
were
observed
at
both
dates.
In
addition,
corn
source
had
a
significant
effect
on
feed
conversion
during
the
grower
period
(
16­
31
days)
in
the
NC
2003
group
that
resulted
in
cumulative
effects
at
31
and
49
days
of
age.
There
were
significant
differences
in
survival
due
to
sex,
with
faster­
growing
males
exhibiting
higher
14
mortality
during
the
finisher
period
(
31­
49
days)
and
cumulatively
(
0­
49),
as
is
expected
under
normal
conditions.
Carcass
analysis
showed
that
among
the
female
chickens
of
the
MIR604
transgenic
treatment
produced
greater
relative
thigh
meat
than
the
MIR604
non­
transgenic
or
NC
2003.
Among
the
males,
the
NC
2003
group
had
larger
Pectoralis
minor
than
either
MIR604
group.
Overall
carcass
yield
was
not
significantly
different
among
treatment
groups.

Discussion:
However,
the
measured
concentrations
of
mCry3A
in
the
formulated
diets
provided
in
the
study
report
could
not
be
verified.
The
study
report
references
a
Syngenta
report
that
quantifies
and
characterizes
mCry3A
in
the
chicken
diet
as
being
in
progress,
but
BPPD
has
not
received
such
a
report.

Conclusion:
This
study
is
acceptable.
The
study
shows
that
feed
made
from
MIR604
transgenic
corn
does
not
adversely
affect
broiler
chickens.
15
References
Milofsky,
T.,
"
Environmental
Risk
Assessment
for
modified
Cry3A
(
mCry3A)
Bacillus
thuringiensis
protein
and
the
genetic
material
necessary
for
its
production
in
Event
MIR604
corn",
February
2006.

Tomimatsu,
G.
S.,
"
Ecological
risk
assessment
for
67979­
EUP­
U,
from
Syngenta,
Inc.
for
corn
containing
Bacillus
thuringiensis
Cry3A
protein
for
control
of
corn
root
worm
(
Diabrotica
spp.)",
February
15,
2005.
16
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nontarget
Insect
Testing,
Tier
I
(
885.4340)

MRID
NO:
46265603
DP
BARCODE:
DP302786
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCry3A­
0102
STUDY
NO:
Mambo­
Tox
Study/
Report
No.
SYN­
03­
50
Syngenta
Project
No.
2033032
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Mambo­
Tox
Ltd.,
Southampton,
UK
TITLE
OF
REPORT:
A
Laboratory
Test
of
the
Toxicity
of
Modified
Cry3A
Protein
(
MCRY3A­
0102)
to
Larvae
and
Adults
of
the
Ladybird
Beetle,
Coccinella
septempunctata
(
Coleoptera:
Coccinellidae)

AUTHOR:
Waterman,
L.

STUDY
COMPLETED:
April
6,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
compliant
with
OECD
and
UK
GLP
regulations.
17
STUDY
SUMMARY:
In
a
laboratory
study,
4­
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
5.5
mg
of
MCRY3A­
0102
(
equivalent
to
50
mg
of
mCry3A
protein).
This
amount
is
approximately
equivalent
to
10X
the
amount
of
mCry3A
expressed
in
maize
leaves.
The
test
item
was
a
sample
of
mCry3A
protein
purified
from
cells
of
recombinant
E.
coli
(
MCRY3A­
0102).
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,
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
0102
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
0102
treatments,
but
the
mean
number
of
days
to
adult
emergence
was
significantly
lower
in
the
MCRY3A­
0102
group.

CLASSIFICATION:
Acceptable
Test
Material
The
test
substance,
MCRY3A­
0102,
active
ingredient
mCry3A
protein,
Batch
Number
MCRY3A­
0102,
was
supplied
by
the
sponsor
with
a
reported
purity
of
90.3%
w/
w
and
an
expiration
date
of
December
3,
2003.
The
test
substance
was
purified
from
cells
of
recombinant
E.
coli.
The
test
substance
was
stored
at
­
30E
to
­
20EC
prior
to
use.
The
study
was
conducted
prior
to
the
test
substance
expiration
date.
The
MCRY3A­
0102
concentration
in
the
treated
aphids
used
in
this
test
was
determined
in
a
separate
study
(
MRID
46265604)
to
be
9
Fg
mCry3A/
g
of
aphid
(
9
ng
mCry3A/
aphid).

Test
Methods
Four­
day­
old
ladybird
beetle
larvae
(
Coccinella
septempunctata,
from
an
in­
house
colony),
nominally
in
the
second
instar,
were
placed
in
ventilated,
5­
cm
diameter
plastic
Petri
dishes
(
1/
dish)
at
the
start
of
the
test.
The
larvae
were
fed
daily
with
live
pea
aphids
(
Acyrthosiphon
pisum)
that
had
been
immersed
for
30
seconds
in
a
solution
containing
5.5
mg
of
the
test
material
(~
50
mg
MCRY3A­
0102)
in100
mL
of
0.004%
v/
v
Agral
90
(
a
non­
ionic
surfactant)
in
deionized
water
and
allowed
to
dry
for
30
minutes.
This
amount
(
50
Fg
mCry3A­
0102)
is
approximately
equivalent
to
10X
the
concentration
of
mCry3A
expressed
in
maize
leaves.
Aphids
fed
to
the
negative
control
group
were
immersed
in
the
Agral
90
18
solution
only,
those
fed
to
the
positive
control
group
were
immersed
in
a
solution
of
0.5
mL
of
Nemolt
(
teflubenzuron)/
L
of
0.004%
v/
v
Agral
90.
Each
treatment
was
replicated
40
times.
Each
replication
consisted
of
one
larvae/
adult.
Freshly­
treated
live
aphids
were
provided
daily
until
the
larvae
pupated.
The
number
of
aphids
provided
increased
with
age
of
the
larvae
(
Table
1).
No
other
food
or
water
source
was
provided.
The
test
was
conducted
in
a
room
at
21­
27EC,
35%­
92%
relative
humidity,
and
16
hrs
light/
8
hrs
darkness.

TABLE
1.
Number
of
treated
aphids
supplied
to
ladybeetle
larvae
Age
of
larvae
Approximate
number
(
and
instar)
of
aphids
provided
daily
4
days
20
(
I
and
II)

5
and
6
days
30
(
III
and
IV)

7
and
8
days
40
(
IV
and
adult)

Data
from
p.
14,
MRID
46265603
After
adult
beetles
emerged
from
the
pupae,
they
were
moved
into
clean
dishes
(
1
male
and
1
female/
dish,
where
numbers
allowed)
and
fed
approximately
50
treated
pea
aphids
3
times/
week
for
14
days.
The
adults
were
assessed
for
condition
at
each
feeding.

Results
Summary
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
(
Table
3).
The
study
authors
speculated
that
the
faster
development
might
have
been
related
to
the
additional
dietary
protein
provided
by
the
test
substance.

TABLE
2.
Mortality
Results
Pre­
Imaginal
Mortality
Adult
Mortality
Treatment
Concentration
%
Mortality
a
Corrected
%
Mortality
%
Mortality
Corrected
%
Mortality
Control
Agral
90
0
­
7.5
­
MCRY3A­
0102
50
µ
g
mCry3A/
mL
Agral
solution
0
0
15.0
8.1
Nemolt
0.5
mL/
L
Agral
solution
100***
100
­
­

a)
Results
for
individual
treatments
were
compared
to
the
control
using
Chi­
Square
test
and
means
marked
with
asterisks
differed
significantly
from
the
control
(***
P
<
0.001)
19
TABLE
3.
Developmental
time
(
days)
of
4­
day
old
C.
septempunctata
larvae
Treatment
Larvae
to
pupae
Mean
±
std
deviation
Larvae
to
adult
Mean
±
std
deviation
Negative
control
5.48
±
0.50
9.80
±
0.68
MCRY3A­
0102
5.33
±
0.47
9.48*
±
0.59
Mean
development
time
was
compared
between
the
test
item
and
control
treatment
using
t­
tests
for
unmatched
pairs
*
Significantly
different
from
control
P<
0.05
Study
Author's
Conclusions
The
study
author
concluded
that
prolonged
dietary
exposure
to
MCRY3A­
0102
via
application
to
aphids
at
a
nominal
rate
equivalent
to
50
Fg
mCry3A/
mL
of
Agral
solution
was
not
harmful
to
C.
septempunctata
larvae
or
adults
20
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nonguideline
MRID
NO:
46265604
DP
BARCODE:
DP303605
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCRY3A­
0102
STUDY
NO:
SSB­
004­
04
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Syngenta
Biotechnology,
Inc.,
Research
Triangle
Park,
NC
TITLE
OF
REPORT:
Analysis
of
Test
Diet
Used
to
Expose
Coccinella
septempunctata
to
Modified
Cry3A
Protein:
Supplement
to
Report
Titled
"
A
Laboratory
Test
of
The
Toxicity
of
Modified
Cry3A
Protein
(
MCRY3A­
0102)
to
Larvae
and
Adults
of
the
Ladybird
Beetle
Coccinella
septempunctata
(
Coleoptera:
Coccinellidae)";
Mambo­
Tox
Study/
Report
No.
SYN­
03­
50
AUTHOR:
Graser,
G.

STUDY
COMPLETED:
April
26,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
EPA
GLP
compliant.
21
STUDY
SUMMARY:
Samples
of
pea
aphids
(
Acyrthosiphon
pisum)
that
had
been
immersed
in
a
wetting
agent
solution
containing
MCRY3A­
0102
at
a
concentration
nominally
equivalent
to
50
Fg
MCRY3A­
0102
/
mL
and
provided
as
diet
in
a
ladybird
beetle
(
Coccinella
septempunctata)
toxicity
study
(
MRID
46265603)
were
analyzed
for
MCRY3A­
0102
protein
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA).
The
integrity
of
the
mCry3A
in
the
treated
pupae
was
determined
using
Western
blot
analysis.
ELISA
results
showed
that
Cocccinella
larvae
and
adults
in
the
toxicity
study
were
exposed
to
9
Fg
MCRY3A­
0102
/
g
of
aphid
(
9
ng
MCRY3A­
0102
/
aphid),
and
the
Western
blot
analysis
showed
that
intact
mCry3A
was
present
in
the
aphids.

CLASSIFICATION:
Acceptable
Test
Material
The
test
substance
was
a
sample
of
pea
aphids
(
Acyrthosiphon
pisum)
that
had
been
treated
in
the
same
manner
as,
and
in
parallel
with,
pea
aphids
that
were
used
as
treated
diet
in
a
previous
non­
target
insect
toxicity
study
with
Coccinella
septempunctata
(
MRID
46265603).
The
pea
aphids
in
that
study
were
immersed
in
a
wetting
agent
solution
containing
MCRY3A­
0102
at
a
concentration
nominally
equivalent
to
50
Fg
MCRY3A­
0102
/
mL
and
provided
as
diet
to
the
coccinnelids.
A
sample
of
control
aphids
not
treated
with
MCRY3A­
0102
was
also
submitted
for
analysis.
The
samples
were
frozen
immediately
after
preparation
in
the
original
study
and
remained
frozen
until
analysis.

Test
Methods
The
test
substance
was
quantitatively
analyzed
for
MCRY3A­
0102
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA),
and
the
integrity
of
the
mCry3A
was
determined
using
Western
blot
analysis.
Standard
operating
procedures
were
referenced
in
the
report.
Two
mL
of
extraction
buffer
were
added
to
0.156
g
of
the
mCry3A­
treated
aphids
(
150
aphids)
and
to
0.176
g
of
the
untreated
aphids
(
140
aphids).
Each
sample
was
mixed,
incubated
on
ice
for
one
hour,
homogenized,
centrifuged
for
15
minutes
at
2600
x
g,
and
the
supernatants
were
collected.
The
pellets
were
extracted
a
second
time
using
the
same
procedure,
and
a
third
extraction
was
done
in
an
overnight
incubation
at
4EC.
The
supernatants
from
the
second
and
third
extractions
were
combined
and
centrifuged
again
for
10
minutes
at
13,000
x
g
prior
to
analysis.
These
extracts
were
used
for
the
ELISA,
while
only
the
first
extract
of
each
sample
was
used
for
the
Western
blot
analysis.

For
the
ELISA,
the
extracts
were
analyzed
for
MCRY3A­
0102
using
immuno­
affinity
purified
rabbit
anti­
mCry3A
polyclonal
antibodies
and
immuno­
affinity
purified
goat
anti­
Btt
(
native
Cry3A
from
Bacillus
thuringiensis
subsp.
tenebrionis)
polyclonal
antibodies.
22
For
the
Western
blot
analysis,
two
aliquots
of
the
treated
aphid
extract,
containing
6
and
12
ng
mCry3A,
respectively
(
as
determined
by
ELISA),
were
subjected
to
SDS­
PAGE
using
an
8­
16%
polyacrylamide
gradient
gel.
Two
aliquots
of
a
solution
of
MCRY3A­
0102
(
positive
control)
containing
6
and
12
ng
of
mCry3A,
respectively,
and
one
aliquot
of
the
untreatedaphid
extract
(
negative
control)
were
run
concurrently
on
the
same
gel.
After
electroblotting,
the
membrane
was
probed
with
immuno­
affinity
purified
goat
anti­
Btt
polyclonal
antibodies.
Donkey
anti­
goat
IgG
linked
to
alkaline
phosphatase,
diluted
in
blocking
buffer,
was
used
to
bind
to
the
primary
antibody
and
visualized
by
development
with
alkaline
phosphatase
substrate
solution.
The
Western
blot
was
examined
for
the
presence
of
intact
immunoreactive
mCry3A
(
ca.
67,000
molecular
weight)
and
immunoreactive
MCRY3A­
0102
polypeptides
of
lower
molecular
weight.

Results
Summary
ELISA
of
the
treated
aphid
extract
indicated
a
recovery
of
9
Fg
MCRY3A­
0102
/
g
of
aphid
(
9
ng
MCRY3A­
0102
/
aphid).
No
mCry3A
was
detected
in
the
untreated
aphid
extract.
Western
blot
analysis
of
the
treated
aphid
extract
showed
a
single
immunoreactive
band
corresponding
to
the
predicted
molecular
weight
of
MCRY3A­
0102
(
ca.
67,700
Da).
No
immunoreactive
material
was
detected
in
the
untreated
aphids.

Study
Author's
Conclusions
The
study
author
concluded
that
the
ELISA
results
showed
that
Coccinella
larvae
and
adults
in
the
toxicity
study
were
exposed
to
about
9
Fg
MCRY3A­
0102
/
g
of
aphid
diet
throughout
the
larval
and
adult
feeding
stages,
and
the
Western
blot
analysis
showed
that
intact
MCRY3A­
0102
was
present.
23
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nontarget
Insect
Testing,
Tier
I
(
885.4340)

MRID
NO:
46265605
DP
BARCODE:
DP303605
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCry3A­
0102
STUDY
NO:
Mambo­
Tox
Study/
Report
No.
SYN­
03­
54
Syngenta
Project
No.
2033034
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Mambo­
Tox
Ltd.,
Southampton,
UK
TITLE
OF
REPORT:
A
Laboratory
Toxicity
Test
of
the
Modified
Cry3A
Protein
(
MCRY3A­
0102)
to
Larvae
of
the
Ground­
Dwelling
Beetle,
Poecilus
cupreus
(
Coleoptera:
Carabidae)

AUTHOR:
Vinall,
S.

STUDY
COMPLETED:
April
7,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
compliant
with
OECD
and
UK
GLP
regulations.
24
STUDY
SUMMARY:
In
a
laboratory
study,
ground­
dwelling
beetle
(
Poecilus
cupreus)
larvae
were
fed
daily
with
blowfly
(
Calliphora
vomitoria)
pupae
that
had
been
injected
with
MCRY3A­
0102
at
a
nominal
rate
equivalent
to
50
Fg
mCry3A
protein/
g
of
fly
pupa.
This
nominal
amount
is
approximately
equivalent
to
10X
the
amount
of
mCry3A
protein
expressed
in
maize
leaves.
The
test
item,
MCRY3A­
0102,
was
purified
from
cells
of
recombinant
E.
coli.
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.
An
additional
study
analyzed
the
actual
amount
of
mCry3A
in
fly
pupa.
This
amount
was
determined
to
be
12
Fg
MCRY3A­
0102
/
g
pupae.

CLASSIFICATION:
Acceptable
Test
Material
The
test
substance,
MCRY3A­
0102,
active
ingredient
mCry3A
protein,
Batch
Number
MCRY3A­
0102,
was
supplied
by
the
sponsor
with
a
reported
purity
of
90.3%
w/
w
and
an
expiration
date
of
July
15,
2004.
The
MCRY3A­
0102
test
substance
was
purified
from
cells
of
recombinant
E.
coli.
The
test
substance
was
stored
at
­
30E
to
­
20EC
prior
to
use.
The
study
was
conducted
prior
to
the
test
substance
expiration
date.
The
mean
concentration
of
MCRY3A­
0102
in
the
fly
pupae
used
in
this
test
was
determined
in
a
separate
study
(
MRID
46465606)
to
be
12
Fg
MCRY3A­
0102
/
g
pupa,
or
approximately
0.82
Fg
mCry3A/
pupa
(
24%
of
nominal,
calculated
by
primary
reviewer).

Test
Methods
Larvae
of
the
ground­
dwelling
beetle
(
Poecilus
cupreus,
obtained
from
Bio
Test
Labor
GmbH,
Sagerheide,
Germany)
were
fed
blowfly
(
Calliphora
vomitoria)
pupae
that
had
been
injected
with
the
test
substance
at
a
rate
equivalent
to
50
Fg
mCry3A/
g
of
fly
pupa.
The
test
substance
solution
was
prepared
by
diluting
0.0399
g
of
MCRY3A­
0102
(~
0.03601
g
mCry3A)
to
10
mL
with
deionized
water
to
give
a
final
solution
concentration
of
3.601
Fg
MCRY3A­
0102
/
0.072
g
fly
pupa.
Each
pupa
was
injected
with
1
FL
of
the
test
solution.
A
negative
control
of
fly
pupae
injected
with
deionized
water
only,
and
a
positive
control
of
fly
25
pupae
injected
with
teflubenzuron
(
0.664
ng
a.
i./
g
of
fly
pupa)
were
also
used
in
the
test.
At
test
start,
the
beetle
larvae
were
24
to
48
hours
old.

The
test
containers
were
glass
tubes
filled
with
25
g
(
dry
wt)
of
LUFA
2.1
standard
sandy
soil
maintained
at
approximately
35%
of
its
water
holding
capacity.
One
fly
pupa
was
placed
in
each
container,
which
was
capped
with
a
ventilated
polyethylene
stopper.
The
remaining
treated
pupae
were
frozen
at
­
20E
to
­
30EC
until
needed
each
day.
Each
treatment
was
replicated
40
times
(
each
replication
consisting
of
one
beetle).
The
tubes
were
kept
upright
in
plastic
boxes
with
ventilated
lids
and
maintained
in
an
environmental
chamber
at
19E
to
22EC
and
64%
to
90%
relative
humidity
in
constant
darkness.
The
fly
pupae
were
replaced
daily
with
freshly­
defrosted
pupae
until
the
beetle
larvae
were
seen
to
have
prepared
and
entered
a
pupating
chamber
in
the
soil,
at
which
point
the
feeding
was
stopped.
The
condition
of
the
beetle
larvae
was
assessed
3
times/
week
for
the
first
two
weeks
of
the
study,
and
2
times/
week
thereafter
until
day
32
following
treatment.
At
day
32
and
afterward,
the
test
containers
were
checked
daily
and
the
number
of
emerging
adults
was
recorded.
The
emerged
adults
were
weighed
and
sexed,
and
overall
mortality
and
final
adult
weights
were
analyzed
statistically.

Results
Summary
There
was
no
significant
difference
in
the
percent
pre­
imaginal
mortality
or
mean
weight
of
emerged
adults
between
the
MCRY3A­
0102
and
the
negative
control
groups
(
Table
1).
Treatment
with
the
positive
control
produced
100%
pre­
imaginal
mortality.

TABLE
2.
Larval
mortality
and
adult
weight
of
P.
cupreus
fed
treated
and
untreated
fly
pupae.

Treatment
%
Mortality
%
Corrected
mortality
a
Mean
weight
of
adults
(
mg)
b
MCRY3A­
0102
10
0
82.9
Negative
control
20
­
81.5
Positive
control
100
100
­

a)
A
Chi­
square
test
was
used
to
compare
mortality
in
the
negative
control
and
mCry3A­
0102
treatment.
b)
A
t­
test
was
used
to
compare
the
mean
weight
of
the
emerging
beetles
between
the
negative
control
and
mCry3A­
0102
treatment.
No
significant
differences
occurred
(
P
>
0.05)
Data
from
p.
16,
MRID
46265605
Study
Author's
Conclusions
The
study
author
concluded
that
continuous
oral
exposure
to
MCRY3A­
0102
during
development
did
not
affect
survival
of
P.
cupreus
larvae
or
affect
the
weight
of
adult
beetles
that
emerged
after
pupation
of
the
treated
larvae.
26
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nonguideline
MRID
NO:
46265606
DP
BARCODE:
DP303605
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCRY3A­
0102
STUDY
NO:
SSB­
007­
04
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Syngenta
Biotechnology,
Inc.,
Research
Triangle
Park,
NC
TITLE
OF
REPORT:
Analysis
of
Test
Diet
Used
to
Expose
Poecilus
cupreus
to
Modified
Cry3A
Protein:
Supplement
to
Report
Titled
­
"
A
Laboratory
Toxicity
Test
of
the
Modified
Cry3A
Protein
(
MCRY3A­
0102)
to
larvae
of
the
Ground­
Dwelling
Beetle
Poecilus
cupreus
(
Coleoptera:
Carabidae)";
Mambo­
Tox
Study/
Report
No.
SYN­
03­
54
AUTHOR:
Graser,
G.

STUDY
COMPLETED:
April
26,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
EPA
GLP
compliant.
27
STUDY
SUMMARY:
Samples
of
blowfly
(
Calliphora
vomitoria)
pupae
that
had
been
injected
with
MCRY3A­
0102
at
a
concentration
nominally
equivalent
to
50
Fg
mCry3A/
g
pupae
and
used
as
diet
in
a
ground­
dwelling
beetle
(
Poecilus
cupreus)
toxicity
study
(
MRID
46265605)
were
analyzed
for
mCry3A
protein
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA).
The
integrity
of
the
MCRY3A­
0102
in
the
treated
pupae
was
determined
using
Western
blot
analysis.
ELISA
results
showed
that
Poecilus
larvae
in
the
toxicity
study
were
exposed
to
about
12
Fg
MCRY3A­
0102
/
g
pupae
throughout
their
development
and
the
Western
blot
analysis
showed
that
intact
MCRY3A­
0102
was
present
in
the
pupae.

CLASSIFICATION:
Acceptable
Test
Material
The
test
substances
were
samples
of
blowfly
(
Calliphora
vomitoria)
pupae
that
had
been
treated
in
the
same
manner
as,
and
in
parallel
with,
treated
pupae
that
were
used
as
diet
in
a
previous
non­
target
insect
toxicity
study
with
Poecilus
cupreus
(
MRID
46265605).
The
pupae
in
that
study
were
injected
with
MCRY3A­
0102
at
a
nominal
concentration
equivalent
to
50
Fg
MCRY3A­
0102
/
g
fly
pupae.
Samples
of
control
pupae
(
injected
with
deionized
water
instead
of
MCRY3A­
0102)
used
in
that
study
were
also
submitted
for
analysis.
The
samples
were
frozen
immediately
after
preparation
in
the
original
study
and
remained
frozen
until
analysis.

Test
Methods
The
test
substance
was
quantitatively
analyzed
for
MCRY3A­
0102
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA),
and
the
integrity
of
the
MCRY3A­
0102
was
determined
using
Western
blot
analysis.
Standard
operating
procedures
were
referenced
in
the
report.
The
pupae
were
ground
to
a
fine
powder
in
liquid
nitrogen
using
a
mortar
and
pestle.
Two
mL
of
extraction
buffer
were
added
to
0.208
g
of
the
MCRY3A­
0102
­
treated
pupae
(
3
pupae)
and
to
0.213
g
of
the
untreated
pupae
(
3
pupae).
Each
sample
was
mixed,
incubated
on
ice
for
one
hour,
homogenized,
centrifuged
for
15
minutes
at
2600
x
g,
and
the
supernatants
were
collected.
The
pellets
were
extracted
a
second
time
using
the
same
procedure,
and
a
third
extraction
was
done
in
an
overnight
incubation
at
4EC.
The
supernatants
from
the
second
and
third
extractions
were
combined
and
centrifuged
again
for
10
minutes
at
13,000
x
g
prior
to
analysis.
These
extracts
were
used
for
the
ELISA,
while
only
the
first
extract
of
each
sample
was
used
for
the
Western
blot
analysis.
28
For
the
ELISA,
the
extracts
were
analyzed
for
MCRY3A­
0102
using
immuno­
affinity
purified
rabbit
anti­
mCry3A
polyclonal
antibodies
and
immuno­
affinity
purified
goat
anti­
Btt
(
native
Cry3A
from
Bacillus
thuringiensis
subsp.
tenebrionis)
polyclonal
antibodies.

For
the
Western
blot
analysis,
five
aliquots
of
the
fly
pupae
extracts,
containing
0.075,
0.150,
0.375,
0.750,
and
1.500
ng
mCry3A,
respectively
(
as
determined
by
ELISA),
were
subjected
to
SDS­
PAGE
using
an
8­
16%
polyacrylamide
gradient
gel.
Three
aliquots
of
a
solution
of
MCRY3A­
0102
(
positive
control),
containing
0.2,
0.4,
and
0.8
ng
of
mCry3A,
respectively,
and
three
aliquots
of
the
extract
from
the
untreated
fly
pupae
(
negative
control)
were
run
concurrently
on
the
same
gel.
After
electroblotting,
the
membrane
was
probed
with
immunoaffinity
purified
goat
anti­
Btt
polyclonal
antibodies.
Rabbit
anti­
goat
IgG
linked
to
horseradish
peroxidase,
diluted
in
blocking
buffer,
was
used
to
bind
to
the
primary
antibody
and
visualized
by
development
with
SuperSignal
®
West
Pico
Chemiluminescent
Substrate
(
Pierce)
solution.
The
Western
blot
was
examined
for
the
presence
of
intact
immunoreactive
MCRY3A­
0102
(
ca.
67,000
molecular
weight)
and
immunoreactive
MCRY3A­
0102
polypeptides
of
lower
molecular
weight.

Results
Summary
ELISA
of
the
treated
blowfly
pupae
extracts
recovered
approximately
12
Fg
mCry3A/
g
of
pupae
(
0.82
Fg
MCRY3A­
0102
/
pupa),
for
about
24%
recovery
of
mCry3A
that
had
been
injected
into
the
pupae
(
nominally
50
Fg
MCRY3A­
0102
/
g
pupae).
No
mCry3A
was
detected
in
the
untreated
blowfly
pupae.

Western
blot
analysis
of
the
treated
blowfly
pupae
showed
a
single
immunoreactive
band
corresponding
to
the
predicted
molecular
weight
of
MCRY3A­
0102
(
ca.
67,700
Da).
No
immunoreactive
material
was
detected
in
the
untreated
blowfly
pupae.

Study
Author's
Conclusions
The
study
author
concluded
that
the
ELISA
results
showed
that
Poecilus
larvae
in
the
toxicity
study
were
exposed
to
about
12
Fg
MCRY3A­
0102
/
g
of
pupae
diet
throughout
their
development,
and
Western
blot
analysis
showed
that
intact
MCRY3A­
0102
was
present.
29
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nontarget
Insect
Testing,
Tier
I
(
885.4340)

MRID
NO:
46265607
DP
BARCODE:
DP303605
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCRY3A­
0102
STUDY
NO:
Mambo­
Tox
Study/
Report
No.
SYN­
03­
53
Syngenta
Project
No.
2033036
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Mambo­
Tox
Ltd.,
Southampton,
UK
TITLE
OF
REPORT:
A
Laboratory
Toxicity
Test
of
Modified
Cry3A
Protein
(
MCRY3A­
0102)
to
the
Rove
Beetle,
Aleochara
bilineata
(
Coleoptera:
Staphlinidae)

AUTHOR:
Vinall,
S.

STUDY
COMPLETED:
November
26,
2003
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
compliant
with
OECD
and
UK
GLP
regulations.
30
STUDY
SUMMARY:
In
a
laboratory
study,
mortality
was
determined
for
adult
rove
beetles
(
Aleochara
bilineata)
given
continuous
access
to
beef
paste
diet
containing
MCRY3A­
0102
at
a
nominal
rate
equivalent
to
50
Fg
mCry3A/
g
of
diet
for
35
days.
The
test
substance,
MCRY3A­
0102,
was
purified
from
cells
of
recombinant
E.
coli.
In
an
additional
study
(
46265608)
it
was
determined
that
91.7%
of
the
MCRY3A­
0102
protein
mixed
into
the
test
diet
was
recovered
and
it
was
found
to
be
bioactive.
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.

CLASSIFICATION:
Acceptable
Test
Material
The
test
substance,
MCRY3A­
0102,
active
ingredient
mCry3A
protein,
Batch
Number
MCRY3A­
0102,
was
supplied
by
the
sponsor
with
a
reported
purity
of
90.3%
w/
w
and
an
expiration
date
of
December
3,
2003.
The
test
substance
was
purified
from
cells
of
recombinant
E.
coli.
The
test
substance
was
stored
at
­
30E
to
­
20EC
prior
to
use.
The
study
was
conducted
prior
to
the
test
substance
expiration
date.
The
concentration,
stability,
and
activity
of
the
treated
diet
used
in
the
test
were
determined
in
a
separate
study
(
MRID
46265608).
Recovery
of
mCry3A
from
the
treated
diet
was
91.7%
of
the
nominal
concentration.

Test
Methods
The
test
insects
(
A.
bilineata)
were
obtained
from
De
Groene
Vlieg,
Nieuwe
Tonge,
The
Netherlands
in
advance
of
the
bioassay
as
parasitized
pupae
of
the
onion
fly
(
D.
antiqua).
Adult
beetles
that
had
emerged
over
a
24
hour
period
were
individually
confined
and
provided
with
a
pellet
of
raw
minced
beef
for
food.
The
beetles
were
placed
in
a
dark
cold
room
(
0­
8

C)
for
six
days
to
delay
their
development.
Three
days
before
the
bioassay
started,
beetles
were
moved
back
to
a
controlled
environment
room
maintained
at
23­
25
º
C
31
and
64­
79%
relative
humidity,
under
a
16
hour
photoperiod.
At
the
start
of
the
bioassay,
beetles
were
physiologically
4
days
old.
Groups
of
10
male
and
10
female
adult
rove
beetles
were
fed
beef
paste
diet
containing
the
test
material
at
a
rate
equivalent
to
50
Fg
MCRY3A­
0102
/
g
of
diet.
The
treated
diet
was
prepared
by
diluting
0.0554
g
of
MCRY3A­
0102
in
100
mL
of
deionized
water
and
mixing
10
mL
of
the
resulting
solution
into
90
g
of
previouslycooked
pureed
beef.
Negative
control
beetles
were
fed
diet
mixed
with
deionized
water
only,
and
positive
control
beetles
were
fed
diet
mixed
with
teflubenzuron
at
0.01
mg
a.
i./
g
of
diet.
Diets
were
prepared
at
one
time,
with
the
remainder
frozen
at
­
20EC
until
use.
Each
treatment
of
20
beetles
was
replicated
4
times.

For
the
first
7
days
of
the
test
the
beetles
were
housed
in
plastic
pots
with
ventilated,
clip­
on
lids
in
order
to
encourage
regular
contact
with
the
diet.
The
beetles
were
then
transferred
to
polystyrene
boxes
with
ventilated
lids
for
the
remaining
28
days
of
the
test.
The
boxes
contained
a
4­
cm
layer
of
washed
and
graded
sand
that
was
kept
wet
with
distilled
water.
Approximately
0.2
g
of
the
treated
diet
was
placed
in
the
boxes
and
replaced
daily
with
diet
allowed
to
defrost
for
one
hour.
Condition
of
the
beetles
was
assessed
on
days
1
and
7
of
the
test.
The
test
occurred
in
a
controlled
environment
room
maintained
at
20E
to
24EC
and
61%
to
88%
relative
humidity
(
with
minor
fluctuations)
with
a
photoperiod
of
16
hrs
light/
8
hrs
darkness.

To
assess
the
effects
of
treatment
on
fecundity,
approximately
500
host
onion
fly
pupae
were
placed
under
the
surface
of
the
wet
sand
on
days
14,
21,
and
28
of
the
study.
On
day
35,
live
beetles
were
removed
and
assessed
for
condition.
The
sand
was
allowed
to
dry
for
approximately
one
week,
after
which
the
onion
fly
pupae
were
sieved
out
and
transferred
to
plastic
emergence
chambers
with
ventilated
lids.
The
bottom
of
the
chambers
contained
netting
that
acted
as
a
sieve
through
which
emerging
adult
beetles
would
fall
into
a
collecting
chamber
below.
The
emergence
chambers
were
maintained
in
a
controlled
environment
room
for
a
further
34
days
to
permit
the
F1
generation
of
rove
beetles
to
emerge,
and
the
number
of
emergents
recorded
every
1
to
4
days.
The
test
ended
when
the
number
of
emergents/
replicate
fell
below
2.0/
day
in
the
control
treatment.

Results
Summary
There
was
no
statistically
significant
difference
in
day
35
mortality
among
the
groups
(
Table
1).
Although
the
control
mortality
was
34%,
the
study
author
stated
that
A.
bilineata
mortality
in
similar
28­
day
lab
tests
is
generally
20
to
40%.
There
was
also
no
statistically
significant
difference
in
the
mean
number
of
progeny
produced
by
the
negative
control
and
the
treated
groups.
Reproduction
in
the
positive
control
group
was
significantly
reduced.
32
TABLE
1.
Mortality
and
number
of
progeny
of
A.
bilineata
supplied
MCRY3A­
0102
for
35
days
Treatment
%
Mortality
at
35
daysa
%
Corrected
Mortalityb
Mean
no.
of
F1
Progenyc
%
Effect
on
Reproductiond
MCRY3A­
0102
(
50
Fg
mCry3A/
g
diet)
31
0
663
±
219
­
2.5
Negative
control
34
­
647
±
169
­

Teflubenzuron
(
10
Fg/
g
diet)
35
2
3***
±
5
99.5
a)
The
overall
number
of
adult
beetles
that
were
found
dead
or
were
missing
at
35
DAT
were
compared
by
Fisher's
Exact
test.
None
of
the
treatment
results
different
significantly
from
the
control
(
P
>
0.05)
b)
Corrected
using
Abbotts
formula
c)
The
numbers
of
progeny
emerging
per
replicate
were
compared
by
one­
way
ANOVA
and
Dunnett's
test
(
prior
to
analysis,
a
log
10
(
n+
1)
transformation
was
applied
to
the
data).
d)
The
percentage
reduction/
increase
in
numbers
of
F1
progeny,
relative
to
the
control,
was
calculated
using
the
formula
R=
(
1­(
Rt/
Rc))
x
100,
where
Rt
and
Rc
are
the
numbers
of
offspring
observed
in
the
treatment
and
control
groups,
respectively.
A
negative
value
indicates
an
increase
and
a
positive
value
a
decrease.
***
Signficantly
different
from
control
p#
0.001
Data
from
pp.
18­
19,
MRID
46265607
Study
Author's
Conclusions
The
study
author
concluded
that
MCRY3A­
0102
did
not
adversely
affect
survival
or
reproductive
capacity
of
A.
bilineata
when
offered
continuously
in
the
diet
at
a
rate
equivalent
to
50
Fg
mCry3A/
g
of
diet.
33
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nonguideline
MRID
NO:
46265608
DP
BARCODE:
DP303605
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCRY3A­
0102
STUDY
NO:
SSB­
006­
04
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Syngenta
Biotechnology,
Inc.,
Research
Triangle
Park,
NC
TITLE
OF
REPORT:
Analysis
of
Test
Diet
Used
to
Expose
Aleochara
bilineata
to
Modified
Cry3A
Protein:
Supplement
to
Report
Titled
­
"
A
Laboratory
Toxicity
Test
of
Modified
Cry3A
Protein
(
MCRY3A­
0102)
to
the
Rove
Beetle,
Aleochara
bilineata
(
Coleoptera:
Staphylinidae)";
Mambo­
Tox
Study/
Report
No.
SYN­
03­
53
AUTHOR:
Graser,
G.

STUDY
COMPLETED:
April
23,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
EPA
GLP
compliant.
34
STUDY
SUMMARY:
Samples
of
formulated
diet
treated
with
MCRY3A­
0102
and
used
in
a
rove
beetle
(
Aleochara
bilineata)
toxicity
study
(
MRID
46265607)
were
analyzed
for
MCRY3A­
0102
protein
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA).
The
integrity
of
the
MCRY3A­
0102
in
the
treated
diet
was
determined
using
Western
blot
analysis,
and
the
bioactivity
of
MCRY3A­
0102
in
the
treated
diet
was
determined
in
a
bioassay
using
Colorado
potato
beetle
(
CPB)
larvae.
ELISA
of
the
treated
test
diet
recovered
91.7%
of
the
nominal
mCry3A
diet
concentration
used
in
the
original
study,
indicating
MCRY3A­
0102
was
present
at
the
intended
concentration.
Western
blot
analysis
of
the
treated
diet
indicated
that
mCry3A
did
not
degrade
under
conditions
of
the
original
test
diet
preparation
or
storage,
and
the
CPB
bioassay
confirmed
that
the
original
nontarget
insect
test
group
(
A.
bilineata)
had
been
exposed
to
active
MCRY3A­
0102.

CLASSIFICATION:
Acceptable
Test
Material
The
test
substances
were
samples
of
a
formulated
beef
paste
diet
containing
MCRY3A­
0102
(
active
ingredient,
mCry3A
protein)
that
had
been
used
in
a
previous
non­
target
insect
toxicity
study
with
Aleochara
bilineata
(
MRID
46265607).
The
nominal
active
ingredient
concentration
in
the
diet
for
that
study
was
50
Fg
MCRY3A­
0102
/
g
of
diet.
Samples
of
control
diet
(
with
deionized
water
instead
of
MCRY3A­
0102)
used
in
that
study
were
also
submitted
for
analysis.
The
samples
were
frozen
immediately
after
preparation
in
the
original
study
and
remained
frozen
until
analysis.

Test
Methods
The
test
substance
was
quantitatively
analyzed
for
MCRY3A­
0102
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA),
and
the
integrity
of
the
MCRY3A­
0102
was
determined
using
Western
blot
analysis.
Standard
operating
procedures
were
referenced
in
the
report.
The
bioactivity
of
MCRY3A­
0102
in
the
test
substance
was
determined
in
a
bioassay
using
Colorado
potato
beetle
(
CPB)
(
Leptinotarsa
decemlineata)
larvae.

For
the
ELISA
and
Western
blot
analysis,
2
mL
of
extraction
buffer
were
added
to
approximately
0.35
g
of
the
Aleochara
test
or
control
diet.
Each
sample
was
mixed,
incubated
on
ice
for
one
hour,
homogenized,
centrifuged
for
10
minutes
at
2600
x
g,
and
the
supernatants
were
collected.
The
pellets
were
extracted
a
second
time
using
the
same
procedure,
and
a
third
extraction
was
done
in
an
overnight
incubation
on
ice.
All
three
extracts
of
each
sample
were
centrifuged
again
for
10
minutes
at
13,000
x
g
prior
to
analysis.
35
These
extracts
were
used
for
the
ELISA,
while
only
the
first
extract
of
each
sample
was
used
for
the
Western
blot
analysis.

For
the
ELISA,
the
extracts
were
analyzed
for
MCRY3A­
0102
using
immuno­
affinity
purified
rabbit
anti­
mCry3A
polyclonal
antibodies
and
immuno­
affinity
purified
goat
anti­
Btt
(
native
Cry3A
from
Bacillus
thuringiensis
subsp.
tenebrionis)
polyclonal
antibodies.

For
the
Western
blot
analysis,
two
aliquots
of
the
Aleochara
test
diet
extract,
containing
15
and
49
ng
mCry3A,
respectively
(
as
determined
by
ELISA),
were
subjected
to
SDS­
PAGE
using
an
8­
16%
polyacrylamide
gradient
gel.
Two
aliquots
of
a
solution
of
MCRY3A­
0102
(
positive
control),
containing
15
and
50
ng
of
MCRY3A­
0102,
respectively,
and
one
aliquot
of
the
Aleochara
control
diet
(
negative
control)
were
run
concurrently
on
the
same
gel.
After
electroblotting,
the
membrane
was
probed
with
immuno­
affinity
purified
goat
anti­
Btt
polyclonal
antibodies.
Donkey
anti­
goat
IgG
linked
to
alkaline
phosphatase,
diluted
in
blocking
buffer,
was
used
to
bind
to
the
primary
antibody
and
visualized
by
development
with
alkaline
phosphatase
substrate
solution.
The
Western
blot
was
examined
for
the
presence
of
intact
immunoreactive
MCRY3A­
0102
(
ca.
67,000
molecular
weight)
and
immunoreactive
MCRY3A­
0102
polypeptides.

For
the
CPB
bioassay,
diets
containing
10%
and
20%
of
the
Aleochara
diet
were
prepared
by
adding
0.522
and
0.464
g,
respectively,
of
stock
CPB
diet
to
0.058
and
0.116
g,
respectively,
of
the
Aleochara
diet.
Control
CPB
diet
was
prepared
in
the
same
manner
using
the
Aleochara
control
diet.
Positive
control
diets
were
also
prepared
by
adding
MCRY3A­
0102
to
stock
CPB
diet
at
concentrations
equivalent
to
12.5
and
50
Fg
MCRY3A­
0102
/
g.
As
a
control
for
the
positive
control
diet,
CPB
diet
was
prepared
using
the
same
volume
of
water
as
was
used
to
add
the
positive
control
solution
to
the
CPB
diet.
An
untreated
CPB
diet
control
was
also
included.
A
piece
of
the
supplemented
diets
(
10%
or
20%
Aleochara
test
diet
or
10%
or
20%
Aleochara
control
diet),
was
placed
in
a
Petri
dish
with
10
newlyhatched
CPB
larvae.
Similarly,
groups
of
10
larvae
were
provided
with
stock
CPB
diet
(
untreated
control),
stock
CPB
diet
treated
directly
with
MCRY3A­
0102
(
positive
control),
or
stock
CPB
diet
treated
with
water
(
control
for
the
positive
control).
Each
treatment
was
replicated
3
times.
The
dishes
were
covered
and
maintained
at
ambient
conditions
for
96
hours.
Diets
were
replaced
after
approximately
24
and
48
hrs
with
fresh
diet
prepared
in
the
same
manner.
Mortality
was
assessed
daily.

Results
Summary
ELISA
of
the
Aleochara
test
diet
extract
showed
a
recovery
of
91.7%
of
the
nominal
50
Fg
MCRY3A­
0102
/
g
of
diet
concentration
used
in
the
original
Aleochara
study.
No
mCry3A
was
detected
in
the
Aleochara
control
diet.

Western
blot
analysis
of
the
Aleochara
test
diet
showed
a
single
immunoreactive
band
corresponding
to
the
predicted
molecular
weight
of
MCRY3A­
0102
(
ca.
67,700
Da).
No
immunoreactive
material
was
detected
in
the
Aleochara
control
diet.
36
In
the
CPB
bioassay,
the
diets
prepared
with
10%
and
20%
of
the
original
Aleochara
test
diet
produced
57%
and
77%
larval
mortality,
respectively,
after
96
hours.
The
positive
controls
produced
similar
mortality.
The
Aleochara
control
diet
produced
10%
mortality,
as
did
the
control
CPB
diets.

Study
Author's
Conclusions
The
study
author
concluded
that
a)
the
high
proportion
of
MCRY3A­
0102
recovered
by
ELISA
(
91.7%)
indicated
MCRY3A­
0102
was
present
in
the
Aleochara
test
diet
at
the
intended
concentration,
b)
the
Western
blot
analysis
showed
that
MCRY3A­
0102
did
not
degrade
under
conditions
of
the
Aleochara
test
diet
preparation
or
storage,
and
c)
the
bioassay
results
confirmed
that
the
Aleochara
test
group
was
exposed
to
active
MCRY3A­
0102.
37
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nontarget
Insect
Testing,
Tier
I
(
885.4340)

MRID
NO:
46265609
DP
BARCODE:
DP303605
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCRYA­
0102
STUDY
NO:
Mambo­
Tox
Study/
Report
No.
SYN­
03­
52
Syngenta
Project
No.
2033033
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Mambo­
Tox
Ltd.,
Southampton,
UK
TITLE
OF
REPORT:
A
Laboratory
Toxicity
Test
of
Modified
Cry3A
Protein
(
MCRY3A­
0102)
to
the
Predatory
Bug,
Orious
insidiosus
(
Heteroptera:
Anthocoridae)

AUTHOR:
Vinall,
S.

STUDY
COMPLETED:
November
19,
2003
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
compliant
with
OECD
and
UK
GLP
regulations.
38
STUDY
SUMMARY:
In
a
laboratory
study,
predatory
bug
(
Orius
insidiosus)
nymphs
were
provided
artificial
diet
containing
MCRY3A­
0102
at
a
nominal
rate
equivalent
to
50
Fg
mCry3A/
g
diet
for
21
days.
The
test
substance
was
purified
from
cells
of
recombinant
E.
coli.
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
significantly
increased.

CLASSIFICATION:
Acceptable
Test
Material
The
test
substance,
MCRY3A­
0102,
active
ingredient
modified
Cry3A
protein,
Batch
Number
MCRY3A­
0102,
was
supplied
by
the
sponsor
with
a
reported
purity
of
90.3%
w/
w
and
an
expiration
date
of
December
3,
2003.
The
test
substance
was
purified
from
cells
of
recombinant
E.
coil.
The
test
substance
was
stored
at
­
30E
to
­
20EC
prior
to
use.
The
study
was
conducted
prior
to
the
test
substance
expiration
date.
The
concentration
and
activity
of
MCRY3A­
0102
in
the
treated
diet
were
determined
in
a
separate
study
(
MRID
46265610).
Recovery
of
MCRY3A­
0102
from
the
test
diet
was
95.6%
of
the
nominal
concentration.

Test
Methods
Predatory
bug
nymphs(
Orius
insidiosus,
reared
in­
house
from
adults
obtained
from
Koppert
UK
Ltd.,
Suffolk,
UK)
were
provided
artificial
diet
containing
MCRY3A­
0102
at
a
rate
equivalent
to
50
Fg
MCRY3A­
0102/
g
diet
for
21
days.
The
base
diet
was
a
paste
composed
of
beef,
lamb,
yeast,
and
honey.
Diet
for
the
test
substance
group
was
prepared
by
diluting
0.0554
g
(~
0.05
g
mCry3A)
of
MCRY3A­
0102
to
100
mL
with
deionized
water,
and
mixing
20
mL
of
this
preparation
with
180
g
of
diet.
The
negative
control
group
received
the
artificial
diet
mixed
with
deionized
water
only,
and
the
positive
control
group
received
diet
containing
teflubenzuron
(
0.01
mg
a.
i./
g
of
diet).
The
prepared
diet
was
stored
frozen
until
needed.

The
test
chambers
were
plastic
pots
with
ventilated
lids.
The
tops
of
the
cylinders
were
coated
with
Fluon
to
prevent
the
bugs
from
crawling
upwards.
One
2
to
3­
day­
old
nymph
was
placed
in
each
chamber
with
approximately
0.2
g
of
treated
diet
in
a
plastic
cup
covered
with
Parafilm.
The
nymphs
pierced
the
Parafilm
to
feed.
Fresh
diet
was
provided
daily
after
being
allowed
to
defrost
for
approximately
one
hour.
A
cotton
ball
moistened
with
tap
water
provided
drinking
water.
39
The
study
was
carried
out
in
an
environment
controlled
room
maintained
at
25
to
26EC
and
61
to
87%
relative
humidity.
The
photoperiod
was
16
hrs
light/
8
hrs
darkness.
The
nymphs
were
monitored
for
condition
daily
until
they
became
adults
or
until
21
days
after
test
initiation,
whichever
came
first.
At
the
end
of
the
study,
the
percent
pre­
imaginal
mortality
for
each
treatment
was
calculated
and
the
data
were
analyzed
statistically.

Results
Summary
There
was
no
significant
difference
in
the
percent
pre­
imaginal
mortality
between
the
MCRY3A­
0102
and
negative
control
groups
(
Table
1).
Mortality
in
the
positive
control
group
was
significantly
increased.

TABLE
1.
Mortality
of
O.
insidiosus
supplied
with
treated
diet
Treatment
%
Pre­
imaginal
Mortalitya
%
Corrected
Mortalityb
MCRY3A­
0102
(
50
Fg
mCry3A/
g
diet)
18
0
Negative
control
23
­

Teflubenzuron
(
10
Fg/
g
diet)
98***
97
a)
Results
for
individual
treatments
were
compared
to
the
control
by
Fisher's
Exact
Test
b)
Corrected
using
Abbotts
formula
***
Signficantly
different
from
control
p#
0.001
Data
from
p.
16,
MRID
46265609
Study
Author's
Conclusions
The
study
author
concluded
that
MCRY3A­
0102
was
not
harmful
to
O.
insidiosus
when
offered
continuously
in
the
diet
at
a
rate
equivalent
to
50
Fg
MCRY3A­
0102/
g
of
diet.
40
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nonguideline
MRID
NO:
46265610
DP
BARCODE:
DP303605
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCRY3A­
0102
STUDY
NO:
SSB­
005­
04
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Syngenta
Biotechnology,
Inc.,
Research
Triangle
Park,
NC
TITLE
OF
REPORT:
Analysis
of
Test
Diet
Used
to
Expose
Orius
insidiosus
to
Modified
Cry3A
Protein:
Supplement
to
Report
Titled
­
"
A
Laboratory
Toxicity
Test
of
Modified
Cry3A
Protein
(
MCRY3A­
0102)
to
the
Predatory
Bug,
Orius
insidiosus
(
Heteroptera:
Anthocoridae)";
Mambo­
Tox
Study/
Report
No.
SYN­
03­
52
AUTHOR:
Graser,
G.

STUDY
COMPLETED:
April
23,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
EPA
GLP
compliant.
41
STUDY
SUMMARY:
Samples
of
formulated
meat
diet
treated
with
MCRY3A­
0102
and
used
in
a
predatory
bug
(
Orius
insidiosus)
toxicity
study
(
MRID
46265609)
were
analyzed
for
MCRY3A­
0102
protein
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA).
The
integrity
of
the
mCry3A
in
the
treated
diet
was
determined
using
Western
blot
analysis,
and
the
bioactivity
of
MCRY3A­
0102
in
the
treated
diet
was
determined
in
a
bioassay
using
Colorado
potato
beetle
(
CPB)
larvae.
ELISA
of
the
treated
diet
recovered
95.6%
of
the
nominal
MCRY3A­
0102
diet
concentration
used
in
the
original
study
(
50
Fg/
g),
indicating
MCRY3A­
0102
was
present
at
the
intended
concentration.
Western
blot
analysis
of
the
treated
diet
indicated
that
MCRY3A­
0102
did
not
degrade
under
conditions
of
the
original
test
diet
preparation
or
storage,
and
the
CPB
bioassay
confirmed
that
the
original
nontarget
insect
test
group
(
O.
insidiosus)
had
been
exposed
to
active
MCRY3A­
0102.

CLASSIFICATION:
Acceptable
Test
Material
The
test
substances
were
samples
of
a
formulated
meat­
based
diet
containing
MCRY3A­
0102
(
active
ingredient,
mCry3A
protein)
that
had
been
used
in
a
previous
non­
target
insect
toxicity
study
with
Orius
insidiosus
(
MRID
46265609).
The
nominal
active
ingredient
concentration
in
the
diet
for
that
study
was
50
Fg
MCRY3A­
0102/
g
of
diet.
Samples
of
control
diet
(
with
deionized
water
instead
of
MCRY3A­
0102)
used
in
that
study
were
also
submitted
for
analysis.
The
samples
were
frozen
immediately
after
preparation
in
the
original
study
and
remained
frozen
until
analysis.

Test
Methods
The
test
substance
was
quantitatively
analyzed
for
MCRY3A­
0102
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA),
and
the
integrity
of
the
MCRY3A­
0102
was
determined
using
Western
blot
analysis.
Standard
operating
procedures
were
referenced
in
the
report.
The
bioactivity
of
mCry3a
in
the
test
substance
was
determined
in
a
bioassay
using
Colorado
potato
beetle
(
CPB)
(
Leptinotarsa
decemlineata)
larvae.

For
the
ELISA
and
Western
blot
analysis,
3
mL
of
extraction
buffer
were
added
to
approximately
1
g
of
the
Orius
test
or
control
diet.
Each
sample
was
mixed,
incubated
on
ice
for
one
hour,
homogenized,
centrifuged
for
10
minutes
at
2600
x
g,
and
the
supernatants
were
collected.
The
pellets
were
extracted
a
second
time
using
the
same
procedure,
and
a
third
extraction
was
done
in
an
overnight
incubation
on
ice.
All
three
extracts
of
each
sample
42
were
centrifuged
again
for
10
minutes
at
13,000
x
g
prior
to
analysis.
These
extracts
were
used
for
the
ELISA,
while
only
the
first
extract
of
each
sample
was
used
for
the
Western
blot
analysis.

For
the
ELISA,
the
extracts
were
analyzed
for
MCRY3A­
0102
using
immuno­
affinity
purified
rabbit
anti­
mCry3A
polyclonal
antibodies
and
immuno­
affinity
purified
goat
anti­
Btt
(
native
Cry3A
from
Bacillus
thuringiensis
subsp.
tenebrionis)
polyclonal
antibodies.

For
the
Western
blot
analysis,
two
aliquots
of
the
Orius
test
diet
extract,
containing
19
and
57
ng
MCRY3A­
0102,
respectively
(
as
determined
by
ELISA),
were
subjected
to
SDS­
PAGE
using
an
8­
16%
polyacrylamide
gradient
gel.
Two
aliquots
of
a
solution
of
MCRY3A­
0102
(
positive
control),
containing
20
and
60
ng
of
MCRY3A­
0102,
respectively,
and
one
aliquot
of
the
Orius
control
diet
(
negative
control)
were
run
concurrently
on
the
same
gel.
After
electroblotting,
the
membrane
was
probed
with
immuno­
affinity
purified
goat
anti­
Btt
polyclonal
antibodies.
Donkey
anti­
goat
IgG
linked
to
alkaline
phosphatase,
diluted
in
blocking
buffer,
was
used
to
bind
to
the
primary
antibody
and
visualized
by
development
with
alkaline
phosphatase
substrate
solution.
The
Western
blot
was
examined
for
the
presence
of
intact
immunoreactive
mCry3A
(
ca.
67,000
molecular
weight)
and
immunoreactive
MCRY3A­
0102
polypeptides.

For
the
CPB
bioassay,
diets
containing
10%
and
20%
of
the
Orius
diet
were
prepared
by
adding
0.9
and
0.8
g,
respectively,
of
stock
CPB
diet
to
0.1
and
0.2
g,
respectively,
of
the
Orius
diet.
Control
CPB
diet
was
prepared
in
the
same
manner
using
the
Orius
control
diet.
Positive
control
diets
were
prepared
by
adding
MCRY3A­
0102
to
stock
CPB
diet
at
concentrations
equivalent
to
12.5
and
50
Fg
MCRY3A­
0102/
g.
As
a
control
for
the
positive
control
diet,
CPB
diet
was
prepared
using
the
same
volume
of
water
as
was
used
to
add
the
positive
control
solution
to
the
CPB
diet.
An
untreated
CPB
diet
control
was
also
included.
A
piece
of
the
supplemented
diet
(
10%
or
20%
Orius
test
diet
or
10%
or
20%
Orius
control
diet),
was
placed
in
a
Petri
dish
with
10
newly­
hatched
CPB
larvae.
Similarly,
groups
of
10
larvae
were
provided
with
stock
CPB
diet
(
untreated
control),
stock
CPB
diet
treated
directly
with
MCRY3A­
0102
(
positive
control),
or
stock
CPB
diet
treated
with
water
(
control
for
the
positive
control).
Each
treatment
was
replicated
3
times.
The
dishes
were
covered
and
maintained
at
ambient
conditions
for
96
hours.
Diets
were
replaced
after
approximately
24
and
48
hrs
with
fresh
diet
prepared
in
the
same
manner.
Mortality
was
assessed
daily.

Results
Summary
ELISA
of
the
Orius
test
diet
extract
showed
a
recovery
of
95.6%
of
the
nominal
50
Fg
MCRY3A­
0102/
g
of
diet
concentration
used
in
the
Orius
study.
No
MCRY3A­
0102
was
detected
in
the
Orius
control
diet.

Western
blot
analysis
of
the
Orius
test
diet
showed
a
single
immunoreactive
band
corresponding
to
the
predicted
molecular
weight
of
MCRY3A­
0102
(
ca.
67,700
Da).
No
immunoreactive
material
was
detected
in
the
Orius
control
diet.
43
In
the
CPB
bioassay,
the
diets
prepared
with
10%
and
20%
of
the
original
Orius
test
diet
produced
83%
and
90%
larval
mortality,
respectively,
after
96
hours.
The
positive
controls
produced
similar
mortality.
The
Orius
control
diet
produced
13%
mortality,
similar
to
the
control
CPB
diets
(
10%).

Study
Author's
Conclusions
The
study
author
concluded
that
a)
the
high
proportion
of
MCRY3A­
0102
recovered
by
ELISA
(
95.6%)
indicated
mCry3A
was
present
in
the
Orius
test
diet
at
the
intended
concentration,
b)
the
Western
blot
analysis
showed
that
MCRY3A­
0102
did
not
degrade
under
conditions
of
the
Orius
test
diet
preparation
or
storage,
and
c)
the
bioassay
results
confirmed
that
the
Orius
test
group
was
exposed
to
active
mCry3A.
44
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Honey
Bee
Testing,
Tier
I
(
885.4380)

MRID
NO:
46155818
DP
BARCODE:
DP300338
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCry3A­
0102
STUDY
NO:
SYN­
03­
57
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Mambo­
Tox
Ltd.,
Southampton,
UK
TITLE
OF
REPORT:
A
Semi­
Field
Test
to
Evaluate
the
Effects
of
the
Modified
Cry3A
Protein
(
MCRY3A­
0102)
on
Brood
Development
of
the
Honeybee,
Apis
mellifera
(
Hymenoptera:
Apidae)

AUTHOR:
Halsall,
N.

STUDY
COMPLETED:
December
2,
2003
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
OECD
and
UK
GLP
compliant.
45
STUDY
SUMMARY:
Honeybees
(
Apis
mellifera)
were
exposed
via
in­
hive
feeders
to
either
a
nominal
concentration
of
50
Fg
MCRY3A­
0102/
g
of
sucrose
solution,
480
g
of
diflubenzuron
insect
growth
regulator/
L
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.

CLASSIFICATION:
Acceptable
Test
Material
The
test
substance,
MCRY3A­
0102,
active
ingredient
modified
Cry3A
protein,
Batch
Number
MCRY3A­
0102,
was
supplied
by
the
sponsor
with
a
reported
purity
of
90.3%.
For
the
test,
the
test
substance
was
suspended
in
200
mL
of
50%
w/
v
sucrose
solution
at
a
concentration
of
50
Fg
mCry3A
protein/
g
of
sucrose
solution.
The
test
substance
was
purified
from
cells
of
recombinant
E.
coli.

Test
Methods
The
test
occurred
in
a
private
garden
in
Chilworth,
Hampshire,
UK,
with
no
areas
of
flowering
crops
within
foraging
distance
(
distance
not
specified).
Three
days
prior
to
the
study,
16
healthy
hives
of
honeybees
(
Apis
mellifera)
in
similar
condition
were
transferred
to
the
test
site.
The
distance
between
test
hives
was
not
noted
in
the
study
write
up.
At
the
test
start,
a
sufficient
number
of
frames
were
removed
from
each
hive
to
record
the
position
of
100
cells
containing
eggs
and
100
cells
containing
1­
to
3­
day­
old
larvae.
The
cells
were
mapped
on
an
acetate
sheet
overlaid
on
each
frame.
A
dead
bee
trap
was
fitted
to
the
entrance
of
each
hive.

The
treatment
solutions
were
supplied
to
each
treated
hive
using
a
1­
L
capacity
commercial
bee
feeder.
Hives
received
either
50
Fg
of
the
test
material/
g
of
sucrose
solution,
a
negative
control
of
50%
w/
v
sucrose
solution,
or
a
positive
control
of
480
g/
L
diflubenzuron
insect
growth
regulator
in
sucrose
solution.
Each
treatment
was
replicated
4
times.
The
treatment
solutions
were
prepared
fresh
daily
and
replaced
in
each
hive
daily
over
5
days.
The
diet
was
not
analyzed
in
any
additional
studies.
46
Brood
development
was
monitored
for
21
days
after
the
beginning
of
treatment.
Egg
cells
were
assessed
at
days
4,
6,
12,
19,
and
21.
Larval
cells
were
assessed
at
days
4,
6,
12,
and
19.
The
dead
bee
traps
were
assessed
at
study
start
and
every
1
to
3
days
thereafter
for
the
duration
of
the
study.
Dead
adults,
larvae,
and
pupae
were
discarded
after
counting.
The
hives
were
assessed
for
general
condition
[
the
amount
of
brood
and
food
(
pollen
and
nectar
reserves)]
on
days
0
and
22.

The
percent
mortality
among
treatments
was
compared
using
a
Chi­
square
test,
both
before
and
after
applying
Abbott's
formula
to
correct
for
control
mortality.
Hive
condition
before
and
after
treatments
was
compared
using
ANOVA.
The
level
of
significance
used
was
5%.

Results
Summary
All
the
treatment
solutions
were
consumed
each
day.
Egg
cell
mortality
in
the
negative
control,
test,
and
positive
control
groups
was
28.5%,
27.3%,
and
100%,
respectively.
Larval
cell
mortality
was
6.0%,
6.8%,
and
100%,
respectively.
There
was
no
significant
difference
in
mortality
between
the
test
and
negative
control
groups.
There
was
also
no
significant
difference
in
pre­
and
post­
test
hive
condition
between
the
test
and
negative
control
treatments.
Results
for
the
positive
control
treatment
were
significantly
different
from
the
other
treatments
for
both
mortality
and
hive
condition.
The
number
of
dead
bees
found
during
the
test
was
too
low
for
meaningful
comparison
among
treatments.

Table
1.
Summary
of
Development
of
Egg
Cells,
Larval
Cells,
and
of
Brood
Count
Per
Frame
Treatment
Brood
Development
Assessmentsa
Mean
%
Brood
per
Frame
b(
before
and
after
treatment)
Egg
Cells
Larvae
Cells
Before
After
%
Mortality
%
Corrected
Mortality
%
Mortality
%
Corrected
Mortality
Treatment
28.5
­
6.0
­
36.4
49.6
MCRY3A­
0102
27.3
0
6.8
0.9
34.2
40.7
Dimilin
Flo
100.0***
100.0
100.0
100***
34.5
27.7**
a)
For
the
brood
development
assessments,
mortality
in
individual
treatments
was
compared
to
the
control
using
Chi­
square
tests.
Results
marked
with
asterisks
differed
significantly
from
the
control
(***
P
<
0.01)
b)
For
the
brood
percent
assessments,
a
one­
way
ANOVA
was
used
to
compare
mean
percent
brood
on
each
side
of
each
frame
between
treatments.
Prior
to
analysis
an
angular
transformation
was
applied
to
the
data.
No
significant
differences
occurred
between
any
of
the
treatments
before
treatment
(
P
>
0.05).
After
treatment,
however,
the
mean
percentage
area
of
each
frame
occupied
by
brood
was
significantly
lower
in
the
Diminlo
Flo
treatment
when
compared
to
the
control
Study
Authors'
Conclusions
The
study
authors
concluded
that
MCRY3A­
0102
fed
to
honeybees
for
5
days
at
a
concentration
nominally
equivalent
to
50
Fg
MCRY3A­
0102
protein/
g
of
sucrose
solution
did
not
adversely
affect
condition
or
survival
of
larvae
in
brood
cells,
and
had
no
effect
on
survival
of
exposed
adult
worker
bees.
47
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nonguideline
MRID
NO:
46265602
DP
BARCODE:
DP303605
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCry3A­
0102
STUDY
NO:
Brixham
Environmental
Laboratory
Report
Project
ID
03­
0294/
A
Syngenta
Seeds
Biotechnology
Report
No.
SSB­
001­
04
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Brixham
Environmental
Laboratory,
Brixham,
Devon
TQ5
8BA,
UK
TITLE
OF
REPORT:
Characterization
of
Fish
Feed
Test
Substance
(
FFMIR604­
0103)
Prepared
From
Event
MIR604­
Derived
Maize
Grain
AUTHOR:
Graser,
G.

STUDY
COMPLETED:
April
26,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
USEPA
GLP
compliant.

STUDY
SUMMARY:
Formulated
fish
feed
containing
50%
by
weight
transgenic
maize
(
Event
MIR604)
and
used
as
diet
in
a
28­
day
rainbow
trout
feeding
study
was
analyzed
for
mCry3A
protein
content
by
enzyme­
linked
immunosorbent
assay.
The
mCry3A
was
uniformly
distributed
in
the
formulated
feed
at
a
concentration
of
0.09
±
005
Fg/
g
fresh
weight.

CLASSIFICATION:
Acceptable
48
Test
Material
The
test
substance,
FFMIR604­
0103,
was
a
formulated
fish
feed
(
Zeigler
Bros.,
Inc.)
prepared
from
Event
MIR604
maize
grain,
which
contains
mCry3A
protein.
The
nutritionally
balanced
feed
contained
50%
w/
w
maize
grain,
and
was
"
cold"
pelleted
to
minimize
thermal
degradation
of
mCry3A.
A
fish
feed
control
substance,
FFMIR604­
0103C,
was
prepared
in
the
same
manner,
using
grain
from
a
non­
transgenic
isoline.
The
test
and
control
substances
were
used
in
a
previous
28­
day
rainbow
trout
feeding
study
(
MRID
46155617),
and
samples
were
frozen
until
analysis.
The
ingredients
and
nutritional
composition
of
the
formulated
fish
feeds
are
provided
in
Appendix
A
of
MRID
46465602,
and
the
method
used
to
prepare
the
feeds
is
provided
in
Appendix
B.
A
sample
of
the
ground
transgenic
maize
grain
(
KMIR604­
0103)
used
to
prepare
the
test
substance,
and
a
sample
of
the
non­
transgenic
maize
grain
(
KMIR604­
0103C)
used
in
the
control
feed,
were
also
analyzed.

Test
Methods
The
test
substances
were
quantitatively
analyzed
for
mCry3A
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA).
Standard
operating
procedures
were
referenced
in
the
report.
Three
mL
of
extraction
buffer
were
added
to
approximately
1
g
samples
of
the
ground
maize
grains
and
to
three
separate,
random
aliquots
of
the
fish
feed
samples.
The
samples
were
then
mixed,
incubated
on
ice
for
30
minutes,
and
homogenized
prior
to
centrifugation
for
15
minutes
at
2500
x
g.
The
supernatants
were
quantitatively
analyzed
by
ELISA
using
immuno­
affinity
purified
rabbit
anti­
mCry3A
polyclonal
antibodies
and
immuno­
affinity
purified
goat
anti­
Btt
(
native
Cry3A
from
Bacillus
thuringiensis
subsp.
tenebrionis)
polyclonal
antibodies.
The
homogeneity
of
the
fish
feed
test
substance
FFMIR604­
0103
was
assessed
by
comparing
the
mCry3A
concentrations
in
the
three
samples.

Results
Summary
The
mCry3A
concentration
in
the
ground
transgenic
grain
(
KMIR604­
0103)
was
0.3
Fg
mCry3A/
g
fresh
weight.
The
mean
mCry3A
concentration
in
the
fish
feed
test
substance
(
FFMIR604­
0103)
was
0.09
±
0.005
Fg
mCry3A/
g
fresh
weight.
The
mCry3A
concentrations
in
the
three
random
fish
feed
test
substance
samples
were
0.09,
0.09,
and
0.10
Fg
mCry3A/
g
fresh
weight,
indicating
that
mCry3A
was
present
in
the
feed
at
a
uniform
concentration.
No
mCry3A
was
detected
in
the
ground
control
maize
grain
(
KMIR604­
0103C)
or
in
the
control
fish
feed
(
FFMIR604­
1013C).
It
should
be
noted
that
the
concentration
in
the
fish
food
was
significantly
less
than
10
Fg.
It
was
not
stated
the
concentration
of
mCry3A
expressed
in
the
corn
itself.

Study
Author's
Conclusions
The
study
author
concluded
that
mCry3A
protein
was
present
in
the
fish
feed
test
substance
(
FFMIR604­
0103)
that
was
used
as
test
diet
in
the
previous
28­
day
rainbow
trout
feeding
49
study.
The
mCry3A
concentration
in
the
feed
was
0.09
Fg/
g
fresh
weight,
and
mCry3A
appeared
to
be
uniformly
distributed
in
the
feed.
50
DATA
EVALUATION
RECORD
Primary
Reviewers:
Lewis,
E.
B.,
A.
Q.
Armstrong,
R.
H.
Ross,
L.
A.
Wilson
EPA
Secondary
Reviewers:
G.
S.
Tomimatsu,
Ph.
D,
Mika
J.
Hunter
STUDY
TYPE:
Earthworm
Subchronic
Toxicity
Test;
OECD
Guideline
Study
No.
207
MRID
NO:
462656011
DP
BARCODE:
DP303605
TEST
MATERIAL:
mCry3A­
0102
STUDY
NO:
Mambo­
Tox
Study/
Report
No.
SYN­
03­
55
Syngenta
Project
No.
2033037
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Mambo­
Tox
Ltd.,
Southampton,
UK
TITLE
OF
REPORT:
Determination
of
Acute
Toxicity
of
Modified
Cry3A
Protein
(
MCRY3A­
0102)
to
the
Earthworm
Eisenia
fetida
in
an
Artificial
Soil
Substrate
AUTHOR:
Vinall,
S.
STUDY
COMPLETED:
April
7,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
compliant
with
OECD
and
UK
GLP
regulations.

STUDY
SUMMARY:
In
a
laboratory
test,
earthworms
(
Eisenia
fetida)
in
artificial
soil
were
exposed
to
MCRY3A­
0102
at
a
nominal
concentration
of
370
Fg/
g
dry
soil
(
334
Fg
MCRY3A­
0102
protein/
g
dry
soil)
for
14
days.
A
negative
control
of
deionized
water
and
a
positive
control
of
10,
20,
30,
40,
or
50
mg
2­
chloroacetamide/
kg
of
dry
soil
were
also
used
in
the
test.
At
test
end,
MCRY3A­
0102
treated
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%.
Positive
control
worms
exposed
to
 
30
mg
2­
chloroacetamide/
kg
of
dry
soil
had
a
mortality
rate
of
100%.
CLASSIFICATION:
Acceptable
Test
Material
The
test
substance,
MCRY3A­
0102,
active
ingredient
modified
Cry3A
protein,
Batch
Number
MCRY3A­
0102,
was
supplied
by
the
sponsor
with
a
reported
purity
of
90.3%
w/
w
and
an
expiration
date
of
July
15,
2004.
The
test
substance,
MCRY3A­
0102,
was
purified
from
cells
of
recombinant
E.
coil.
The
test
substance
was
stored
at
­
30E
to
­
20EC
prior
to
use.
The
study
was
conducted
prior
to
the
test
substance
expiration
date.
The
presence
and
activity
of
mCry3A
in
the
artificial
soil
used
in
the
test
were
confirmed
in
a
separate
study
51
(
MRID
46265612).
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.

Test
Methods
Adult
earthworms
with
clitellum
(
Eisenia
fetida,
from
Blades
Biological,
Edenbridge,
Kent,
UK)
were
exposed
to
MCRY3A­
0102
in
artificial
soil
,
a
negative
control
(
deionized
water)
or
a
positive
control
(
2­
chloroacetamide)
for
14
days.
The
test
material
was
prepared
by
adding
184.6
mg
of
MCRYC3A­
0102
to127.5
mL
of
deionized
water
and
mixing
it
with
500
g
(
dry
wt)
of
artificial
soil,
for
a
concentration
of
370
Fg/
g
dry
soil
(
334
Fg
MCRY3A­
0102
protein/
g
dry
soil)
21;
as
clarified
in
a
detailed
report,
"
Characterization
of
Modified
Cry3A
Test
Substance
(
MCRY3A­
0102)
and
Certificate
of
Analysis
(
Joseph
and
Graser,
2003;
Syngenta
Seeds
Biotechnology
Report
No.
SSB­
025­
03).
The
negative
control
group
received
127.5
mL
of
deionized
water
in
500
g
of
dry
soil.
The
positive
control
was
a
rate
response
test
using
concentrations
equivalent
to
10,
20,
30,
40,
or
50
mg
of
2­
choroacetamide/
kg
of
dry
soil.
Worms
with
a
wet
weight
of
300
to
600
mg
each
was
used
for
testing
and
was
acclimated
for
about
24
hrs.

The
artificial
soil
in
each
test
chamber
was
composed
of
10%
peat,
20%
clay,
and
70%
sand
(
total
of
498.5
g)
with
1.5
g
CaCO3
added
to
adjust
the
pH
to
6.0
±
0.5.
During
the
test
the
soil
was
maintained
at
approximately
50%
water
holding
capacity.
The
test
chambers
were
1­
L
glass
jars
with
hinged
glass
lids.
At
study
start,
10
adult
earthworms
(
wet
weight
300
to
500
mg
each)
were
placed
on
the
surface
of
the
treated
soil
in
each
test
chamber
and
the
lids
were
fitted.
The
test
material
and
negative
control
treatments
were
replicated
4
times;
the
positive
control
treatments
were
replicated
3
times.
Mortality
was
assessed
at
days
7
and
14,
and
surviving
worms
in
the
test
material
and
negative
control
treatments
were
individually
weighed
at
test
end.
The
test
was
conducted
in
a
controlled
environment
of
18
to
20EC
and
57%
to
89%
relative
humidity,
with
constant
overhead
light
(
485­
569
lux;
ELE
Single
Channel
Light
Measuring
System,
Skye
Instruments
Ltd.,
Powys,
UK).
The
soil
pH
was
measured
at
study
start
and
end
(
Hydrus
300
pH
meter).

Frozen
samples
of
treated
soils
were
shipped
to
Syngenta
Biotechnology
(
SBI)
for
confirmation
of
the
presence/
absence
of
MCRY3A­
0102
protein.

Results
Summary
Mortality
(
based
on
motility
in
response
to
a
mechanical
stimulus)
and
mean
weight
loss
were
comparable
between
the
mCRY3A­
0102
and
negative
control
treatments
(
Table
1).
Day
7
mortality
in
the
positive
control
treatment
was
100%
for
all
groups
receiving
$
30
mg
1Based
on
the
sponsor's
determination
that
the
average
concentration
of
Event
MIR604
hybrids
was
~
0.25Fg/
g
dry
weight
roots;
and
the
extraction
efficiency
for
roots
was
75%
and
after
correcting
for
purity
(
90.3%).
52
2­
chloroacetamide/
kg
soil,
indicating
the
assay
was
appropriate.
The
14­
day
LC50
for
2­
chloroacetamide
was
calculated
by
probit
analysis
to
be
18
mg/
kg.

TABLE
1.
Mortality
and
weight
loss
of
E.
fetida
Cumulative
mortality
(%)
Treatment
Day
7
Day
14
14­
Day
Weight
loss
(%)

MCRY3A­
0102
2.5
5.0
5.8
Negative
control
0
0
11.4
Data
from
p.
16,
MRID
46265611
TABLE
2.
Cumulative
Percent
Mortality
of
Worms
for
Each
Rate
of
the
Toxic
Reference
at
the
7
and
14
DAT
Assessments
Concentration
of
2­
chloroacetamide
(
mg/
kg[
dry
weight]
or
artificial
soil
Assessment
Time
10
20
30
40
50
Estimate
of
LC50
a
7
DAT
0
60
100
100
100
­
14
DAT
3
60
100
100
100
18
mg/
kg
a
Probit
analysis
was
performed
on
14­
day
mortality
for
the
2­
chloroacetamide
treatment
rates
to
derive
the
LC50.
Prior
to
analysis,
the
dose
rates
were
log10­
transformed.

Range
of
pH
values
were
recorded
and
appeared
consistent
throughout
the
test:
pH
6.0
±
0.2
amongst
untreated
and
treated
soils.

Study
Author's
Conclusions
The
study
author
concluded
that
a
14­
day
exposure
of
earthworms
to
the
test
material
at
a
rate
equivalent
to
334
Fg
MCRY3A­
0102/
g
dry
soil
did
not
adversely
affect
earthworm
weight
or
survival.
53
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nonguideline
MRID
NO:
46265612
DP
BARCODE:
DP303605
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MCRY3A­
0102
STUDY
NO:
SSB­
008­
04
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
TESTING
FACILITY:
Syngenta
Biotechnology,
Inc.,
Research
Triangle
Park,
NC
TITLE
OF
REPORT:
Analysis
of
Artificial
Soil
Used
to
Expose
Eisenia
fetida
to
Modified
Cry3A
Protein:
Supplement
to
Report
Titled
"
Determination
of
Acute
Toxicity
of
Modified
Cry3A
Protein
(
MCRY3A­
0102)
to
the
Earthworm
Eisenia
fetida
in
an
Artificial
Soil
Substrate";
Mambo­
Tox
Study/
Report
No.
SYN­
03­
55
AUTHOR:
Graser,
G.

STUDY
COMPLETED:
April
23,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided.
The
study
was
US
EPA
GLP
compliant.
54
STUDY
SUMMARY:
Samples
of
artificial
soil
treated
with
MCRY3A­
0102
at
a
rate
nominally
equivalent
to
334
Fg
MCRY3A­
0102
protein/
g
dry
soil
and
used
in
an
earthworm
(
Eisenia
fetida)
toxicity
study
(
MRID
46265611)
were
analyzed
for
MCRY3A­
0102
protein
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA).
The
integrity
of
the
MCRY3A­
0102
in
the
treated
soil
was
determined
using
Western
blot
analysis,
and
the
bioactivity
of
MCRY3A­
0102
in
the
treated
soil
was
determined
in
a
bioassay
using
Colorado
potato
beetle
(
CPB)
larvae.
ELISA
of
the
treated
soil
recovered
about
18%
of
the
nominal
MCRY3A­
0102
soil
concentration
on
day
0.
Extraction
on
days
3,
7,
and
14
resulted
in
14.8%,
6.3%,
and
7.6%
of
the
nominal
concentration
mixed
in
the
soil.
The
author
attributed
the
low
recovery
to
the
physico­
chemical
characteristics
of
the
soil.
Western
blot
analysis
of
treated
soil
indicated
that
MCRY3A­
0102
was
present
in
the
soil
throughout
the
14­
day
study,
and
that
some
degradation
to
lower
molecular
weight
polypeptides
occurred,
beginning
at
day
3.
The
CPB
bioassay
confirmed
that
MCRY3A­
0102­
treated
soil
was
bioactive
at
all
sampling
times,
resulting
in
larvae
mortality
of
60%
and
83%
after
120
hours
for
the
CPB
diets
prepared
with
5%
and
10%
treated
soil.

CLASSIFICATION:
Acceptable
Test
Material
The
test
substance
was
artificial
soil
prepared
in
parallel
in
the
same
manner
and
maintained
under
the
same
conditions
as
soil
treated
with
MCRY3A­
0102
(
active
ingredient,
mCry3A
protein)
that
was
used
in
a
previous
non­
target
organism
toxicity
study
with
Eisenia
fetida
(
MRID
46265611).
For
that
study,
the
artificial
soil
was
treated
with
MCRY3A­
0102
at
a
nominal
rate
equivalent
to
334
Fg
MCRY3A­
0102
/
g
dry
weight.
Samples
of
the
treated
soil
were
collected
directly
after
preparation
(
day
0),
and
at
3,
7,
and
14
days
after
treatment.
A
sample
of
untreated
artificial
soil
from
the
same
batch
was
used
as
a
control.
All
samples
were
frozen
and
remained
frozen
until
analysis.

Test
Methods
The
test
substance
was
quantitatively
analyzed
for
MCRY3A­
0102
using
an
enzyme­
linked
immunosorbent
assay
(
ELISA),
and
the
integrity
of
the
MCRY3A­
0102
was
determined
using
Western
blot
analysis.
Standard
operating
procedures
were
referenced
in
the
report.
The
bioactivity
of
mCry3a
in
the
test
substance
was
determined
in
a
bioassay
using
Colorado
potato
beetle
(
CPB)
(
Leptinotarsa
decemlineata)
larvae.
55
For
the
ELISA
and
Western
blot
analysis,
10
mL
of
extraction
buffer
were
added
to
approximately
2
g
of
treated
or
control
soil.
Each
sample
was
mixed,
incubated
on
ice
for
one
hour,
centrifuged
for
15
minutes
at
2500
x
g,
and
the
supernatants
were
collected.
The
pellets
were
extracted
a
second
and
a
third
time
for
30
minutes
under
constant
rotation.
A
fourth
extraction
was
done
overnight
in
the
same
way.
All
four
extracts
for
each
sample
were
centrifuged
again
for
10
minutes
at
13,000
x
g
prior
to
analysis.
These
extracts
were
used
for
the
ELISA,
while
only
the
first
extract
of
each
sample
was
used
for
the
Western
blot
analysis.

For
the
ELISA,
the
extracts
were
analyzed
for
mCry3A
using
immumo­
affinity
purified
rabbit
anti­
mCry3A
polyclonal
antibodies
and
immuno­
affinity
purified
goat
anti­
Btt
(
native
Cry3A
from
Bacillus
thuringiensis
subsp.
tenebrionis)
polyclonal
antibodies.

For
the
Western
blot
analysis,
two
aliquots
of
the
treated
soil
extract,
containing
approximately
8.5
and
17
ng
MCRY3A­
0102
respectively
(
as
determined
by
ELISA),
were
subjected
to
SDS­
PAGE
using
an
8­
16%
polyacrylamide
gradient
gel.
Two
aliquots
of
a
solution
of
MCRY3A­
0102
(
positive
control),
containing
10
and
20
ng
of
MCRY3A­
0102,
respectively,
and
one
aliquot
of
the
control
soil
extract
(
negative
control)
were
run
concurrently
on
the
same
gel.
After
electroblotting,
the
membranes
were
probed
with
immuno­
affinity
purified
goat
anti­
Btt
(
native
Cry3A
from
Bacillus
thuringiensis
subsp.
tenebrionis)
polyclonal
antibodies.
Donkey
anti­
goat
IgG
linked
to
alkaline
phosphatase,
diluted
in
blocking
buffer,
was
used
to
bind
to
the
primary
antibody
and
visualized
by
development
with
alkaline
phosphatase
substrate
solution.
The
Western
blot
was
examined
for
the
presence
of
intact
immunoreactive
MCRY3A­
0102
(
ca.
67,000
molecular
weight)
and
immunoreactive
MCRY3A­
0102
polypeptides.

For
the
CPB
bioassay,
diets
containing
10%
and
5%
(
w/
w)
treated
artificial
soil
were
prepared
by
adding
4.50
and
4.75
g,
respectively,
of
stock
CPB
diet
to
0.50
and
0.25
g,
respectively,
of
the
MCRY3A­
0102
­
treated
soil.
Control
CPB
diet
was
prepared
in
the
same
manner
using
the
untreated
artificial
soil,
to
which
250
FL
of
deionized
water/
g
of
soil
was
added.
Positive
control
diets
were
also
prepared
by
adding
MCRY3A­
0102
to
stock
CPB
diet
at
concentrations
equivalent
to
12.5
and
50
Fg
MCRY3A­
0102
/
g.
As
a
control
for
the
positive
control
diet,
CPB
diet
was
prepared
using
the
same
volume
of
water
as
was
used
to
add
the
positive
control
solution
to
the
CPB
diet.
An
untreated
CPB
diet
control
was
also
included.
A
piece
of
the
supplemented
diets
(
5%
or
10%
control
soil
or
5%
or
10%
MCRY3A­
0102
­
treated
soil)
was
placed
in
a
Petri
dish
with
10
newly­
hatched
CPB
larvae.
Similarly,
groups
of
10
larvae
were
provided
with
stock
CPB
diet
(
untreated
control),
stock
CPB
diet
treated
directly
with
MCRY3A­
0102
(
positive
control),
or
stock
CPB
diet
treated
with
water
(
control
for
the
positive
control).
Each
treatment
was
replicated
3
times.
The
dishes
were
covered
and
maintained
at
ambient
conditions
for
120
hours.
Diets
were
replaced
every
24
hrs
with
fresh
diet
prepared
in
the
same
manner.
Mortality
was
assessed
daily.
56
Results
Summary
ELISA
of
the
treated
artificial
soil
extracts
showed
recoveries
of
18.1%,
14.8%,
6.3%,
and
7.6%
for
the
0­,
3­,
7­,
and
14­
day
samples,
respectively.
No
MCRY3A­
0102
was
detected
in
the
untreated
control
soil.

Western
blot
analysis
of
the
MCRY3A­
0102­
treated
artificial
soil
collected
at
all
sampling
times
showed
a
single
immunoreactive
band
corresponding
to
the
predicted
molecular
weight
of
mCry3A
(
ca.
67,700
Da).
Beginning
with
the
3­
day
samples,
one
or
more
bands
representing
MCRY3A­
0102
degradation
products
were
seen;
these
were
more
prominent
in
the
7­
and
14­
day
samples.
The
major
degradation
products
appeared
as
a
doublet
band
at
ca.
57,000
Da.
No
immunoreactive
material
was
detected
in
extracts
of
the
untreated
control
artificial
soil.

In
the
CPB
bioassay,
the
MCRY3A­
0102
­
treated
soil
showed
bioactivity
at
all
sampling
times,
producing
60­
83%
larval
mortality
after
120
hrs
for
the
diets
prepared
with
5%
and
10%
treated
soil.
The
positive
controls
produced
87%
and
97%
mortality.
The
negative
control
soils
produced
13
and
20%
mortality,
similar
to
the
control
CPB
diets
(
20%).

Study
Author's
Conclusions
The
study
author
concluded
that
active
MCRY3A­
0102
was
present
in
the
artificial
soil
used
in
the
Eisenia
fetida
toxicity
study.
While
only
about
18%
of
the
nominal
mCry3A
concentration
in
the
day
0
treated
soil
was
recovered,
the
study
author
attributed
the
low
recovery
to
the
physico­
chemical
properties
of
the
soil
matrix.
Western
blot
analysis
indicated
that
intact
MCRY3A­
0102
was
present
in
the
soil
throughout
the
study,
and
that
some
degradation
to
lower
molecular
weight
polypeptides
occurred
during
the
study.
Finally,
the
study
author
concluded
that
MCRY3A­
0102
­
treated
soil
samples
collected
at
days
0,
3,
7,
and
14
of
the
CPB
bioassay
were
highly
bioactive
against
CPB
larvae.
57
DATA
EVALUATION
RECORD
Primary
Reviewer:
Eric
B.
Lewis
EPA
Secondary
Reviewer:
Mika
J.
Hunter
STUDY
TYPE:
Nonguideline
MRID
NO:
46265615
DP
BARCODE:
303605
CASE
NO:
Not
provided
SUBMISSION
NO:
Not
provided
TEST
MATERIAL:
MIR604
(
Bacillus
thuringiensis
mCry3A
protein)

STUDY
NO:
MIR604
SPONSOR:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
27709
TESTING
FACILITY:
North
Carolina
State
University,
Chicken
Educational
Unit,
Raleigh,
NC
27603
TITLE
OF
REPORT:
Evaluation
of
Transgenic
Corn
(
Maize)
MIR604
in
Broiler
Chickens
AUTHOR:
Brake,
J.
T.

STUDY
COMPLETED:
April
14,
2004
CONFIDENTIALITY
CLAIMS:
None
GOOD
LABORATORY
PRACTICE:
A
signed
GLP
statement
was
provided
stating
the
study
was
not
GLP
compliant,
but
was
conducted
according
to
accepted
scientific
methods.
58
STUDY
SUMMARY:
Chickens
were
fed
formulated
diets
containing
either
transgenic
MIR604
corn
with
mCry3A
protein,
an
MIR604
isoline
non­
transgenic
corn,
or
a
non­
transgenic
NC
2003
commercial
corn
for
49
days.
The
concentration
of
mCry3A
protein
in
the
transgenic
MIR604
diets
was
reported
to
be
0.04
Fg/
g
dry
wt,
0.06
Fg/
g
dry
wt,
and
0.08
Fg/
g
dry
wt
in
the
starter,
grower,
and
finisher
diets,
respectively.
Chicken
mortality
in
all
treatments
was
comparable.
Mean
body
weight
and
feed
conversion
ratios
for
birds
in
the
NC
2003
treatment
were
significantly
lower
than
those
for
birds
in
either
MIR604
treatment
at
days
31
and
49,
probably
as
a
result
of
a
slightly
lower
protein
concentration
in
the
NC
2003
diet.
Overall
carcass
yield
was
similar
for
all
treatments,
although
thigh
weight
was
significantly
lower
in
the
NC
2003
treatment
males.

CLASSIFICATION:
Supplemental
Test
Material
The
test
substance
was
MIR604
Positive
(
maize
grain
containing
Bacillus
thuringiensis
mCry3A
protein),
supplied
by
the
sponsor
and
formulated
into
broiler
diet.
The
concentration
of
mCry3A
protein
in
MIR604
Positive
grain
was
reported
to
be
0.23
±
0.035
Fg/
d
dry
wt.
The
concentration
of
mCry3A
protein
in
the
formulated
diets
used
in
the
test
were
reported
to
be
0.04
±
0.005
Fg/
g
dry
wt
in
the
starter
diet,
0.06
±
0.005
Fg/
g
dry
wt
in
the
grower
diet,
and
0.08
±
0.013
Fg/
g
dry
wt
in
the
finisher
diet.

Test
Methods
Chickens
(
Gallus
domesticus,
Ross
344
males
and
Ross
308
females,
from
eggs
produced
and
incubated
in­
house)
were
fed
diets
containing
grain
from
a
transgenic
corn
hybrid
(
MIR604
Positive),
a
non­
transgenic
isoline
corn
hybrid
(
MIR604
Negative),
or
a
nontransgenic
locally­
grown
commercial
corn
(
NC
2003)
for
49
days.
Day­
old
chicks
were
randomly
assigned
to
the
treatments.
Each
pen
contained
25
birds
of
the
same
sex.
Each
treatment
group
contained
12
cages
of
birds,
6
cages
of
males
and
6
cages
of
females.
(
900
birds
total
in
the
study).
The
pens
were
4
ft
x
12
ft
x
8
ft
wood
frames
with
plastic­
coated
wire
mesh
and
were
maintained
in
a
curtain­
sided
house
that
was
heated
or
ventilated
as
necessary.
Illumination
was
from
incandescent
lighting
for
23
hrs/
day
for
days
1­
7,
21
hrs/
day
from
days
8­
21,
and
from
natural
daylight
thereafter.
The
house
was
checked
twice/
day
for
temperature
and
lighting,
and
the
birds
were
checked
twice/
day
for
clinical
signs
and
mortality.

Diets
formulated
with
the
different
corn
types
were
standardized
to
be
of
relatively
similar
nutrient
composition
(
nutrient
analyses
were
provided
in
the
report).
Starter,
grower,
and
finisher
diets
were
formulated
with
57.5%,
63.0%,
and
67.5%
corn,
respectively.
Starter
diet
59
was
provided
until
day
16,
at
which
time
grower
diet
was
added
to
any
remaining
starter
in
the
feeders
and
provided
until
day
31.
On
day
31,
any
remaining
grower
diet
was
discarded
and
finisher
diet
was
provided
until
16
hours
prior
to
slaughter
on
day
50.
Diet
and
well
water
were
provided
ad
libitum.
Pen
weights
were
collected
on
days
1,
16,
31,
and
49.
Feed
consumption
data/
pen
was
determined
on
days
16,
31,
and
49.
On
day
51
a
random
sample
of
2
birds/
pen
was
collected
and
processed
to
determine
carcass
yield.

Body
weight,
feed
conversion,
and
survival
data
were
analyzed
using
the
General
Linear
Model
procedure
by
SAS,
with
a
significance
level
of
5%.
These
variables
were
analyzed
for
the
starter
period
(
days
0­
16),
grower
period
(
days
16­
31),
finisher
period
(
days
31­
41),
and
a
cumulative
period
(
0­
49
days).

Results
Summary
There
were
no
significant
adverse
clinical
signs
noted,
and
no
statistically
significant
difference
in
mortality
was
found
among
any
of
the
treatments.
Mean
body
weight
and
feed
conversion
ratios
for
birds
in
the
MIR604
Positive
and
MIR604
Negative
treatments
were
comparable
(
Tables
1
and
2).
Mean
body
weight
and
feed
conversion
ratios
for
birds
in
the
NC2003
treatment
were
significantly
lower
than
those
for
birds
in
either
MIR604
treatment
at
days
31
and
49,
probably
as
a
result
of
a
slightly
lower
protein
concentration
(
6­
8%,
calculated
by
reviewer)
in
the
NC2003
grower
and
finisher
diets.
Overall
carcass
yields
for
males
and
females
of
all
treatments
were
comparable,
although
thigh
weights
of
males
in
both
MIR604
treatments
were
significantly
higher
than
those
of
NC2003
males
(
Table
3).
When
part
weights
were
considered
as
a
percent
of
live
weight,
there
were
some
significant
differences
among
the
treatments
for
thighs
in
females
and
pectoralis
minor
in
males.
However,
differences
between
the
MIR604
Positive
and
NC2003
treatments
were
not
significant
in
either
sex.

Table
1.
Mean
Body
Weight
and
Standard
Error
of
Each
Corn
Diet
and
Sex
Treatment
Body
weight
(
hatch)

Grams
Body
weight
(
day
16)

Grams
Body
weight
(
day
31)

Grams
Body
weight
(
day
49)

Grams
MIR604
Positive
44.60
±
0.19
547.8
±
9.6
1684.1
±
50.6a
3468.4
±
129.5a
MIR604
Negative
44.66
±
0.18
563.0
±
5.4
1690.5
±
40.2a
3469.4
±
113.1a
NC
2003
44.70
±
0.19
551.1
±
9.6
1634.0
±
44.3b
3365.2
±
117.6b
Male
44.70
±
0.17
574.0
±
5.07
A
1814.0
±
13.27
A
3825.9
±
26.65
A
Female
44.54
±
0.13
534.0
±
4.93
B
1525.1
±
9.51
B
3042.7
±
15.56
B
MIR604
Negative­
Male
44.76
±
0.17
576.4
±
6.38
1817.7
±
23.00
3831.3
±
58.73
MIR604
Negative­
Female
44.56
±
0.21
549.7
±
3.95
1563.2
±
10.72
3107.5
±
16.98
MIR604
Positive­
Male
44.53
±
0.33
573.4
±
10.42
1848.1
±
19.11
3895.5
±
22.93
60
MIR604
Positivefemale
44.67
±
0.21
522.3
±
6.24
1520.1
±
11.86
3041.3
±
34.15
NC
2003
 
Male
45.01
±
0.25
572.1
±
10.52
1776.2
±
19.55
3750.9
±
34.15
NC
2003
 
Female
44.40
±
0.25
530.0
±
10.66
1491.9
±
12.30
2979.5
±
13.34
a,
b
Means
with
different
superscripts
differ
significantly
(
p#
0.05)
A,
B
Means
that
possess
different
superscripts
differ
significantly
(
P
#
0.01)
Data
from
p.
23,
MRID
46265615
TABLE
2.
Mean
feed
conversion
ratio1
(
g:
g)
of
surviving
birds
Treatment
Starter
FCR
(
days
0­
16)
Grower
FCR
(
days
16­
31)
Finisher
FCR
(
days
31­
49)
Cumulative
FCR
(
days
0­
31)
Cumulative
FCR
(
days
0­
49)

MIR604
Positive
1.32
±
0.0
1.55
±
0.0b
2.00
±
.0
1.48
±
0.0b
1.75
±
0.0b
MIR604
Negative
1.31
±
0.0
1.56
±
0.0b
2.01
±
0.0
1.48
±
0.0b
1.75
±
0.0b
NC
2003
1.35
±
0.0
1.60
±
0.0a
2.05
±
0.0
1.52
±
0.0a
1.79
±
0.0a
1Total
feed
consumed/
total
body
weight
a,
bMeans
with
different
superscripts
differ
significantly
(
p#
0.05)
Data
from
p.
24,
MRID
46265615
61
TABLE
3.
Carcass
and
parts
yield
at
day
51
Males
Treatment
Dressed
carcass1
(
g)
Thighs
(
g)
Pectoralis
major
(
g)
Pectoralis
minor
(
g)

MIR604
Positive
2947.2
±
57.44
526.3
±
14.05a
638.8
±
19.18
150.8
±
4.63
MIR604
Negative
2936.7
±
56.88
484.3
±
15.10ab
609.6
±
16.73
145.7
±
4.57
NC
2003
2812.9
±
61.49
454.6
±
20.30b
605.1
±
23.09
151.3
±
4.97
Dressed
carcass2
(%)
Thighs
(%)
Pectoralis
major
(%)
Pectoralis
minor
(%)

MIR604
Positive
74.08
±
0.50
13.23
±
0.26
16.06
±
0.39
3.80
±
0.11ab
MIR604
Negative
74.97
±
0.27
12.37
±
0.34
15.57
±
0.33
3.71
±
0.07b
NC
2003
75.14
±
0.37
12.12
±
0.42
16.14
±
0.45
4.03
±
0.07a
Females
Dressed
carcass
(
g)
Thighs
(
g)
Pectoralis
major
(
g)
Pectoralis
minor
(
g)

MIR604
Positive
2248.3
±
45.53
380.8
±
7.6
515.4
±
18.9
126.2
±
3.3
MIR604
Negative
2291.0
±
41.4
361.9
±
9.2
511.8
±
13.8
128.0
±
3.5
NC
2003
2204.2
±
54.8
366.2
±
11.3
487.1
±
22.0
125.9
±
3.4
Dressed
carcass
(%)
Thighs
(%)
Pectoralis
major
(%)
Pectoralis
minor
(%)

MIR604
Positive
75.16
±
0.41
12.74
±
0.12a
17.22
±
0.48
4.23
±
0.11
MIR604
Negative
75.05
±
0.51
11.87
±
0.28b
16.76
±
0.35
4.19
±
0.10
NC
2003
75.27
±
0.34
12.51
±
0.25ab
16.59
±
0.53
4.30
±
0.06
1Fresh
carcass
without
head,
neck,
feet,
feathers,
viscera,
blood
2Expressed
as
percent
of
live
body
weight
a,
bMeans
with
different
superscripts
differ
significantly
(
p#
0.05)
Data
from
pp.
27
and
28,
MRID
46265615
Study
Author's
Conclusions
The
study
author
concluded
that
transgenic
MIR604
hybrid
diets
supported
broiler
chicken
growth
at
low
mortality
rates
and
very
good
feed
conversion
ratios
without
large
differences
in
overall
carcass
yield.
The
study
author
also
concluded
that
while
it
was
unclear
if
the
small
differences
seen
could
be
attributed
to
the
corn
source
per
se
or
to
slight
differences
in
the
formulated
diets,
the
transgenic
corn
clearly
had
no
deleterious
effects
in
this
study.
