Section
2.0
2
­
1
2
­
Health
Effects
2
­
Comment
Excerpts
Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
17
Excerpt
Text:
Human
Health
and
Food
Safety
­
Reviews
by
EPA
and
FDA
establish
that
corn
containing
Cry3Bb1
protein
is
safe
for
consumption
by
humans
and
animals.

­
The
Cry3
class
of
Bt
proteins,
including
Cry3Bbl
that
has
85%
amino
acid
similarity
to
Cry3A
in
Newleaf
potatoes,
has
an
established
history
of
safe
consumption
by
humans.
­
No
adverse
effects
were
observed
in
mice
following
exposure
to
a
maximum
achievable
acute
oral
dose
of
Cry3Bb1
protein,
a
dose
which
is
many
orders
of
magnitude
higher
than
upper
bound
estimates
for
human
dietary
exposure.
­
Exposure
to
Cry3Bbl
via
other
routes,
such
as
dermal
or
inhalation,
is
considered
by
EPA
to
be
unlikely
because
the
protein
is
contained
within
corn
plant
cells.
­
The
extremely
limited
potential
for
horizontal
gene
transfer
of
the
intact,
functional
antibiotic
resistance
gene
nptII
from
genetically
modified
corn
to
bacteria
poses
minimal
risk
to
the
environment
and
human
health.
­
A
10
base
pair
sequence
of
DNA
present
within
the
Cry3B1
and
nptII
transformation
cassette
was
lost
upon
stabile
integration
of
the
cassette
into
the
plant
genome
and
no
evidence
was
found
for
its
incorporation
elsewhere
in
the
genome.

EPA
Response:
EPA
agrees
with
these
comments
which
are
reflected
in
the
MON863
Biopesticide
Regulatory
Action
Document
(
BRAD)
in
section
IIA
and
IIB.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
22
Excerpt
Text:
Comment
or
Question:
There
is
a
lack
of
acute
toxicity
data
for
corn
event
MON
863,
through
dermal,
inhalation
and
dietary
routes
of
exposure.

Scientific
Background:
Cry3Bbl
protein
resides
within
plant
tissues,
thus
limiting
dermal
and
inhalation
routes
of
exposure.
The
EPA
has
previously
stated
for
other
Cry
proteins
produced
in
corn
that
"...
exposure
via
the
skin
or
inhalation
is
not
likely
since
the
CrylAb
and
Cry1F
proteins
are
contained
within
corn
plant
cells
which
essentially
eliminates
or
reduces
exposure
routes
to
negligible."
[
Footnote
23:
EPA
Biopesticides
Registration
Action
Document,
Bt
Plant
Incorporated
Protectants,
Oct
15,
2001.]
There
is'
no
evidence
to
suggest
that
Cry
proteins
should
have
a
greater
capacity
for
contact
or
respiratory
Section
2.0
2
­
2
sensitization
than
any
other
protein
found
in
corn
or
other
food
crops.

EPA
Response:
EPA
agrees
with
these
comments
which
are
reflected
in
the
MON863
Biopesticide
Regulatory
Action
Document
(
BRAD)
in
section
IIA
and
IIB.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
23
Excerpt
Text:
On
the
other
hand,
there
is
the
potential
for
dietary
exposure
to
low
levels
of
Cry3Bb
I
protein
from
consumption
of
corn
­
rain
and
processed
corn
food
products;
consequently,
the
safety
of
Cry3Bb1
protein
has
been
assessed
via
a
high
dose
acute
gavage
study
in
rodents.
There
was
no
evidence
of
toxicity
in
mice
dosed
with
up
to
3,200
mg/
kg
Cry3Bbl
protein,
the
highest
dose
achievable.
This
exposure
level
is
more
than
five
orders
of
magnitude
higher
than
upper
bound
estimates
for
human
dietary
consumption.
Similar
results
'
were
obtained
for
the
closely
related
Cry3A
protein;
no
toxicity
was
observed
in
mice
dosed
with
up
to
5,200
mg/
kg,
the
highest
dose
tested.

EPA
Response:
EPA
agrees
with
these
comments
which
are
reflected
in
the
MON863
Biopesticide
Regulatory
Action
Document
(
BRAD)
in
section
IIA
and
IIB.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
106000
Excerpt
Number:
13
Excerpt
Text:
Human
Health
and
Food
Safety
­
Monsanto
has
utilized
current,
appropriate
and
scientifically
robust
protocols
for
the
simulated
gastric
digestion
and
sequence
matching
tests.

EPA
Response:
EPA
agrees
with
these
comments
which
are
reflected
in
the
MON863
Biopesticide
Regulatory
Action
Document
(
BRAD)
in
section
IIA
and
IIB.
Section
2.1
2
­
3
2.1
­
Human
Health
2.1
­
Comment
Excerpts
Commenter
Name:
Berg,
Mark
Commenter
Organization
Name:
Foundation
E.
A.
R.
T.
H.
Comment
Number:
30509­
078000
Excerpt
Number:
5
Excerpt
Text:

Based
on
the
1999
Doane
information
provided
by
Foundation
E.
A.
R.
T.
H.,
corn
rootworm
insecticide
users
in
the
ECI
and
WCB
grew
17.4
million
acres
of
corn
in
1999.
Almost
60%
of
those
acres
were
treated
with
an
insecticide
to
control
corn
rootworm.
The
results
of
this
research
suggest
that
the
introduction
of
CRW
corn
may
reduce
the
acres
treated
by
44%
,
representing
4.6
million
acres
in
the
first
year.
Since
the
current
average
use
rate
of
corn
rootworm
is
0.5
pounds
of
active
ingredient
per
acre,
this
would
result
in
a
reduction
of
2.4
million
pounds
of
soil­
applied
insecticide
active
ingredient.

Commenter
Name:
Berg,
Mark
Commenter
Organization
Name:
Foundation
E.
A.
R.
T.
H.
Comment
Number:
30509­
078000
Excerpt
Number:
6
Excerpt
Text:

­
It
is
apparent
that
these
producers
have
concerns
with
usage
of
insecticides.
While
12%
have
personally
become
ill
due
to
exposure
to
insecticides,
20%
claim
that
a
family
member
or
other
associate
has
such
an
experience.

­
By
indicating
either
a
8,
9,
or
10,
exposure
to
the
insecticide
when
filling
insecticide
boxes
(
76%)
and
exposure
of
other
family
members
to
insecticides
(
72%)
surfaced
as
the
primary
concerns
of
growers
with
current
soil
applied
insecticides
­
as
they
are
well
aware
of
the
potential
health
hazards
associated
with
these
chemicals.
About
half
are
extremely
concerned
about
the
impact
of
insecticide
on
the
environment
(
53%)
and
exposure
to
livestock
(
47%).

EPA
Response:
EPA
agrees
with
the
commenter
that
the
MON863
corn
PIP
should
alleviate
some
of
the
concerns
of
producers
who
worry
about
the
effects
of
the
soil
insecticides
typically
employed
to
control
corn
rootworm.
This
should
especially
be
the
case
for
exposure
of
producers
to
the
soil
insecticides
whose
use
may
be
reduced
with
this
particular
PIP.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
11
Excerpt
Text:
Section
2.1
2
­
4
Human
health
assessment
We
also
noted
that
for
the
human
safety
assessment
much
of
the
safety
conclusions
are
either
based
on
human
safety
trials
conducted
with
various
old
microbial
Bt
products
conducted
prior
to
1989
or
simply
on
lack
of
data
and
evidence.

'
The
extremely
low
mammalian
toxicity
of
Bt­
based
insecticide
products
has
been
demonstrated
in
numerous
safety
studies
conducted
prior
to
1989.'
(
Monsanto
Document,
page
21
of
56)

EPA
Response:
EPA
disagrees
with
the
statement
by
Greenpeace
that
the
human
safety
assessment
for
the
MON863
corn
PIP
is
based
on
"
various
old
microbial
Bt
products."
The
safety
assessment
for
MON
863
is
based
on
the
data
submitted
by
the
company
specifically
utilizing
the
proteins
expressed
in
MON863
corn.
The
tests
and
their
results
are
found
in
the
appropriate
section
(
IIB)
of
the
Biopesticide
Registration
Action
Document
(
BRAD).
The
tests
are
intended
to
answer
questions
regarding
the
potential
of
the
MON863
PIP
proteins
to
display
dietary
toxicity
or
potential
allergenicity.
These
tests
are
based
in
part
on
the
current
guidelines
for
microbial
pesticides
and
input
from
Scientific
Advisory
Panels
convened
to
address
issues
of
protein
toxicity
and
allergenicity.
While
information
about
the
known
previous
exposure
to
the
proteins
expressed
in
MON863
corn
may
be
considered
as
part
of
the
safety
assessment,
the
basis
of
the
assessment
continues
to
be
the
specific
test
results
and
information
submitted
by
the
company.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
12
Excerpt
Text:
'
The
conclusion
of
reasonable
certainty
of
no
harm
and
the
resultant
tolerance
exemptions
for
this
wide
array
of
Bt.
mixtures
and
Cry
proteins
in
food
or
feed
were
based
on
the
lack
of
adverse
effects
to
mammals
in
numerous
toxicological
studies.
This
conclusion
is
supported
by
a
history
of
safe
use
in
agriculture
for
over
40
years
..'
(
Monsanto
Document,
page
21
of
56)

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
33
Excerpt
Text:
There
is
little
known
about
the
degree
of
similarity
or
differences
between
Cry3Bb
proteins
and
other
Cry
proteins
in
terms
of
the
health
effects
of
greatest
concern,
which
would
include
allergenicity
and
digestive
system
impacts.

EPA
Response:
EPA
disagrees
with
the
statement
of
the
Union
of
Concerned
Scientists
that
little
is
known
about
the
Cry3Bb1
protein's
health
effects
in
terms
of
allergenicity
and
digestive
system
impacts.
The
test
results
that
have
been
submitted
for
three
variants
of
the
Cry3Bb1
protein
expressed
in
MON863
all
have
negative
results
for
expected
dietary
toxicity
and
potential
allergenicity.
The
results
are
found
in
section
II.
B.
1.
of
the
BRAD.
Section
2.1
2
­
5
Commenter
Name:
K
Kreed
Commenter
Organization
Name:
K
Kreed
Comment
Number:
30509B­
035000
Excerpt
Number:
2
Excerpt
Text:
I
have
serious
concerns,
as
the
mother
of
a
child
with
food
allergies,
regarding
the
effect
of
these
foods
on
allergic
individuals.
There
seems
to
be
an
increase
in
the
number
of
children
with
severe
food
and
respiratory
allergies
(
and
the
two
are
inter­
related
as
eating
allergenic
foods
can
cause
severe
respiratory
problems)
and
many
are
concerned
that
our
food
supply
is
contributing.
I
did
not
have,
or
was
not
aware
that
I
had
any
food
allergies,
until
recently
when
I
had
a
severe
reaction,
apparently
to
wheat
gluten.
It
is
extremely
scary
when
you
or
especially
your
child,
has
a
severe
reaction
to
a
substance.
Do
we
know
for
sure
what
effects
splicing
genes
from
different
species
will
have
on
allergies?
What
labeling
will
be
required
for
these
products?
I
am
concerned
for
example
that
one
who
is
allergic
to
tomatoes
may
eat
corn
that
has
been
genetically
engineered
using
tomato
genes
and
have
a
reaction.

Commenter
Name:
K
Kreed
Commenter
Organization
Name:
K
Kreed
Comment
Number:
30509B­
035000
Excerpt
Number:
3
Excerpt
Text:
As
far
as
suggested
research,
I
would
like
to
state
that
allergies
may
take
several
years
to
develop
to
a
substance.
Many
people
who
acquire
pets
have
no
allergy
to
the
animals
until
they
have
had
them
for
several
years,
then
severe
allergies
can
"
suddenly"
develop.
Therefore
short
duration
tests
to
determine
whether
people
can
tolerate
genetically
foods
may
not
be
accurate.
Corn
is
a
staple
food,
difficult
to
avoid
in
our
diets,
so
I
would
urge
you
to
move
slowly
in
approving
substances
affecting
the
entire
population.

EPA
Response:
EPA
sympathizes
with
the
commenter
who
has
a
child
with
a
food
allergy
and
suffers
herself
with
gluten
sensitivity.
One
of
the
difficulties
with
food
allergy
is
that
it
may
appear
over
time
and
there
is
no
effective
way
to
predict
when
or
if
one
will
develop
an
allergic
sensitivity
to
a
normally
tolerated
food
protein.
As
the
commenter
indicates
allergies
develop
over
time
by
a
process
of
sensitization
then
appear
suddenly.
The
sensitization
process
is
probably
the
least
understood
of
the
multiple
steps
involved
in
allergenicity.
Unlike
other
areas
of
concern
for
pesticide
dietary
exposure,
food
allergy
does
not
have
a
validated
animal
model
test
system
that
can
predict
a
protein's
allergenic
potential.
However,
EPA
believes
that
utilizing
the
best
scientific
methods
currently
available,
the
PIP
proteins
expressed
in
MON863
corn
have
a
reasonable
certainty
of
causing
no
harm
including
dietary
toxicity
and
potential
allergenicity.
The
basis
of
the
assessment
are
the
specific
test
results
and
information
submitted
by
the
company
about
the
PIP
proteins
found
in
MON863.
The
results
are
found
in
section
II.
B.
1.
IIB
of
the
BRAD
and
suggest
that
the
PIP
proteins
do
not
present
a
potential
allergenicity
concern.

Commenter
Name:
K
Kreed
Commenter
Organization
Name:
K
Kreed
Comment
Number:
30509B­
035000
Excerpt
Number:
4
Section
2.1
2
­
6
Excerpt
Text:
I
would
also
urge
requiring
labeling
of
ALL
products
utilizing
genetic
engineering,
so
that
the
millions
of
people
with
allergies
can
avoid
these
products
if
they
wish.

EPA
Response:
EPA
is
responsible
for
the
safety
assessment
of
the
PIP
protein
expressed
in
the
MON
863
corn
product.
EPA
does
not
have
regulatory
authority
to
require
labeling
of
food
products.
Regulatory
authority
for
food
labeling
rests
with
the
Food
and
Drug
Administration
(
FDA).
Currently,
FDA
has
determined
that
it
is
not
necessary
to
label
foods
containing
GMO
constituents,
http://
www.
cfsan.
fda.
gov/~
dms/
biolabgu.
html.

Commenter
Name:
Paul
Mitchell
Commenter
Organization
Name:
Texas
A&
M
University
­
Department
of
Agriculture
Economics
Comment
Number:
30509B­
082000
Excerpt
Number:
5
Excerpt
Text:
Lastly,
I
believe
that
the
risks
associated
with
the
registration
and
ensuing
(
widespread)
adoption
of
corn
event
MON
863
are
small
compared
to
the
costs
of
not
doing
so.
Experience
with
the
various
corn
Bt
events
for
corn
borer
control
have
been
acceptable.
No
environmental
or
human
health
problems
have
been
identified
at
this
time,
though
many
have
been
looking
for
such
problems.
No
human
allergic
reactions
have
been
identified
or
other
adverse
human
or
animal
health
impacts.

EPA
Response:
EPA
agrees
with
the
commenter
as
to
the
current
experience
with
corn
borer
PIP
products.
EPA
agrees
with
these
comments
which
are
reflected
in
the
MON863
Biopesticide
Regulatory
Action
Document
(
BRAD)
in
section
IIA
and
IIB.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
7
Excerpt
Text:
Comment
or
Question:
Dietary
exposure
of
Cry3Bb1
in
corn
event
MON
863
exceeds
that
of
other
food
crops
treated
with
microbial
products
and
may
pose
a
greater
risk
of
allergenic
and
digestive
system
impacts.

Scientific
Background:
Cry3Bb1
has
been
evaluated
using
the
best
available
hazard
assessment
methodology.
The
Cry3Bb1
gene
was
obtained
from
a
microbial
source
that
has
no
history
of
allergenicity.
The
Cry3Bb1
protein
lacks
sequence
similarity
to
known
allergens,
it
is
rapidly
digested
using
recommended
assays,
and
is
found
at
levels
that
are
orders
of
magnitude
lower
than
levels
of
allergens
commonly
found
in
food.
The
EPA
has
granted
a
tolerance
exemption
for
the
production
of
Cry3Bb
I
protein
in
corn
plants
(
40
CFR
180.1214).
Consequently,
any
residues
of
Cry3Bb1
protein
that
may
be
present
in
corn
grain
are
considered
safe
for
human
consumption.
Also,
a
recently
completed
consultation
with
FDA
established
that
corn
varieties
containing
event
MON
863
are
not
materially
different
in
composition,
safety
and
other
relevant
parameters
from
corn
currently
on
the
market.
Section
2.1
2
­
7
EPA
has
granted
tolerance
exemptions
for
other
Cry
proteins
that
either
may
be
introduced
into
food
crops
through
genetic
engineering,
or
applied
topically
to
plant
surfaces
as
a
component
of
Bt
microbial
pesticide
formulations.
EPA
completed
a
comprehensive
review
of
safety
studies
carried
out
on
Bt
microbial
formulations
that
have
been
registered
for
several
decades.
Based
on
their
review
of
the
data,
EPA
concluded
that
"...
toxicology
studies
submitted
to
the
U.
S.
Environmental
Protection
Agency
to
support
the
registration
of
B
thuringiensis
subspecies
have
failed
to
show
any
significant
adverse
effects
in
body
weight
gain,
clinical
observations
or
upon
necropsy."
[
Footnote
20:
McClintock,
J.
T.,
C.
R.
Schaffer
and
R.
D.
Sjoblad
(
1995).
A
comparative
review
of
the
mammalian
toxicity
of
Bacillus
thuringiensis
­
based
pesticides.
Pestic.
Sci.
45:
95­
105.]
As
stated
in
a
World
Health
Organization
International
Program
on
Chemical
Safety
Environmental
Health
Criteria
monograph
on
Bt,
"...
owing
to
their
specific
mode
of
action,
Bt
products
are
unlikely
to
pose
any
hazard
to
humans
or
other
vertebrates..."
and
"
Bt
has
not
been
documented
to
cause
ally
adverse
effects
on
human
health
when
present
in
drinking
water
or
food."
[
Footnote
21:
WHO
(
2000).
International
Program
on
Chemical
Safety
(
IPCS)
Environmental
Health
Criteria
217:
Bacillus
thuringiensis.
http://
www.
who.
int/
pcs/
docs/
ehc­
217.
html]

Comment
or
Question:
Human
exposure
to
Cry3Bb
has
been
limited
since
no
foliar
Cry3Bb
product
has
achieved
significant
market
share
in
any
crop
and
breakdown
in
sunlight
quickly
removes
residues
before
products
reach
customers.
Furthermore,
human
exposure
to
the
Cry3A
protein
expressed
in
NewLeaf
potatoes
was
limited
due
to
its
small
market
share.

Scientific
Background:
There
is
a
history
of
safe
consumption
for
Cry3A
protein
and
other
related
Cry3
class
proteins,
stemming
from
the
safe
use
and
consumption
of
NewLeaf
potatoes.
Approximately
71,000
acres
of
NewLeaf
varieties
were
planted
in
North
America
while
the
product
was
on
the
market.
Thus,
there
has
been
a
history
of
safe
consumption
for
the
Cry3A
protein
from
its
consumption
as
a
component
of
NewLeaf
potatoes.
The
Cry3A
protein
is
functionally
and
structurally
related
(
85%
amino
acid
sequence
homology)
to
Cry3Bb
protein.
[
Footnote
22:
Galitsky
et
al.
(
2001).
Acta
Crystallographica
D57:
1101­
1109.]
Assuming
a
mean
concentration
of
1
ug/
gram
of
Cry3A
protein
in
potato
tubers,
and
a
lack
of
degradation
of
Cry3A
protein
during
processing
of
the
NewLeaf
potato
into
human
food,
the
approximate
Cry3A
protein
consumption
is
150
ug/
day
following
consumption
of
a
150
gram
potato.
While
this
may
be
a
conservative
estimate
of
exposure,
it
is
a
reasonable
basis
for
establishing
a
history
of
safe
consumption
for
Cry3
proteins
of
which
the
Cry3Bb1
protein
is
a
member.

Commenter
Name:
ICGA
Commenter
Organization
Name:
ICGA
Comment
Number:
30509B­
093000
Excerpt
Number:
7
Excerpt
Text:
In
addition
to
benefits
for
agriculture,
there
are
benefits
for
the
consumer
as
well.
A
recent
report
from
the
American
Phytopathological
Society
(
APS)
outlines
the
benefits
of
Bt
corn
to
both
human
health
and
the
environment.
According
to
Gary
Munkvold,
plant
pathologist
at
Iowa
State
University
and
an
APS
member,
the
innovative
Bt
technology
has
a
distinct
health
benefit
of
discouraging
the
buildup
of
mycotoxins
in
corn.
Mycotoxin
build­
up
is
directly
related
to
certain
fungal
plant
diseases,
which
can
be
increased
by
insect
damage
in
crops.
Some
of
these
toxins,
such
as
furnonisin,
can
be
fatal
to
horses
and
pigs
and
are
probable
human
carcinogens.
Lower
mycotoxin
concentrations
clearly
represent
a
benefit
to
the
public..
Section
2.1
2
­
8
These
studies
conducted
by
Dr
Munkvold
looked
at
Bt
to
control
the
European
corn
borer.
However,
corn
rootworm
puts
similar
stress
on
the
corn
plant,
making
it
more
susceptible
to
invasion
of
molds
and
mycotoxins.
We
expect
that
rootworm.
Bt
would
also
reduce
the
mycotoxins
in
corn.

EPA
Response:
EPA
does
not
agree
with
the
commenter
that
MON863'
s
corn
rootworm
control
can
be
expected
to
reduce
mycotoxins.
While
there
is
good
evidence
that
the
PIPs
in
corn
that
control
the
European
corn
borer
have
been
responsible
for
reducing
fumonisin
levels
in
corn,
similar
reductions
of
other
mycotoxins
such
as
aflatoxin
in
corn
have
not
been
realized.
It
could
reasonably
be
expected
that
if
insect
damage
was
the
only
means
of
entry
for
these
fungi,
similar
reductions
in
all
mycotoxins
could
be
seen
not
only
in
corn
but
also
cotton.
It
is
always
hoped
that
reduced
colonization
of
mycotoxin
producing
fungi
will
result
with
corn
rootworm
control
but
no
claims
for
such
an
effect
have
been
made
by
the
company
nor
has
data
been
presented
to
indicate
mycotoxin
reductions.
EPA
expects
that
this
will
be
an
area
of
active
research
once
commercial
release
and
cultivation
has
occurred.
However,
the
reduction
of
mycotoxin
levels
has
not
been
a
consideration
for
the
safety
review
of
MON863
corn.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
106000
Excerpt
Number:
8
Excerpt
Text:
Comment
or
Question:
Simulated
gastric
digestion
and
sequence
comparison
studies
to
determine
the
potential
allergenicity
or
toxicity
of
Cry3Bb1
should
be
repeated
using
currently
recommended
protocols.

Scientific
Background:
Monsanto
has
utilized
current,
appropriate
and
scientifically
robust
protocols
for
the
simulated
gastric
digestion
and
sequence
matching
studies.

As
a
matter
of
validation,
the
vast
majority
of
pepsin
digestion
assays
(
i.
e.,
the
current
database
of
information)
have
been
performed
at
pH
1.2
as
outlined
in
the
U.
S.
Pharmacopeia.
[
Footnote
10:
The
United
States
Pharmacopeia
23,
The
National
Formulary
18,
Rockville,
MD:
United
States
Pharmacopial
Convention
Inc,
1995:
2053.]
It
was
deemed
prudent
to
perform
this
assay
at
pH
1.2
to
facilitate
a
direct
comparison
to
data
generated
under
the
exact
same
conditions
for
previously
tested
allergens
and
nonallergens.
[
Footnote
11:
Astwood,
J.
D.,
Leach,
J
and
Fuchs,
R.
L.
1996.
Stability
of
food
allergens
to
digestion
in
vitro.
Nature
Biotechnology
14:
1269­
73]
[
Footnote
12:
de
Val,
G.,
Boihom,
C.
Y.,
Lozano,
R.
M.,
Buchanan,
B.
B.,
Ermel,
R.
W.,
Lee,
Y­
M.,
and
Frick,
O.
L.
1999.
Thioredoxon
treatment
increases
digestibility
and
lowers
allerenicity
of
milk.
J.
of
Allergy
and
Clinical
Immunology
103:
690­
7]

Regarding
sequence
matching
tests,
an
evaluation
of
a
protein
for
clinical
cross­
reactivity
is
best
accomplished
using
a
FASTA
[
Footnote
13:
Algorithm
used
to
find
local
high
scoring
alignments
between
a
pair
of
protein
or
nucleotide
sequences.]
sequence
comparison
that
has
been
shown
to
be
the
most
predictive
of
a
potential
allergen.
[
Footnote
14:
Hileman,
R.,
Silvanovich,
A.,
Hoodman,
R.,
Rice,
R.,
Holleschak,
G.,
Astwood,
J.,
and
Hefle,
S.
2002.
Bioinformatic
methods
for
allergenicity
assessment
using
a
comprehensive
allergen
database.
International
Archives
of
Allergy
and
Immunolology
(
In
Press/
August)]
An
additional
search
for
matches
of
eight
amino
acids
provides
a
margin
of
safety
when
assessing
the
potential
allergenicity
of
a
protein,
but
a
six
amino
acid
search
window
produces
many
Section
2.1
2
­
9
random,
irrelevant
matches.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
3
Excerpt
Text:
1.
Monsanto
has
not
evaluated
potential
allergenicity
or
toxicity
of
Cry3Bb1
with
the
most
appropriate
methods
for
determining
digestive
stability.
The
protocols
used
to
determine
in
vitro
gastric
stability
of
CryBb1
were
not
the
protocols
international
experts
agree
are
most
useful.
Monsanto
should
repeat
gastric
stability
tests
at
pH
2.0,
and
follow
other
guidelines
elaborated
by
Food
and
Agriculture
Organization/
World
Health
Organization
(
FAO/
WHO)
of
the
United
Nations
conducting
those
tests
(
1).

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
14
Excerpt
Text:
1.
Human
Dietary
Safety
Several
safety
tests
performed
by
Monsanto
did
not
use
protocols
that
could
best
assure
detection
of
potential
allergenicity
or
toxicity.
In
particular,
Monsanto
incorrectly
performed
in
vitro
simulated
gastric
digestion
tests
(
hereafter
"
gastric
stability")
and
sequence
comparisons
of
Cry3Bb1
with
allergens
and
toxins.
EPA
should
require
Monsanto
to
repeat
the
in
vitro
digestibility.
and
sequence
matching
tests
using
currently
recommended
protocols.

A.
In
Vitro
Digestibility
Studies
Simulated
gastric
stability
is
important
because
stability
is
a
characteristic
common
to
many
food
allergens
and
anti­
nutrients,
especially
the
most
prevalent
and
serious
food
allergens.
By
contrast,
lack
of
stability
means
that
intact
protein
is
not
exposed
to
the
intestines
where
it
can
be
absorbed
and
act
systemically.
Therefore,
gastric
stability
helps
predict
the
likelihood
of
allergenicity
or
toxicity.
While
Monsanto's
tests
show
that
the
bacterially
produced
MON
863
does
not
possess
gastric
stability,
those
tests
were
not
performed
with
the
most
reliable
protocols.
Monsanto
notes
that
its
gastric
stability
study
was
performed
using
the
protocols
described
by
Astwood
et
al.
in
1996
(
2).
In
2001,
however,
the
FAO/
WHO
convened
an
international
conference
of
experts
to
reconsider
genetically
engineered
food
allergy
assessment,
including
gastric
stability
assays
(
1).
That
conference
recommended
that
pH
2.0
be
used
rather
than
pH
1.2
used
in
the
Astwood
et
al.
protocols
(
2),
since
the
former
is
closer
to
the
pH
of
the
stomach
when
it
contains
food,
while
the
latter
is
more
indicative
of
"
fasting"
pH.
That
difference
in
pH
can
be
critical
in
determining
gastric
stability.
Studies
with
previous
Cry
proteins
have
found
different
stabilities
associated
with
differences
in
pH
(
3,4,5).

While
several
aspects
of
the
FAO/
WHO
gastric
stability
protocols
have
not
been
validated
against
known
food
allergens
or
non­
allergens,
the
rationale
for
the
pH
change
is
sound.
Therefore,
EPA
should
require
Section
2.1
2
­
10
Monsanto
to
repeat
the
gastric
stability
assay
using
pH
2.0
and
should
also
consider
requiring
the
use
of
other
aspects
of
the
updated
FAO/
WHO
protocols.

EPA
Response:
EPA
disagrees
with
CSPI
that
the
company
failed
to
utilize
the
appropriate
protocols
that
would
best
assure
detection
of
potential
allergencity
or
toxicity.
The
basis
of
the
toxicity
assessment
are
the
acute
oral
toxicity
test
in
a
rodent
species
done
with
a
high
dose
of
purified
protein
and
a
full
length
amino
acid
homology
comparison
of
the
introduced
proteins
with
known
protein
toxins.
For
the
allergenicity
assessment,
a
number
of
biochemical
tests
are
utilized:
two
amino
acid
homology
comparisons
for
the
entire
sequence
and
also
a
stepwise
series
of
8
amino
acid
segments,
a
heat
stability
test,
a
test
for
protein
stability
to
digestive
enzymes
and
an
examination
of
the
plant
expressed
protein
for
potential
posttranslational
modifications.
These
results
are
discussed
in
sections
IIA
and
IIB
of
the
BRAD
and
EPA
still
believes
these
data
demonstrate
a
reasonable
certainty
that
the
aggregate
exposure
of
the
PIP
proteins
will
cause
no
harm.

The
specific
comment
by
CSPI
suggests
that
the
FAO/
WHO
conference
held
in
Rome,
Italy
February
2001
is
the
definitive
guidance
on
the
method
for
assessing
allergenicity
of
a
novel
protein.
In
general,
CODEX
is
considered
the
body
that
sets
international
safety
standards
and
a
subsequent
meeting
of
an
Ad
hoc
CODEX
meeting
of
food
allergy
experts
held
in
Vancouver,
Canada,
September
2001
considered
the
FAO/
WHO
recommendations.
The
outcome
of
that
CODEX
meeting
was
that,
while
the
FAO/
WHO
recommendations
should
be
considered
in
a
safety
assessment,
there
were
still
questions
relating
to
what
appropriate
and
valid
methods
were
to
be
used
for
both
amino
acid
sequence
homology
and
pepsin
resistance
(
in
vitro
digestibility).
The
ambiguity
of
the
CODEX
guidance
and
the
demonstrated
false
positive
correlations
of
corn
proteins
with
allergens
arising
with
the
use
of
shorter
than
eight
amino
acid
segments
(
Hileman
et
al.,
2002)
suggest
that
it
is
still
reasonable
to
utilize
the
8
amino
acid
segment
stepwise
analysis
until
further
refinement
of
the
homology
assessment
with
shorter
segments
is
obtained.

The
pH
of
the
pepsin
resistance
(
in
vitro
digestibility)
assay
is
another
question
raised
by
CSPI.
The
commenter
suggests
that
the
pH
utilized
in
the
assay
should
be
2.0
rather
than
1.2
to
better
mimic
the
pH
of
the
stomach
after
ingestion
of
a
meal.
EPA
has
stressed
that
the
pepsin
resistance
assay
is
a
test
for
a
biochemical
characteristic,
not
intended
to
mimic
a
physiological
process
like
gastric
digestion
or
imply
anything
about
toxicity.
While
further
information
from
the
scientific
community
are
expected
to
clarify
the
range
of
conditions
affecting
the
assay
such
as
pH,
enzyme
and
substrate
concentrations,
EPA
believes
the
current
information
is
still
useful
for
making
a
safety
determination.
Section
2.2
2
­
11
2.2
­
Molecular
Biology/
Product
Characterization
2.2
­
Comment
Excerpts
Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
1
Excerpt
Text:
­
Molecular
description
of
expressed
Bt
toxin
and
its
mode
of
action
Although
not
explicitly
stated
in
the
document,
probably
the
74
kDa
size
Cry3B
protein
is
expressed
in
the
transgenic
plants.
This
is
the
natural
size
of
the
protein
in
which
it
is
active.
Hence,
unlike
for
Cry1,
no
inactive
large
protoxin
forms
exist
and,
therefore,
microbial­
derived
proteins
are
at
least
in
a
comparable
biochemical
state
as
plant­
produced
proteins.

EPA
Response:
EPA
agrees
with
this
comment
that
the
Cry3Bb1
protein
is
expressed
as
a
74kD
toxin
and
that
unlike
the
Cry1
family,
activity
is
not
dependent
on
removal
of
a
large
portion
of
a
protoxin
molecule.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
8
Excerpt
Text:
The
MON
863
event
was
created
using
gene
gun
technology
with
a
4,691
base
pair
ZMIR13Lconstruct.
This
construct
incorporates
the
NPTII
neomycin
phosphotransferase
II
marker
gene
directly
adjacent
to
the
cauliflower
mosaic
virus
35S
promoter.
Many
questions
have
been
raised
about
the
wisdom
of
using
antibiotic­
resistance
marker
genes
in
the
development
of
GMO
crop
cultivars.
There
is
a
global
consensus
that
use
of
such
selectable
marker
genes
may
be
risky,
are
no
longer
necessary,
and
hence
should
be
phased
out.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
9
Excerpt
Text:
While
concerns
have
been
raised
before
regarding
human
dietary
exposure
to
NPTII
antibiotic
marker
gene
proteins
in
GMO
foods,
data
in
the
public
arena
suggest
that
the
levels
of
NPTII
expressed
in
edible
portions
of
previously
approved
Bt­
corn
plants
are
very
low.
The
Monsanto
submission
reports
higher
expression
levels
of
NPTII
proteins
than
earlier
Bt­
corn
events.

EPA
Response:
EPA
disagrees
with
the
UCS
commenter
who
believes
there
is
an
unreasonable
risk
associated
with
the
use
of
the
antibiotic
resistance
gene
neomycin
phosphotransfersase
II
(
NPTII)
as
a
selectable
marker,
especially
in
terms
of
horizontal
gene
transfer.
EPA
first
assessed
the
safety
of
the
NPTII
protein
as
a
selectable
Section
2.2
2
­
12
marker
and
published
a
tolerance
exemption
in
1994
for
this
protein
(
40
CFR
180.1134).
The
basis
for
the
tolerance
determination
was
the
lack
of
oral
toxicity
and
absence
of
similarity
to
known
allergens.
EPA
holds
that
the
same
information
on
dietary
safety
still
suffices
to
support
the
tolerance
exemption
for
NPTII.
Further,
EPA
is
not
currently
aware
of
a
demonstrated
"
global
consensus"
on
antibiotic
selectable
marker
genes,
especially
nptII.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
10
Excerpt
Text:
4.
Missing
DNA
Warrants
Further
Study
In
the
study
entitled
"
Molecular
Analysis
of
Corn
Event
MON
863"
(
MRID
#
451568­
01),
Cavato
et
al.
describe
the
techniques
used
to
confirm
that
just
one
copy
of
the
transgene
cassette
was
expressed
and
that
expression
is
stable
in
subsequent
generations
of
plants
bred
from
the
MON
863
line.

The
ZMIR13L
vector
used
in
engineering
MON
863
corn
contains
two
major
sections.
The
first
contains
the
Cauliflower
mosaic
35S
promoter
and
the
NPTII
selectable
marker
gene,
flanked
by
the
NOS
3'
regulatory
nopaline
synthase
gene
of
Agrobacterium
tumefaciens
T­
DNA.
The
purpose
of
this
regulatory
gene
is
to
end
transcription
and
direct
polyadenylation.
Molecular
analysis
of
the
MON
863
event
suggests
that
just
one
copy
of
this
sequence
has
been
expressed
and
that
expression
is
stable.

The
second
section
within
the
ZMIR13L
plasmid
contains
the
Cry3Bb1.11098
gene.
It
is
flanked
to
the
left
by
­

­
An
activating
sequence
(
4­
AS1);
­
"
wt
CAB,"
a
5'
untranslated
leader
of
the
wheat
chlorophyll
a/
b
protein;
and
­
An
intron
from
the
rice
actin
gene.

To
the
right,
the
Cry3Bb1.11098
gene
is
flanked
by
"
tahsp17,"
which
is
described
in
the
submission
as
a
"
3'
nontranslated
region
on
the
coding
sequence
for
wheat
heat
shock
protein
17.3
which
ends
transcription
and
directs
polyadenylation."
This
second
regulatory
sequence
is
coupled
to
a
HindIII
restriction
endonuclease
"
that
cuts
DNA
at
specific
locations,"
as
well
as
a
MluI
restriction
endonuclease,
also
incorporated
to
assist
in
the
proper
placement
and
truncation
of
the
transgene
within
the
corn
genome.

The
Cavato
et
al.
report
states
that
­

"
Genomic
flanking
sequence
indicates
that
while
the
HindIII
site
at
the
3'
end
of
PV­
ZMIR13L
is
missing,
the
entire
tahsp
17
3'
polyadenylation
sequence
is
present
in
event
MON
863."

The
"
missing"
DNA
is
approximately
10
base
pairs
and
includes
the
HindIII
site
and
MluI
half
site.
The
authors
stress
that
despite
the
missing
DNA,
the
Cry3Bb
gene
and
its
regulatory
sequence
are
properly
and
stably
expressed
in
the
MON
863
event.
The
authors
do
not
explain,
however,
what
may
have
happened
to
the
10­
base
pair
segment
of
DNA
containing
the
two
restriction
endonucleases.
Presumably,
Monsanto
explored
whether
this
missing
DNA
lodged
somewhere
else
in
the
transformed
corn
lines.
We
would
expect
Section
2.2
2
­
13
that
the
company
has
developed
probes
capable
of
detecting
these
restriction
endonucleases.
The
lack
of
further
explanation
of
what
they
found
in
their
search
for
the
missing
DNA
raises
important
questions.

Possible
implications
have
been
suggested
in
research
by
Nielsen
and
colleagues.
Their
work
focused
on
the
impacts
of
transgenic
sugar
beets
on
bacterial
gene
flow
in
the
soil.
The
transgene
used
to
develop
the
modified
sugar
beets
included
several
of
the
same
promoter
and
marker
genes
as
the
Cry
3Bb
transgene.
They
found
in
their
work
that
the
"
presence
in
transgenic
plants
of
various
prokaryotic
markers
and
vector
sequences
may
facilitate
additive
insertion
of
foreign
genetic
material
into
bacterial
hosts
after
homologybased
heteroduplex
formation"
(
Nielsen
et
al.,
2000).

The
restriction
enzymes
missing
in
Cry
3Bb
transformed
corn
lines
could
trigger
unexpected
and
possibly
detrimental
pleiotrophic
effects,
depending
on
whether
and
where
they
are
lodged
in
the
genome
of
various
corn
hybrids
including
the
MON
863
transgene.
Monsanto
must
develop
a
mechanism
to
reliably
determine
the
fate
of
this
missing
DNA
and
make
it
widely
and
freely
available.
Lacking
such
a
probe
and
ongoing
scrutiny
of
all
the
hybrids
into
which
the
MON
863
event
is
placed,
the
U.
S.
corn
industry
will
be
vulnerable
to
a
loss
in
consumer
confidence
and
export
sales
if,
a
few
years
down
the
road,
it
is
discovered
that
this
missing
DNA
had,
in
certain
varieties
under
certain
circumstances,
been
expressed
in
a
way
leading
to
detectable
changes
in
corn
grain
or
forage
composition
or
product
performance.

EPA
Response:
EPA
disagrees
with
the
UCS
commenter
that
more
effort
should
be
made
to
account
for
the
loss
of
the
10
base
pair
section
of
DNA
not
found
in
the
MON863
corn
integration.
The
integrated
genes
function
as
expected.
It
is
highly
unlikely
that
the
10
base
pair
DNA
section,
which
basically
codes
for
restriction
enzyme
recognition
sites,
would
insert
elsewhere,
would
be
detectable
if
inserted
elsewhere
or
would
have
a
significant
effect
that
would
not
be
discovered
by
a
comparison
of
the
agronomic
performance
or
nutritional
characteristics
of
the
MON863
corn
plant
compared
to
traditional
corn
cultivars.
As
none
of
these
features
have
been
altered
except
for
the
intentionally
introduced
traits,
further
consideration
of
the
10
base
pair
of
lost
DNA
is
not
necessary.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
14
Excerpt
Text:
In
addition,
we
fully
expect
that
Monsanto
also
intends
to
incorporate
the
Cry
3Bb
gene
in
corn
varieties
engineered
to
be
Roundup
Ready
via
modification
of
the
EPSPS
gene,
a
critical
regulatory
protein
within
the
shikimate
pathway
of
all
plants.
The
aromatic
amino
acids
are
synthesized
within
this
pathway
and
play
multiple,
critical
roles
in
plant
growth
and
development
and
plant
defense
responses
to
pest
attack
and
abiotic
stress.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
15
Excerpt
Text:
Section
2.2
2
­
14
Recent
evidence
has
emerged
that
in
some
Roundup
Ready
plants,
changes
in
the
levels
of
expression
of
phenylalanine
(
Padgett
et
al.,
1995),
or
other
aromatic
amino
acids
or
their
secondary
products
(
Sidhu
et
al.,
2000),
may
be
impairing
plant
defense
mechanisms
and
retarding
or
weakening
the
systemic
acquired
resistance
(
SAR)
response
of
soybean
and
corn
plants
(
see
Benbrook,
2001).

In
corn
varieties
with
stacked
Bt
and
Roundup
Ready
genes,
it
is
probable
that
different
combinations
of
pest
and
abiotic
stresses
will
trigger
a
wide
array
of
unusual
protein
expression
patterns.
Some
of
these
combinations
will
surely
impact
product
performance
and
the
efficacy
of
resistance
management
plans,
while
others
may
pose
allergenicity
or
other
food
safety
risks.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
16
Excerpt
Text:
For
this
reason,
EPA
must
make
it
clear
in
response
to
this
and
other
applications
that
additional
data
must
be
submitted
on
each
proposed
stacked
gene
product
sufficient
to
settle
uncertainty
regarding
the
unique
risks
posed
when
a
given
variety
is
genetically
engineered
in
two
or
more
ways,
especially
when
the
modifications
target
different
physiological
processes
and
pathways
that
are
known
to
interact,
as
the
case
with
Bt
and
Roundup
Ready
corn
varieties.

EPA
Response:
The
comments
concerning
potential
adverse
physiological
effects
of
transformations
conferring
tolerance
to
glyphosate
are
outside
the
scope
of
this
regulatory
action.
In
the
future,
should
EPA
be
presented
with
a
registration
application
for
a
product
expressing
both
pesticidal
and
other
traits,
EPA
will
continue
to
coordinate
its
review
of
the
pesticidal
traits
of
such
plant
with
USDA­
APHIS'
review
of
non­
pesticidal
traits.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
35
Excerpt
Text:
Nielsen
and
colleagues
point
out
in
a
recent
article
in
Applied
and
Environmental
Microbiology
that
­

"
Of
the
15
different
resistance
genes
incorporated
into
plants,
several
encode
resistance
to
clinically
used
antibiotics....
Recently,
uptake
of
transgenic
plantharbored
DNA
fragments
by
bacteria
based
on
restoration
of
a
partially
deleted
(
10­
or
317­
bp
internal
deletion)
bacterial
kanamycin
(
KM)
resistance
gene
(
nptII)
after
recombination
with
transgenic
plant­
inserted
homologues
was
demonstrated"
(
Nielsen
et
al.,
2000).

Recent
evidence
documenting
how
freely
bacteria
can
exchange
genes
has
rekindled
concerns
over
the
emergence
of
new
strains
of
antibiotic
resistant
bacteria
as
a
result
of
use
of
antibiotic
selectable
marker
genes.
The
Cry
3Bb
gene
cassette
contains
the
0.79
Kb
nptII
gene
from
a
transposon
isolated
from
E.
coli.
It
confers
resistance
to
kanamycin
and
neomycin.
[
Footnote
2:
Kanamycin
and
neomycin
are
antibiotics
used
to
control
disease­
causing
bacteria
(
Berkow,
1992).
FDA
has
approved
a
food
additive
petition
from
Section
2.2
2
­
15
Calgene,
Inc.,
(
now
owned
by
Monsanto)
for
use
of
the
nptII
gene
in
genetically
engineered
crops.

EPA
Response:
EPA
disagrees
with
the
UCS
commenter
on
the
issue
of
gene
transfer
form
genetically
engineered
plants
to
environmental
bacteria.
There
have
been
a
number
of
scientific
questions
raised
about
the
use
of
antibiotic
resistance
markers
in
genetically
engineered
plants
since
the
initial
approvals
which
contained
antibiotic
marker
genes.
Little
of
the
scientific
data
generated
on
this
topic
demonstrates
a
risk
scenario
that
would
translate
into
a
detectable
adverse
effect
in
the
environment.
The
major
concern
has
been
the
possibility
that
the
presence
of
the
NPTII
gene
in
genetically
engineered
plants
may
lead
to
an
increase
in
the
level
of
antibiotic
resistance
in
bacteria,
particularly
clinically
important
bacteria.
The
commenter
cites
several
scientific
papers
(
refs.?)
that
purport
to
demonstrate
the
horizontal
gene
transfer
of
the
NPTII
gene
from
genetically
engineered
plant
DNA
by
transformation
into
a
soil
bacterium.
What
is
actually
demonstrated
is
the
reversal
of
a
deletion
mutation
by
homologous
recombination.
The
soil
bacterium
had
an
endogenous
NPTII
gene
which
was
inactivated
by
a
deletion
mutation
of
312
base
pairs,
a
removal
of
some
of
the
gene
to
render
the
gene
product
inactive.
The
researchers
were
able
to
show
that
high
concentrations
of
purified
DNA
isolated
from
genetically
engineered
sugar
beet
was
able
through
transformation
to
repair
the
existing
inactivated
NPTII
gene
and
restore
the
NPTII
kanamycin
resistance
phenotype
in
the
recipient
bacterium.

While
this
thought­
provoking
work
is
able
to
demonstrate
the
previously
theoretical
possibility
of
horizontal
gene
transfer
from
plant
DNA
to
a
bacterium,
the
results
do
not
change
EPA's
safety
findings.
The
experiments
were
done
in
a
sterile
soil
system,
with
high
concentrations
of
purified
plant
DNA
unlikely
to
be
found
in
nature,
with
a
naturally
transformation
competent
bacterium
that
had
a
pre­
existing
NPTII
gene.
These
conditions
describe
several
very
low
probability
contingencies
that
we
expect
would
probably
have
to
coincide
for
this
situation
to
occur
in
nature.
Horizontal
gene
transfer
is
theoretically
possible
and
has
been
shown
to
occur
in
these
highly
manipulated
systems
but
given
the
low
levels
detected
in
these
experimental
systems
it
remains
extremely
unlikely
such
transfer
would
occur
in
nature
and
that
such
a
transfer
would
result
in
any
impact
on
relatively
high
background
kanamycin/
neomycin
antibiotic
resistance
in
the
bacterial
microflora.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
36
Excerpt
Text:
When
the
first
Bt­
transgenic
plant
applications
were
reviewed,
there
was
little
evidence
that
antibiotic
marker
genes
might
trigger
resistance
problems.
Now
the
transfer
of
antibiotic
marker
genes
in
plant
cells
to
soil
bacteria
has
been
demonstrated
(
Gerbhard
and
Smalla,
1998).
In
a
key
article
in
the
journal
Applied
and
Environmental
Microbiology,
Gebhard
and
Smalla
hypothesize
that:

" 
the
introduction
of
bacterial
genes
into
the
plant
genome
leads
to
a
higher
probability
of
gene
transfer
from
plants
to
bacteria
due
to
the
presence
of
homologous
sequences.
However,
until
now,
there
has
been
a
lack
of
clear
experimental
evidence
that
successful
gene
transfer
from
plants
to
bacteria
can
occur
at
all."
(
Gebhard
and
Smalla,
1998).

Commenter
Name:
Charles
Benbrook
Section
2.2
2
­
16
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
38
Excerpt
Text:
Also
contrary
to
conventional
wisdom,
genes
that
confer
antibiotic
resistance
have
been
shown
to
move
readily
between
people
and
from
bacteria
in
the
gut
of
farm
animals
to
people
(
Tschape,
1994).
A
team
in
Denmark
has
shown
that
indigenous
soil
bacteria
can
serve
as
a
sink
for
plasmid­
borne
antibiotic
resistance
traits
from
E.
coli
entering
agricultural
soils
from
animal
manure
or
other
wastes
(
Sorensen
et
al.,
1999).

Until
recently
it
was
also
thought
that
foreign
DNA,
such
as
the
gene
constructs
in
Cry
3Bb
corn,
would
pass
through
the
mammalian
digestive
without
being
activated
and
without
consequence.
The
transfer
of
foreign
DNA
in
food
into
the
blood
and
organ
systems
in
mice
has
now
been
demonstrated
(
Schubbert
et
al.,
1997).
In
addition
some
of
the
transgenic
DNA
was
found
to
covalently
bind
to
mouse
DNA,
in
effect
becoming
a
part
of
the
mouse
genome.
The
authors
modestly
summed
up
this
article
by
stating
­­
"
The
medical
and
evolutionary
implications
of
these
observations
may
be
considerable"
(
Schubbert
et
al.,
1997).

EPA
Response:
EPA
disagrees
with
the
assertion
that
mammals
effectively
take
up
and
incorporate
DNA
from
the
digestive
system.
While
recent
experimental
evidence
has
indicated
that
certain
DNA
vaccine
therapies
may
have
promise,
none
have
been
developed
that
could
encompass
the
scenario
provided
by
consuming
genetically
engineered
foods.
Moreover,
there
is
no
indication
that
consumption
of
traditional
foods
has
resulted
in
transfer
and
incorporation
of
any
traits
from
food
plants.
The
scientific
article
cited
by
the
commenter
showed
that
there
was
a
transient
detection
of
the
M13
phage
DNA
in
mouse
organs
and
blood
when
introduced
by
the
oral
route.
However,
it
is
unclear
that
the
claimed
incorporation
of
the
M13
Phage
DNA
into
mouse
organs
and
tissues
is
a
real
phenomenon
let
alone
a
stable
incorporation
of
foreign
DNA.
These
results
are
controversial
and
have
not
been
repeated
or
confirmed
by
another
laboratory.
It
is
unlikely
that
the
amount
of
DNA
typically
associated
with
food
consumption
represents
a
DNA
exposure
similar
to
the
very
high
concentrations
described
and
employed
in
the
articles.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
9
Excerpt
Text:
Comment
or
Question:
In
the
safety
assessment,
it
is
inappropriate
to
compare
the
Cry3Bb1
protein
produced
in
corn
event
MON
863
to
the
Cry3Bb
protein
present
in
the
commercial
microbial
Bt
pesticide
trademarked
Raven.
This
product
contains
a
longer
Cry3Bb
protein
than
produced
in
MON
863.

Scientific
Background:
The
Cry3Bb1
protein
that
is
produced
in
corn
plants
is
structurally
identical
to
that
produced
in
Raven
(
differing
by
only
seven
amino
acids).
Therefore,
the
comparison
is
relevant.

Comment
or
Question:
Molecular
analysis
of
MON
863
reveals
a
"
missing"
10
base
pair
sequence
that
could
have
unpredictable
effects.
Furthermore,
there
is
a
lack
of
publicly
available
data
and
methods
to
determine
whether
the
"
missing"
DNA
may
have
inserted
into
another
location
of
the
genome.
Section
2.2
2
­
17
Scientific
Background:
A
10
base
pair
DNA
sequence
present
within
the
transformation
cassette
was
lost
upon
stable
integration
of
the
cassette
into
the
plant
genome.
Southern
blot
analyses
using
probe
combinations
spanning
the
deleted
10
base
pair
region
did
not
detect
any
integration
of
the
10
base
pair
segment
elsewhere
in
the
genome.
The
10
base
pair
DNA
segment
most
likely
was
deleted
during
transformation
and
degraded
by
intracellular
nucleases.
Such
deletions
commonly
occur
as
a
consequence
of
DNA
repair
that
takes
place
at
the
integration
site,
as
well
as
in
regions
of
natural
strand
breakage
and
repair.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
19
Excerpt
Text:
Comment
or
Question:
Adaptation
of
CRW
populations
to
the
Cry3Bb1
protein
expressed
in
MON
863
could
limit
the
longevity
of
future
biopesticide
formulations
of
Cry3Bb,
other
Cry3Bb
events
or
Bt
events
with
a
different
mode
of
action.

Scientific
Background:
The
Cry3Bb
protein
has
never
been
used
in
any
formulated
Bt
microbial­
based
product
to
control
CRW
larvae
for
two
important
reasons:
delivery
and
timing.
CRW
larvae
feed
on
corn
roots
over
a
largely
unpredictable
period
of
three
to
eight
weeks,
and
the
Bt
insecticidal
protein
must
be
ingested
to
ellicit
a
pesticidal
physiological
response.
Thus,
in
order
for
CRW
larvae
to
come
into
contact
with
a
microbial
Bt
formulation,
it
would
need
to
be
applied
so
as
to
constantly
cover
the
root
tissue
and
remain
active
for
a
prolonged
period
of
time.
Neither
of
these
issues
can
be
addressed
with
current
Bt
microbial
formulation
technology.
Thus,
it
is
reasonable
to
assume
that
use
of
MON
863
will
not
pose
an
unreasonable
risk
to
a
future,
hypothetical
Bt
microbial­
based
product
for
the
control
of
CRW.
Studies
also
indicate
that
there
is
little
cross­
resistance
between
Bt
plant
and
foliar
sprays
with
similar
proteins
because
of
the
presence
of
other
critical
ingredients,
such
as
spores,
in
the
spray
formulations.

Corn
event
MON
863
produces
the
Cry3Bbl
protein
at
levels
sufficient
to
minimize
CRW
larval
root
damage.
Regulatory
approval
and
commercial
use
of
this
technology
will
require
the
deployment
of
a
scientifically
sound
and
feasible
IRM
plan
that
suppresses
the
emergence
of
insect
resistance
in
targeted
pest
populations.
In
the
event
that
future
Cry3Bbl
events
are
introduced,
EPA
will
require
and
determine
the
acceptability
of
appropriate
IRM
plans
for
all
products.

Event
MON
863
represents
a
negligible
risk
to
other
Bt
events
that
work
via
a
different
mode
of
action.
It
is
well
understood
that
Bt
proteins
having
similar
modes
of
action
are
at
a
greater
risk
of
exhibiting
crossresistance
Conversely,
Bt
proteins
that
differ
in
their
mode
of
action
represent
a
minimal
risk
of
crossresistance
It
is
for
this
reason
that
EPA
and
the
scientific
community
recommend
that
developers
of
Bt
crops
develop
and
express
Bt
proteins
with
differing
modes
of
action
as
a
means
to
further
reduce
the
likelihood
of
resistance
development
in
pest
populations.

The
generally
recognized
mode
of
action
of
Bt
proteins
has
several
distinct
steps
after
ingestion
by
the
insect.
These
steps
include:
solubilization,
proteolytic
stability,
binding
to
the
insect
midgut
epithelium,
formation
of
ion
channels
in
the
insect
midgut
cells,
and
finally
analysis
of
these
cells.
Each
of
these
steps
is
governed
by
the
sequence­
dependent
structure
of
the
protein
and
the
insect­
specific
midgut
environment.
Section
2.2
2
­
18
Thus,
the
more
two
proteins
differ
structurally,
the
more
likely
they
are
to
differ
in
their
mode
of
action
and
would
exhibit
differing
mechanisms
of
resistance.
The
sequence
dissimilarity
between
two
Bt
proteins
is
an
empirical
measure
of
their
likelihood
to
act
by
different
modes
of
action.
Recently
Cry34Abl
and
Cry35Ab1
have
been
listed
as
new
classes
of
novel
Cry
proteins,
which
require
them
to
share
less
than
45%
amino
acid
identity
with
any
other
known
Cry
protein
(
e.
g.,
Cry3Bbl).
The
Cry34
and
Cry35
proteins
expressed
in
CRW­
protected
events
under
development
by
Dow
AgroSciences
and
Pioneer
Hy­
Bred
International
function
as
a
binary
protein
active
against
CRW
larvae.
These
proteins
not
only
are
very
different
from
Cry3Bbl
in
amino
acid
sequence,
but
both
proteins
are
absolutely
required
for
CRW
activity.
Due
to
these
differences
in
protein
sequence
and
mode
of
action,
it
is
unlikely
that
Cry3Bb1
and
the
new
Cry34/
Cry35
binary
protein
have
similar
modes
of
action.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
20
Excerpt
Text:
Comment
or
Question:
The
presence
of
the
nptII
antibiotic
resistance
marker
(
ARM)
gene
in
MON
863
may
pose
a
risk
to
human
and
animal
health
through
horizontal
gene
transfer
(
HGT)
from
genetically
modified
MON
863
corn
to
bacteria
either
in
the
soil
environment,
or
in
the
mammalian
digestive
tract.
The
potential
transfer
of
these
genes
to
bacteria
has
been
suggested
as
an
important
mechanism
for
the
spread
of
antibiotic
resistance
in
bacteria,
which
could
impede
medical
treatment
of
humans
or
animals,
since
the
ARM
used
in
the
transgenic
CRW
plant
encodes
resistance
to
antibiotics
(
kanamycin/
neomycin)
which
are
still
in
use
in
some
clinical
applications.

Scientific
Background:
Researchers
have
never
detected
an
HGT
event
with
the
transfer
of
an
intact
gene
under
field
conditions.
Under
laboratory
conditions,
the
frequency
of
HGT
with
segments
of
DNA
serving
as
model
plant
genes
is
extremely
low.
More
importantly,
the
genes
of
interest
in
MON
863
corn
(
Cry3Bbl
and
npt11)
possess
no
harmful
characteristics
to
the
environment
compared
to
the
background
of
genes
and
gene
products
already
found
in
soil
or
water
borne
microorganisms.
Finally,
the
Cry3Bb1
and
nptII
genes,
and
their
homologues,
are
naturally
present
in
soil
dwelling
microbes.
For
example,
manure
has
been
shown
to
contain
high
numbers
of
bacteria
harboring
the
nptII
gene.
[
Footnote
12:
Nielsen,
K.
M.,
A.
M.
Bones,
K.
Smalla
and
J.
D.
can
Elsas
(
1998).
Horizontal
gene
transfer
from
transgenic
plants
to
terrestrial
bacteria
­
a
rare
event?
FEMS
Microbiol
Reviews
22:
79­
103.]
Since
manure
is
frequently
used
as
a
nitrogen
source
in
agricultural
fields,
the
potential
addition
of
ARMs
to
soil
microbes
via
an
unlikely,
low
frequency
HGT
event
is
trivial
compared
to
the
population
of
antibiotic
resistant
microbes
already
present
in
soil
environments.

Horizontal
gene
transfer
between
bacteria
has
occurred
widely,
primarily
through
the
transfer
of
plasmids
between
members
of
various
species,
and
has
led
to
the
rapid
spread
of
antibiotic
resistance
genes
in
clinical
environments.
[
Footnote
13:
Davidson,
J.
(
1999).
Genetic
exchange
between
bacteria
in
the
environment.
Plasmid
42:
73­
91.]
Bacteria
that
are
already
resistant
to
both
kanamycin
and
neornycin
are
quite
common
in
the
environment
and
there
is
no
data
to
support
the
suggestion
that
HGT
could
increase
the
risk
of
resistance
in
humans
or
livestock.
Furthermore,
the
clinical
use
of
kanamycin
and
neomycin
is
limited
by
human
toxicity
and
the
availability
of
less
toxic,
more
effective
antibiotics.
Substantial
research
efforts
have
tried
but
failed
to
demonstrate
that
ARM
genes
may
be
transferred
from
plants
to
bacteria.
Section
2.2
2
­
19
Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
21
Excerpt
Text:
Bacteria
from
soils
at
field
sites
have
not
been
able
to
accept
and
incorporate
transgenic
plant
DNA
in
controlled
experiments.
[
Footnote
14:
Gebhard,
F.
and
K.
Smalla
(
1998).
Transformation
of
Acinetobacter
sp.
Strain
BD413
by
transgenic
sugar
beet
DNA.
Appl.
Environ.
64:
1550­
1554.]
[
Footnote
15:
Paget,
E,
M.
Lebrun,
G.
Freyssinet
and
P.
Simonet
(
1998).
The
fate
of
recombinant
plant
DNA
in
soil.
Eur.
J.
Soil
Biol.
34:
81­
844.]
Moreover,
attempts
to
demonstrate
in
vitro
transformation.
of
naturally
competent
bacteria
to
antibiotic
resistant
bacteria
using
genomic
DNA
from
transgenic
potato
or
sugar
beet
plants
carrying
an
npt1I
transgene
were
unsuccessful,
[
Footnote
16:
Nielsen,
K.
M.,
F.
Gebhard,
K.
Smalla,
A.
M.
Bones
and
J.
D.
van
Elsas
(
1997).
Evaluation
of
possible
horizontal
gene
transfer
from
transgenic
plants
to
the
soil
bacterium
Acinetobacter
calcoaceticus
BD413.
Theor.
Appl.
Genet.
95:
815­
821.]
as
were
attempts
to
transform
the
bacterial
pathogen
Erwinia
chrysanthemi
with
DNA
from
transgenic
potatoes
carrying
an
ampicillin
resistance
gene.
[
Footnote
17:
Schluter,
K.,
J.
Futterer
and
I.
Potrykus
(
1995).
Horizontal
gene
transfer
from
a
transgenic
potato
line
to
a
bacterial
pathogen
(
Erwinia
chrysanthemi)
occurs­
if
at
all­
at
an
extremely
low
frequency.
Biotechnology
13:
1094­
1098.]
The
highest
potential
transformation
frequencies
in
these
experiments
were
in
the
range
of
10­
13,
10­
17
per
recipient
bacteria.

In
contrast,
in
vitro
HGT
of
plant
transgene
sequences
to
a
model
soil
bacterium
has
been
demonstrated
under
laboratory
conditions
when
sequence
homology
to
the
plant
transgene
was
purposefully
engineered
into
the
recipient
bacterium.
These
studies
utilized
the
naturally
transformable
soil
bacterium
Acinetobacter
strain
BD413
that
carried
a
plasmid
carrying
a
deletion
derivative
of
the
nptII
gene
with
either
a
10
bp
or
317
bp
internal
deletion
as
the
recipient
which
could
be
rescued
after
transformation
with
purified
leaf
DNA
from
transgenic
plants
carrying
an
nptII
transgene
[
canola,
potato,
tobacco,
tomato
and
sugar
beet]
[
Footnote
18:
de
Vries,
J.
and
W.
Wackernagel
(
1998).
Detection
of
nptII
(
kanamycin
resistance)
genes
in
genomes
of
transgenic
plants
by
marker­
rescue
transformation.
Mol.
Gen.
Genet.
257:
606­
613.]
Similar
rescue
experiments
were
also
performed
in
sterile
soil
microcosms
using
a
plasmidborne
317
nptII
deletion
and
purified
transgenic
sugar
beet
DNA
as
gene
donors.
[
Footnote
19:
Nielsen,
K.
M.,
J.
D.
van
Elsas
and
K.
Smalla
(
2000).
Transformation
of
Acinetobacter
sp.
Strain
BD413(
pFG4?
npII)
with
transgenic
plant
DNA
in
soil
microcosm
and
effects
of
kanamycin
on
selection
of
transformants.
Appl.
Environ.
Microbiol.
66:
1237­
1242.]
Transformation
frequencies
of
10­
9
to
10­
10
were
obtained
in
these
experiments.
The
use
of
nonsterile
soil
decreased
the
transformation
frequency
to
undetectable
limits.
Additionally,
the
plasmid­
borne
kanamycin
resistance
phenotype
was
unstable
when
incubated
in
the
microcosm
for
days
without
selection.
Consequently,
while
HGT
has
been
demonstrated
at
very
low
levels
in
laboratory
model
systems
with
engineered
bacteria,
the
HGT
event
was
not
detectable
in
a
nonsterile
soil
microcosm
and
unstable
under
sterile
laboratory
conditions
without
artificial
selection.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
4
Excerpt
Text:
2.
Monsanto
did
not
adequately
compare
the
amino
acid
sequence
of
Cry3Bb1
with
known
allergen
protein
Section
2.2
2
­
20
sequences.
Current
protocols
recommend
testing
for
homology
of
six
identical
contiguous
amino
acids
as
a
possible
indicator
of
allergenicity.
Monsanto
should
repeat
their
analysis
since
international
experts
(
1)
now
agree
that
Monsanto's
use
of
eight
contiguous
amino
acid
homology
may
miss
shorter
matches
that
indicate
allergenicity.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
15
Excerpt
Text:
B.
Protein
Sequence
Comparisons
Between
MON
863
and
Known
Allergens
Monsanto
searched
protein
sequence
databases
for
matches
of
eight
or
more
contiguous
amino
acids
to
indicate
possible
allergenicity,
while
the
recent
FAO/
WHO
protocols
(
1)
call
for
determining
sequence
matches
of
six
contiguous
amino
acids.
Thus,
Monsanto's
test
might
not
detect
similarity
between
MON
863
and
a
known
allergen.

Monsanto
argues
against
using
less
than
eight
contiguous
amino
acids
to
determine
potential
allergenicity.
Monsanto
acknowledges
that
cross­
reactive
allergy
epitopes
as
short
as
five
amino
acids
have
been
found
in
other
proteins,
but
that
they
do
not
bind
antibody
with
affinity
as
high
as
the
original
allergen.
Thus
Monsanto
suggests
that
antibody
affinity
is
an
adequate
measure
of
allergenicity,
and
that
even
if
Cry3Bb
I
had
six
consecutive
amino
acids
in
common
with
an
allergen
epitope,
that
would
not
be
enough
to
cause
an
allergic
reaction.

However,
Monsanto
does
not
give
adequate
support
to
the
use
of
affinity
as
an
accurate
predictor
of
clinical
symptoms.
In
fact,
in
some
cases
of
oral
allergy
syndrome
where
there
is
evidence
that
the
cross
reacting
pollen
allergen
is
the
sensitizing
agent,
the
homologous
cross
reacting
food
allergen
can
cause
clinically
significant
symptoms,
despite
having
no
more
than
six
identical
or
similar
contiguous
amino
acids
in
common
with
the
pollen
(
6,7).
In
addition,
only
a
relatively
small
number
of
cross­
reacting
epitopes
have
been
mapped,
so
it
is
premature
to
dismiss
conclusions
about
the
clinical
importance
of
matches
at
a
six
amino
acid
level
based
on
Monsanto's
limited
examples.

Monsanto
mentions
concern
that
matches
of
six
amino
acids
may
occur
randomly
at
relatively
high
rate.
It
is
possible
that
a
six
(
or
eight)
amino
acid
match
could
occur
with
a
known
allergen
that
does
not
correspond
to
an
IgE
epitope,
and
therefore
is
not
directly
involved
in
the
allergic
response.
In
addition,
because
relatively
few
allergen
epitopes
have
been
determined,
sequence
data
may
not
be
sufficient
to
determine
the
allergenic
importance
of
such
a
match.
The
FAO/
WHO
protocol
(
1)
recognizes
that
possibility,
but
also
recognizes
that
it
can
be
addressed
with
an
assay
using
pooled
serum
from
individuals
known
to
be
allergic
to
the
sequence­
matched
allergen.
EPA
should
require
Monsanto
to
repeat
the
search
for
protein
sequence
matching
at
a
six
amino
acid
level,
which
can
be
done
easily
since
the
databases
are
already
available.

EPA
Response:
EPA
disagrees
with
the
CPSI
commenter
about
the
lack
of
validity
for
the
amino
acid
comparison
of
the
Section
2.2
2
­
21
PIP
expressed
proteins.
For
the
allergenicity
assessment,
two
amino
acid
homology
comparisons
for
the
entire
sequence
and
a
stepwise
series
of
8
amino
acid
segment
comparisons
are
performed
by
computer
analysis.
These
results
are
discussed
in
sections
IIA
and
IIB
of
the
BRAD
and
EPA
still
believes
these
data
demonstrate
a
reasonable
certainty
that
the
aggregate
exposure
of
the
PIP
proteins
will
cause
no
harm
including
potential
allergy
concerns.

The
specific
comment
by
CSPI
suggests
that
the
FAO/
WHO
conference
held
in
Rome,
Italy
February
2001
is
the
definitive
guidance
on
the
method
for
assessing
allergenicity
of
a
novel
protein.
In
general,
CODEX
is
considered
the
body
that
sets
international
safety
standards
and
a
subsequent
meeting
of
an
Ad
hoc
CODEX
meeting
of
food
allergy
experts
held
in
Vancouver,
Canada,
September
2001
considered
the
FAO/
WHO
recommendations.
The
outcome
of
that
CODEX
meeting
was
that,
while
the
FAO/
WHO
recommendations
should
be
considered
in
a
safety
assessment,
there
were
still
questions
relating
to
what
appropriate
and
valid
methods
were
to
be
used
for
amino
acid
sequence
homology
assessments.
The
ambiguity
of
the
CODEX
guidance
and
the
demonstrated
false
positive
correlations
of
corn
proteins
with
allergens
arising
with
the
use
of
shorter
than
eight
amino
acid
segments
(
Hileman
et
al.,
2002)
suggest
that
it
is
still
reasonable
to
utilize
the
8
amino
acid
segment
stepwise
analysis
until
further
refinement
of
the
homology
assessment
with
shorter
segments
is
obtained.
Section
2.3
2
­
22
2.3
­
Data
Needs
and
Methods
2.3
­
Comment
Excerpts
Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
12
Excerpt
Text:
'
The
conclusion
of
reasonable
certainty
of
no
harm
and
the
resultant
tolerance
expemptions
for
this
wide
array
of
B.
t.
mixtures
and
Cry
proteins
in
food
or
feed
were
based
on
the
lack
of
adverse
effects
to
mammals
in
numerous
toxicological
studies.
This
conclusion
is
supported
by
a
history
of
safe
use
in
agriculture
for
over
40
years
..'
(
Monsanto
Document,
page
21
of
56)

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
13
Excerpt
Text:
It
must
be
understood
that
much
of
the
'
history
of
safe
use
in
agriculture
for
over
40
years..'
is
not
based
on
data
but
on
lack
of
data.
But
lack
of
data
does
not
mean
lack
of
evidence.
It
must
further
be
understood
that
the
'
safe
use'
refers
to
the
use
of
microbial
Bt
pesticides
that
were
not
used
widely
in
industrial
agriculture
and
differ
in
a
number
of
important
aspects
from
current
transgenic
Bt
plants:
significantly
extended
temporal
exposure,
degree
of
exposure
(
concentration,
types
of
proteins
in
all
types
of
foods),
biochemical
state
of
Bt
protein.
etc.
As
new
and
different
products
come
on
the
market
and
enter
the
human
food
chain
also
interaction
effects
of
combined
consumption
of
several
different
Bt
toxins
must
be
included
in
human
safety
tests.

'
Extensive
review
of
these
studies
and
a
determination
that
proteins
act
almost
exclusively
through
acute
mechanisms,
resulted
in
the
focusing
of
testing
requirements
for
microbial­
derived
products
to
include
acute
oral,
pulmonary
and
intravenous
toxicity
studies...'
(
Monsanto
Document,
pages
21­
22
of
56)

However,
although
based
primarily
on
previous
studies
conducted
with
various,
very
different
products
in
the
past,
nowhere
in
the
Monsanto
Document
those
studies
are
mentioned
that
did
find
evidence
for
potential
human
health
effects.
For
example,
a
comparatively
recent
EPA­
funded
study
published
in
Environmental
Health
Perspectives
in
1999
on
'
Immune
responses
in
farm
workers
after
exposure
to
Bacillus
thuringiensis
pesticides'
reports
on
evidence
for
potential
allergic
reactions
to
one
of
the
microbial
Bt
pesticides
(
Bernstein
et
al.
1999).
Two
of
123
farm
workers
showed
a
positive
skin­
prick
test
to
the
Bt
spore
extract
containing
the
Bt
protein.
Although
the
authors
do
conclude
that
because
of
this
low
number
of
workers
sensitized
through
respiratory
exposure
it
is
not
likely
that
consumers
would
develop
allergic
reactions
after
oral
exposure.
But
it
does
demonstrate
that
new
and
modern
testing
procedures
are
available
for
human
safety
studies
and
that
occasional
allergic
reactions
cannot
be
ruled
out
once
and
for
all
with
every
Bt
containing
product.
Certainly
such
recent
studies
should
be
cited
and
discussed
in
an
application
package
deriving
much
of
the
safety
conclusion
from
the
published
literature
instead
of
conducted
own
experiments
and
data.
Section
2.3
2
­
23
We
feel
that
the
approach
used
in
the
Monsanto
Document
to
arrive
at
human
safety
conclusions
is
unsatisfying
for
a
comprehensive
human
health
assessment.
In
particular
by
learning
from
the
StarLink
contamination
story,
it
is
clear
that
these
products
will
reach
virtually
everybody
of
the
society
in
some
form
or
another
in
many
products
for
a
long
time
to
come.
In
our
opinion,
this
would
merit
an
extensive
and
transparent
peer
review
by
competent
independent
scientists
of
all
human
safety
studies
conducted
in
the
past
with
Bt
products,
microbial
and
transgenic,
and
establish
to
what
degree
which
of
those
can
be
bridged
for
assessment
of
current
transgenic
plants
and
which
cannot
be
used
as
evidence
and
where
new
human
safety
studies
must
be
initiated.

EPA
Response:
First,
EPA
must
point
out
that
the
Agency
has
not
based
its
regulatory
determination
on
this
PIP
on
claims
of
years
of
experience
with
microbial
biopesticides
utilizing
Bacillus
thuringiensis.
Second,
EPA
disagrees
that
the
dietary
exposure
for
microbial
products
is
practically
nil
and
that
the
scientific
literature
indicates
that
Cry
Proteins
have
induced
positive
immune
responses.
The
data
that
supports
the
safety
determination
is
specific
to
the
PIP
proteins
expressed
in
MON
863
corn.
These
are
toxicity
tests
done
with
high
doses
of
purified
protein
as
well
as
biochemical
tests
done
to
ascertain
protein
characteristics.
These
results
are
discussed
in
sections
IIA
and
IIB
of
the
BRAD.
EPA
believes
these
data
stand
alone
to
demonstrate
a
reasonable
certainty
that
the
aggregate
exposure
of
the
PIP
proteins
will
cause
no
harm.

Additional
data
when
available
can
add
to
the
information
on
dietary
safety
such
as
suggestions
that
there
has
been
a
history
of
dietary
exposure
and
safe
use
of
these
proteins
in
agriculture
due
to
their
expression
in
microbial
biopesticides
utilizing
Bacillus
thuringiensis.
While
definitely
different
in
the
nature
of
exposure,
microbial
pesticides
expressing
similar
Cry
proteins
do
have
significant
dietary
exposure.
There
has
been
signifcant
adoption
of
microbial
biopesticides
utilizing
Bacillus
thuringiensis
in
not
only
organic
agriculture
but
also
conventional
vegetable
production.
This
vegetable
use
would
also
be
expected
to
result
in
potential
dietary
exposure
due
to
uses
which
allow
application
up
until
harvest
and
use
on
crops
that
are
harvested
over
a
period
of
time
such
as
tomatoes
or
cole
crops.
EPA
agrees
that
there
has
probably
been
much
less
exposure
to
the
Cry3
family
of
Bacillus
thuringiensis
toxins
active
against
coleopterans
than
the
lepidopteran
active
toxins.

The
literature
cited
by
the
commenter
(
Bernstein
et
al,
1999)
actually
gives
an
indication
that
the
Cry
proteins
themselves
are
not
responsible
for
the
skin
prick
positive
test
in
agricultural
workers
exposed
to
pesticides
containing
Bacillus
thuringiensis.
It
is
important
to
note
that
none
of
the
workers
examined
in
the
study
reported
a
clinical
allergic
reaction
to
the
Bacillus
thuringiensis
products.
In
addition,
the
spore
extract
preparations
that
tested
positive
by
skin
prick
were
further
purified
for
the
actual
crystal
protein
component.
This
crystal
protein
component
was
shown
by
the
authors
to
not
induce
a
positive
skin
prick
response.
Moreover,
skin
prick
tests
are
notably
inaccurate
giving
a
positive
result
with
an
approximately
50%
correlation
to
actual
clinical
hypersensitivity.
Negative
skin
tests
are
more
indicative
of
clinical
outcome.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
4
Section
2.3
2
­
24
Excerpt
Text:
Future
releases
of
transgenic
plants
In
the
future,
it
is
expected
that
transgenic
plants
containing
genes
that
code
for
so­
called
'
input'
traits,
i.
e.,
traits
that
confer
agronomically
important
characteristics
such
as
insect
resistance
will
be
succeeded
by
a
new
generation
of
transgenic
plants
expressing
so­
called
'
output'
traits,
i.
e.,
traits
that
confer
'
quality'
characters
such
as
altered
oil,
starch,
protein
compositions
of
food
and
feed
crops.
Additionally,
much
research
is
currently
invested
into
the
development
of
transgenic
plants
and
also
transgenic
animals
producing
various
pharmaceuticals,
vaccines,
vitamins,
enzymes
for
medical
and/
or
industrial
purposes.
Field
releases
of
such
transgenic
plants
for
research
purposes
are
being
carried
out
for
several
years
now.
However,
testing
the
environmental
impact
of,
for
example,
vaccine­
producing
transgenic
plants
or
animals
will
be
quite
challenging,
in
particular,
since
these
novel
'
output'
traits
may
well
be
expressed
in
addition
to
the
'
old'
'
input'
traits.
In
order
to
be
able
to
address
these
increasingly
complex
issues
in
a
responsible
manner,
it
is
crucial
that
effective
pre­
release
safety
testing,
hazard
identification
procedures,
risk
assessment
and
management
strategies,
and
sensitive
and
robust
post­
release
monitoring
schemes
are
established
well
before
commercialisation
of
such
potent
novel
plants.

EPA
Response:
EPA
agrees
with
the
Greenpeace
commenter
that
all
genetically
engineered
plants
should
be
subject
to
a
thorough
review
for
environmental
impact
as
required
by
federal
law.
In
the
future,
as
has
been
done
for
all
genetically
engineered
plants
products
to
date,
EPA
looks
forward
to
coordinating
its
environmental
impact
assessment
of
PIP
expressing
plants,
including
those
with
output
traits,
with
USDA­
APHIS.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
32
Excerpt
Text:
3.
Data
Needed
to
Assess
Human
Health
Risks
Are
Inadequate
Bt
foliar
insecticides
have
been
used
safely
for
more
than
two
decades.
Bt's
safety
record
played
a
major
role
in
convincing
the
EPA
that
only
a
cursory
assessment
of
human
and
mammalian
health
risks
from
dietary
ingestion
of
truncated
Bt
endotoxins
in
plant
tissues
was
required
prior
to
approval
of
today's
Cry1
and
Cry2
Bt.
k­
transgenic
plant
varieties.

Monsanto's
current
application
for
Cry3Bb
transgenic
corn
rests
heavily
upon
traditional
toxicity
studies
that
were
originally
submitted
to
the
agency
by
Ecogen
to
support
registration
of
Raven,
a
foliar
Bt
product
containing
Cry3Bb1,
the
endotoxin
from
Bt
tenebrionis
(
Btt).
Btt
studies
on
file
are
routine
toxicological
feeding
experiments
and
were
not
designed
to
detect
mammalian
health
hazards.
In
addition,
the
Ecogen
studies
were
carried
out
with
Btt
derived
from
bacteria
via
fermentation,
which
produces
a
larger
molecule
and
is
different
from
the
truncated
form
of
protein
expressed
in
Cry3Bb
transgenic
corn
plants.

In
the
summary
of
the
registration
application,
Monsanto
states
that
the
safety
of
corn
containing
Cry3Bb
proteins
to
mammals
rests
on:

­
Multiple
studies
showing
very
high
NOELS
("
No
Observable
Effect
Levels")
in
acute
feeding
studies
with
Section
2.3
2
­
25
a
variety
of
Cry
proteins
in
a
number
of
species.

­
Similarity
of
Cry3Bb
proteins
to
other
Cry
proteins
and
Bt
insecticides,
especially
Raven,
which
contains
a
mixture
of
two
Cry3
proteins
and
Cry1Ac.

­
A
40­
year
history
of
safe
use
of
Bt
insecticides
and
Cry
proteins.

There
are
indeed
very
wide
margins
of
safety
in
terms
of
acute
poisoning
when
mammals
ingest
Cry
proteins.
Acute
toxicity
should
still
be
investigated
through
dermal,
inhalation
and
dietary
routes
of
exposure,
given
the
high
expression
levels
in
MON
863
transformed
corn.
Allergenicity
should
also
be
explored
much
more
thoroughly.
The
submission
refers
to
a
review
of
a
database
of
known
allergens,
which
produced
no
evidence
of
a
match.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
33
Excerpt
Text:
There
is
little
known
about
the
degree
of
similarity
or
differences
between
Cry3Bb
proteins
and
other
Cry
proteins
in
terms
of
the
health
effects
of
greatest
concern,
which
would
include
allergenicity
and
digestive
system
impacts.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
34
Excerpt
Text:
The
third
point
is
also
not
reassuring.
The
American
population
has
not
been
extensively
exposed
to
any
Cry
proteins
over
40
years.
Exposure
has
been
almost
nonexistent
to
Cry3
proteins.
No
foliar
Bt
insecticide
containing
Bt
tenebrionis
has
achieved
significant
market
share
in
any
crop.
In
addition,
there
is
very
little
if
any
dietary
exposure
to
residues
left
after
foliar
Bt
applications.
Because
Bt
insecticides
break
down
quickly
when
exposed
to
sunlight
(
within
48
hours
in
most
cases),
residues
are
long­
gone
by
the
time
the
produce
reaches
consumers.

The
only
other
possible
route
of
exposure
since
1997
would
be
transgenic
potatoes
containing
Cry3A
proteins
(
New
Leaf
potatoes),
but
they
have
never
accounted
for
more
than
a
few
percent
of
national
potato
acreage.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
9
Excerpt
Text:
Comment
or
Question:
In
the
safety
assessment,
it
is
inappropriate
to
compare
the
Cry3Bb1
protein
produced
in
corn
event
MON
863
to
the
Cry3Bb
protein
present
in
the
commercial
microbial
Bt
pesticide
Section
2.3
2
­
26
trademarked
Raven.
This
product
contains
a
longer
Cry3Bb
protein
than
produced
in
MON
863.

Scientific
Background:
The
Cry3Bb1
protein
that
is
produced
in
corn
plants
is
structurally
identical
to
that
produced
in
Raven
(
differing
by
only
seven
amino
acids).
Therefore,
the
comparison
is
relevant.

Comment
or
Question:
Molecular
analysis
of
MON
863
reveals
a
"
missing"
10
base
pair
sequence
that
could
have
unpredictable
effects.
Furthermore,
there
is
a
lack
of
publicly
available
data
and
methods
to
determine
whether
the
"
missing"
DNA
may
have
inserted
into
another
location
of
the
genome.

Scientific
Background:
A
10
base
pair
DNA
sequence
present
within
the
transformation
cassette
was
lost
upon
stable
integration
of
the
cassette
into
the
plant
genome.
Southern
blot
analyses
using
probe
combinations
spanning
the
deleted
10
base
pair
region
did
not
detect
any
integration
of
the
10
base
pair
segment
elsewhere
in
the
genome.
The
10
base
pair
DNA
segment
most
likely
was
deleted
during
transformation
and
degraded
by
intracellular
nucleases.
Such
deletions
commonly
occur
as
a
consequence
of
DNA
repair
that
takes
place
at
the
integration
site,
as
well
as
in
regions
of
natural
strand
breakage
and
repair.

EPA
Response:
EPA
disagrees
with
the
Union
of
Concerned
Scientists
commenter
who
claims
that
EPA
relied
on
the
traditional
toxicity
studies
originally
submitted
by
Ecogen
for
the
Raven
Microbial
Product,
a
Bacillus
thuringiensis
strain
that
expresses
the
Cry3Bb1
toxin
for
the
safety
assessment
of
the
PIP
in
MON863
corn.
The
protein
that
was
used
as
test
substance
in
the
toxicity
tests
was
the
same
protein,
Cry3Bb1.11981
(
Q349R),
as
expressed
in
MON863.
The
Cry3Bb1.11981
(
Q349R)
protein
was
produced
in
a
Bacillus
thuringiensis
strain
but
was
shown
to
be
equivalent
to
the
Cry3Bb1
protein
in
corn.
This
information
is
also
presented
in
the
MON863
BRAD
in
Sections
II
A
and
II
B.

This
MON863
corn
plant
expressed
protein
differed
from
the
native
Cry3Bb1
protein
found
in
Bacillus
thuringiensis
var.
kumamotoensis
by
seven
amino
acids
but
was
not
truncated.
The
Cry3
proteins
are
not
like
the
lepidopteran
active
toxins
which
are
usually
expressed
as
an
inactive
protoxin
that
is
subsequently
degraded
or
enzymatically
cleaved
in
the
insect
midgut.
The
Cry3
proteins
are
expressed
in
an
active
form.
Some
confusion
may
have
arisen
from
the
citation
of
Ecogen
data
for
justifying
the
Experimental
Use
Permit
for
the
Cry
3Bb1
expressing
corn
varieties.
Section
2.4
2
­
27
2.4
­
Expression
Levels
2.4
­
Comment
Excerpts
Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
1
Excerpt
Text:
This
application
seeks
registration
of
Cry3Bb
protein
for
"
all
corn
lines
and
varieties,"
including
field
corn,
sweet
corn
and
popcorn.
The
later
two
crops
are
grown
for
direct
human
consumption.
Because
of
the
much
higher
expression
levels
of
this
protein
in
edible
grain
tissues
relative
to
earlier
Bt­
corn
events,
the
EPA
must
proceed
more
cautiously
and
thoroughly
in
its
scientific
assessment
of
the
human
allergenicity
and
food
safety
of
this
technology,
given
that
people
will
be
exposed
to
much
greater
quantities
of
Cry
proteins
than
arising
from
any
previously
reviewed/
approved
Bt
corn
technology.

EPA
Response:
EPA
disagrees
with
the
UCS
commenter
about
the
validity
of
the
safety
assessment
for
the
PIP
proteins
expressed
in
MON863.
The
toxicity
tests
are
done
with
purified
protein
at
levels
much
higher
than
those
expressed
in
the
plant.
EPA
believes
that
the
lack
of
toxicity
at
these
exaggerated
levels
also
covers
the
possibility
of
higher
expression
levels
in
most
PIP
expressing
plants.
See
BRAD
Section
II.
B.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
9
Excerpt
Text:
While
concerns
have
been
raised
before
regarding
human
dietary
exposure
to
NPTII
antibiotic
marker
gene
proteins
in
GMO
foods,
data
in
the
public
arena
suggest
that
the
levels
of
NPTII
expressed
in
edible
portions
of
previously
approved
Bt­
corn
plants
are
very
low.
The
Monsanto
submission
reports
higher
expression
levels
of
NPTII
proteins
than
earlier
Bt­
corn
events.

EPA
Response:
The
levels
of
NPTII
protein
in
other
approved
corn
varieties
is
indeed
very
low
as
stated
by
the
UCS
commenter
since
none
of
those
corn
PIPs
approved
by
EPA
contain
the
NPTII
marker
gene.
The
level
of
NPTII
protein
reported
in
MON863
corn
young
leaves
and
forage
was
similar
or
lower
than
levels
reported
in
other
PIP
products
expressing
NPTII.
The
NPTII
protein
level
reported
for
corn
grain,
the
most
likely
human
dietary
exposure,
is
below
the
limit
of
detection
for
MON
863
corn.
This
means
that
no
NPTII
protein
could
be
found
by
the
assay
which
detected
NPTII
as
low
as
0.76
micrograms/
gram
fresh
weight
tissue.
While
EPA
has
made
the
determination
that
the
aggregate
exposure
to
NPTII
as
a
marker
gene
is
safe
by
issuing
a
tolerance
exemption
(
40
CFR
180.1134),
the
data
presented
by
the
company
indicates
that
probably
no
exposure
to
the
NPTII
will
occur
through
consumption
of
MON863
corn.

Commenter
Name:
Rissler
Section
2.4
2
­
28
Commenter
Organization
Name:
UCS
Comment
Number:
30509B­
089000
Excerpt
Number:
5
Excerpt
Text:
A.
High
Expression
Levels
Pose
Serious
Risk
Concerns.
[
Footnote
11:
See
Appendix
A
for
additional
comments
on
expression
level.]

Cry
3Bb
expression
levels
in
virtually
all
MON
863
corn
tissues
are
several­
fold
higher
than
the
corresponding
levels
of
other
Bt­
endotoxins
in
today's
corn
hybrids
engineered
for
control
of
the
ECB.
The
Cry
3Bb
level
in
leaf
tissue
is
8
times
higher
than
the
level
of
Cry
1Ab
in
MON
810
transformed
corn
and
24.5
times
higher
than
Bt11
Cry
1Ab
(
see
ratios
in
bottom
five
lines
in
Table
2).

In
pollen,
the
level
of
Cry
3Bb
is
a
remarkable
5,391
times
higher
then
the
level
of
Cry
1Ac
in
DeKalb/
Mon
transformed
corn
and
1,240
times
the
level
of
Cry
1Ab
in
the
most
widely
planted
Bt­
ECB
corn
variety,
MON
810.

Table
2
reports
what
is
known
about
expression
levels
in
different
tissues
for
Cry
3Bb
transformed
corn,
as
well
as
most
Bt­
ECB
hybrids.
These
data
lead
to
two
conclusions
with
important
implications
for
risk.
First,
Cry
3Bb
expression
levels
in
leaf
tissue,
pollen,
grain,
and
in
the
whole
plant
exceed
the
levels
in
roots
during
most
of
the
growing
season.
Significantly,
the
level
in
grain
exceeds
the
level
in
roots
almost
threefold
at
harvest
time.
Early
in
the
season
when
levels
need
to
be
highest
in
roots,
levels
in
leaf
tissue
are
40
percent
higher
than
in
the
roots.
Levels
in
pollen
exceed
root
levels,
as
well.

The
high
level
of
Cry
3Bb
protein
in
MON
863
corn
is
not
the
only
expression­
related
issue
that
warrants
careful
review
by
EPA.
As
discussed
in
more
detail
in
subsequent
sections:

­
MON
863
corn
does
not
deliver
a
"
high­
dose"
for
corn
rootworm
resistance
management,
despite
relatively
high
expression
levels
in
several
tissues.

­
High
levels
are
expressed
in
tissues
where
the
toxin
is
not
needed
for
pest
control.

­
Cry
3Bb
toxins
continue
to
be
produced
by
the
plant
for
at
least
two
months
longer
than
needed
for
rootworm
control,
thereby
exposing
nontarget
organisms
to
unnecessary
risks
while
also
needlessly
taxing
the
plant.

­
Essentially
no
data
are
available
on
the
quantity
of
root
exudates
entering
the
soil,
information
that
is
essential
to
fully
assess
the
risks
posed
by
expression
levels
in
the
roots
themselves.

EPA
Response:
Toxicity
tests
are
done
with
purified
protein
at
levels
much
higher
than
those
expressed
in
the
plant.
EPA
believes
that
the
lack
of
toxicity
at
these
exaggerated
levels
also
covers
the
possibility
of
higher
expression
levels
in
most
PIP
expressing
plants.
See
BRAD
Section
II.
B.

The
above
concerns
were
addressed
by
the
maximum
hazard
dose
(
limit
dose)
toxicity
testing
performed
on
representative
beneficials
from
several
taxa
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
3.
a.
iv
and
Section
2.4
2
­
29
C.
I.
B).
Testing
at
the
maximum
hazard
dose
level
takes
into
account
the
variability
in
Cry
Bb1
protein
levels
in
Bt
corn
fields.
The
toxicity
of
the
Cry3Bb1
protein
has
been
evaluated
following
challenge
of
several
species
of
invertebrates
including:
adult
and
larval
honey
bees,
a
parasitic
hymenopteran
(
Nasonia),
green
lacewings,
lady
beetles,
collembola,
monarch
butterfly,
and
earthworms
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
3.
a.
iv
and
C.
I.
B).
Reproductive
and
developmental
observations
were
also
made
on
collembola,
honeybee
and
lady
beetle
larva
maturation
studies
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
3.
a.
iv
and
C.
I.
B).
Insect
host
range
bioassay
and
field
survey
data
were
used
to
assess
the
hazard
to
additional
species
not
directly
tested
in
the
laboratory.

Because
the
Agency
believes
that
additional
studies
would
be
useful
in
completing
the
database
for
long
term
effects
assessment,
it
is
requesting
additional
supplementary
studies
regarding
Cry3Bb1
protein
degradation
and
persistence
in
various
agricultural
soils.

The
Agency
agrees
that
a
high­
dose/
refuge
model
is
not
appropriate
for
MON
863
corn
since
there
is
not
a
"
high
dose"
of
Cry3Bb1
expressed
(
see
BRAD
Dose
Section
pp.
IID
4
to
IID
5).
The
IRM
strategy
currently
recommended
is
based
on
conservative
parameters
and
current
knowledge
of
corn
rootworm
biology
(
see
BRAD
Pest
Biology
Section
pp.
IID
2
to
IID
4
and
Simulation
Models
of
Resistance
Section
pp
IID
5
to
IID
8).
