1
Charge
to
the
Panel:
June
13­
15,
2006
FIFRA
Scientific
Advisory
Panel
Sampling
and
Methodology
1)
The
Panel
is
asked
to
comment
on
the
pheromone
sampling
strategy
employed
by
Monsanto
in
which
only
male
tobacco
budworm
(
TBW)
were
trapped.

Is
this
an
appropriate
sampling
strategy?
Can
inferences
about
female
TBW
be
derived
from
data
gathered
exclusively
with
males?

2)
Monsanto's
TBW
sampling
and
gossypol
analyses
were
conducted
over
a
two
year
period
(
2004
and
2005).
For
several
states
(
Tennessee
and
E.
Texas)
data
were
collected
in
only
one
year.
The
trends
between
seasons
were
generally
consistent,
although
no
statistical/
correlation
analysis
was
performed.

The
Panel
is
asked
to
comment
on
what
uncertainties
exist
from
using
data
collected
from
this
time
period
(
i.
e.,
2
years
for
North
Carolina
and
Georgia
and
1
year
for
Tennessee
and
E.
Texas)
to
adequately
assess
the
potential
of
natural
refuge
(
i.
e.,
non­
cotton
hosts)
as
a
substitute
for
structured
refuge
(
i.
e.,
non­
Bt
cotton)?

3)
In
some
counties/
states,
extremely
low
numbers
of
TBW
were
trapped,
with
some
traps
collecting
only
one
insect.
In
Tennessee,
TBW
numbers
were
so
low
that
data
were
not
reported
at
all
for
2004.
In
addition,
cotton
monitoring
efforts
have
been
recently
hampered
by
low
availability
of
TBW
samples
(
possibly
due
to
a
suppressive
effect
of
Bt
cotton).

Do
low
overall
numbers
of
TBW
trap
captures
in
some
areas
affect
the
ability
to
assess
the
effectiveness
of
natural
refuge
for
IRM?
What
conclusions,
if
any,
should
be
drawn
from
the
failure
to
capture
Bt­
susceptible
TBW
at
particular
sites?

Statistical
Analyses
4)
Monsanto
used
the
Fisher's
Exact
Test
to
determine
whether
the
gossypol
data
could
be
pooled.
Data
were
pooled
for
individual
traps
(
i.
e.
for
multiple
collection
dates
for
each
month)
and
for
counties
(
i.
e.
including
all
traps
within
a
county
for
each
month).

The
Panel
is
asked
to
comment
on
Monsanto's
approach
to
pooling
the
gossypol
data.

5)
Monsanto
did
not
conduct
any
statistical
analyses
comparing
the
two
sampling
years
(
2004
and
2005).
The
Panel
is
asked
to
comment
on
whether
valid
2
comparisons
(
on
a
qualitative
basis)
can
be
made
between
the
two
years
without
statistical
analyses?
Please
describe
any
meta­
statistical
analysis
that
could
improve
the
overall
understanding
of
the
effectiveness
of
natural
refuge
across
locations
and
across
time.

Effective
Refuge
Calculation
and
Modeling
6)
Monsanto
has
corrected
their
calculation
of
effective
refuge
size
presented
in
Gustafson
and
Head,
2004
based
on
the
Agency's
(
BPPD,
2004b)
and
June
2004'
s
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(
FIFRA)
Scientific
Advisory
Panel's
(
SAP)
recommendations
(
SAP,
2004).
Modifications
to
the
calculation
of
the
effective
refuge
size
involved
removing
the
assumption
of
constant
effective
refuge
size
and
explicitly
accounting
for
the
lower
production
of
CBW
and
TBW
in
cotton
where
survival
of
these
insects
is
reduced.
Estimation
of
the
effective
refuge
now
assumes
a
regionally
specific
annual
cycle
of
effective
refuge
size,
according
to
data
collected
in
alternative
host
studies
of
CBW
(
Head
and
Voth,
2004)
and
TBW
(
Head
and
Gustafson,
2005).
These
data
were
combined
with
corn
planting
estimates
on
either
the
regional
scale
for
CBW,
or
county­
scale
for
TBW,
to
estimate
effective
(
i.
e.,
current
(
structured
non­
Bt
cotton
+
non­
cotton)
and
natural
(
non­
cotton
only)
refuge
sizes
for
each
of
what
were
conservatively
assumed
to
be
six
annual
generations
for
each
pest.

a)
Estimation
of
the
relative
number
of
CBW
adult
moths
produced
by
each
of
the
five
sub­
compartments
is
given
by
the
following
equation:
Mij
=
Aij
Eij
LBij
LSij
(
Equation
1).
[
M
is
the
number
of
adult
moths
produced
per
unit
area
of
the
region;
A
is
the
proportion
of
the
region
occupied
by
the
crop
type
of
interest;
E
is
the
relative
(
to
cotton,
i.
e.
Ecotton=
1)
number
of
effective
eggs
(
eggs
that
would
produce
adults
in
the
absence
of
B.
t.
or
pyrethroid
sprays)
laid
in
the
crop
type;
LB
is
the
fraction
of
larvae
surviving
in
the
presence
of
the
B.
t.
crop;
LS
is
the
fraction
of
larvae
surviving
a
pyrethroid
insecticide
spray
on
the
crop;
the
subscript
i
refers
to
the
compartment
(
B
for
B.
t.
or
R
for
refuge);
and
the
subscript
j
refers
to
the
particular
crop
type
within
the
compartment
(
1
=
cotton,
2
=
corn,
3
=
other
C3
host
crop).]

The
effective
refuge,
Reff,
is
defined
as
the
proportion
of
adult
moths
that
would
have
been
produced
in
the
refuge
compartment
(
non­
Bt
cotton,
non­
Bt
corn,
non­
cotton
C3
crops)
in
the
absence
of
any
induced
larval
mortality:

Reff
=
2
1
3
2
1
3
2
1
B
B
R
R
R
R
R
R
M
M
M
M
M
M
M
M
+
+
+
+
+
+
(
Equation
5;
used
when
CBW
populations
were
actively
feedings
in
cotton,
Generations
3­
5)

Effective
refuge
estimations
for
all
of
the
"
non­
cotton"
generations
are
given
by:
3
NC
eff
R
=

2
3
2
3
2
B
R
R
R
R
M
M
M
M
M
+
+
+
(
Equation
6)

The
natural
refuge
component
(
i.
e.,
non­
cotton
C3
crops
+
non­
Bt
corn
components)
of
the
total
effective
refuge
is
as
follows:

CBW
nat
R
=

2
1
3
2
3
2
B
B
R
R
R
R
M
M
M
M
M
M
+
+
+
+
(
Equation
7)

The
Agency
asks
the
SAP
to
comment
on
the
estimated
CBW
effective
and
natural
refuge
calculations.

b)
Pooled,
county­
level
estimates
of
the
percent
cotton­
reared
TBW
moths
were
combined
with
county­
level
landcover
information
to
estimate
the
current
effective
refuge
and
natural
refuge
for
each
county
per
month.
The
relative
TBW
productivity
of
non­
cotton
areas
within
a
county
for
a
specific
month
is
given
as:

ENC
=

NC
NBTC
NBTC
NBTC
A
A
P
A
 
)
/
(
(
Equation
8)

The
current
effective
refuge
(
non­
Bt
cotton
+
non­
cotton
hosts)
for
TBW
is
defined
as
the
proportion
of
TBW
moths
actually
produced
in
the
effective
refuge
compartment
prior
to
selection
by
Bt
cotton:

TBW
eff
R
=
)
(
)
(

NC
NC
NBTC
BTC
NC
NC
NBTC
E
A
A
A
E
A
A
+
+
+
(
Equation
9)

The
estimated
natural
refuge
(
non­
cotton
hosts)
for
TBW
is
given
by
the
following
equation:

TBW
nat
R
=
)
(
NC
NC
NBTC
BTC
NC
NC
E
A
A
A
E
A
+
+
(
Equation
10)

The
Agency
asks
the
SAP
to
comment
on
the
estimated
TBW
effective
and
natural
refuge
calculations.

7)
Monsanto
examined
the
durability
of
each
of
the
three
Bt
cotton
products
(
i.
e.,
Bollgard,
WideStrike,
and
Bollgard
II)
individually
and
together
in
the
marketplace
using
a
three­
gene
model.
The
Bt
protein,
Cry1Ac,
is
common
to
all
three
products.
The
presence
of
each
of
these
products
in
the
marketplace
selects
4
for
potential
resistance
to
Bollgard
cotton,
expressing
only
the
Cry1Ac
protein,
and
also
selects
for
resistance
to
the
other
two
products
through
the
common
selection
for
Cry1Ac
resistance.
The
products
vary
greatly
in
the
rate
at
which
they
select
for
resistance
to
Cry1Ac
because
of
the
presence
of
additional
insecticidal
proteins
in
Bollgard
II
(
Cry2Ab2)
and
in
WideStrike
(
Cry1F).

The
three­
gene
model
for
insect
resistance
evolution
used
in
this
study
is
based
on
a
conceptual
model
similar
to
that
proposed
by
Dow
AgroSciences
(
DAS)
for
its
product,
WideStrike
cotton,
and
was
reviewed
by
a
recent
U.
S.
EPA
Scientific
Advisory
Panel
(
SAP)
(
SAP,
2004).
However,
the
SAP
questioned
some
of
the
mathematical
details
of
the
DAS
model
and
Monsanto
has
made
some
changes
to
address
the
SAP's
concerns.
As
shown
in
schematic
form
in
Figure
3,
Appendix
2,
the
three­
gene
model
is
based
on
the
following
assumptions
concerning
the
mechanism
of
activity
of
the
three
commercial
Bt
cotton
products
(
Bollgard,
Bollgard
II,
and
WideStrike
cotton):

 
The
Cry1Ac
toxin,
present
in
all
three
products,
binds
to
two
receptors,
60%
to
receptor
A
and
40%
to
receptor
B.
 
The
Cry1F
toxin,
present
only
in
WideStrike
cotton,
binds
exclusively
to
receptor
A.
 
The
Cry2Ab2
toxin,
present
only
in
Bollgard
II
cotton,
binds
exclusively
to
receptor
C.

a)
CBW.
Based
both
on
the
intrinsic
durability
of
each
of
the
three
B.
t.
cotton
products
(
Figure
4,
Appendix
2)
and
the
three­
gene
modeling
analyses
for
all
three
Bt
cotton
products
together
in
the
marketplace
(
Table
14,
Appendix
2),
Bollgard
II
retained
the
highest
level
of
efficacy
against
CBW
in
all
scenarios
(
all
regions).
Given
the
assumptions
of
the
three­
gene
model
and
its
limitations,
there
is
likely
enough
effective
natural
refuge
to
be
sufficient
to
delay
the
evolution
of
resistance
to
Bollgard
II
cotton
for
more
than
25
years
(
not
a
precise
number
of
years)
under
all
plausible
scenarios
in
all
four
regions
(
Table
14,
Appendix
2).
This
is
because
of
the
relatively
high
mortality
of
individuals
heterozygous
to
Cry1Ac
resistance
in
the
presence
of
Cry2Ab2,
as
compared
to
WideStrike.
WideStrike
is
intermediate
in
many
scenarios
because
of
the
shared
binding
receptor
between
Cry1F
and
CryAc
and
the
likelihood
of
cross­
resistance
is
greater.
Bollgard
is
weakest
in
all
scenarios
because
there
is
no
high
dose
for
CBW
and
it
is
a
single­
gene
product.
Monsanto's
models
predict
that
CBW
resistance
to
Bollgard
cotton
will
evolve
in
less
than
the
30
year
horizon
in
the
Georgia,
Mississippi,
and
E.
Texas
regions
in
most
scenarios
except
for
2­
C
(
Bollgard
=
0.1;
Bollgard
II
=
0.8;
WideStrike
=
0.1).
Resistance
always
took
at
least
30
years
to
evolve
to
all
three
Bt
cotton
products
in
the
North
Carolina
region
in
all
scenarios,
even
the
natural
refuge
scenarios.
When
Bollgard
cotton
acreage
is
minimized,
Bollgard
II
and
WideStrike
longevity
is
maximized
(
Table
14).
Large
amounts
of
Bollgard
II
cotton
in
the
marketplace
increased
the
durability
of
both
Bollgard
and
WideStrike
(
Table
14,
Appendix
2).
Uncertainties
in
the
pheromone
captures,
estimation
of
adult
productivity,
carbon
5
isotope
analyses,
spatial
analysis,
estimation
of
effective
refuge
calculation,
degree
of
shared
binding
affinity
of
Cry1Ac
to
receptor
A
and
B,
genetics
of
resistance,
resistance
mechanism,
initial
resistance
allele
frequency,
and
other
modeling
assumptions
affect
the
precision
and
accuracy
of
the
modeling
predictions.
Monsanto's
modeling
also
does
not
consider
pre­
selection
for
Cry1Ac
resistance.
Ten
years
of
selection
pressure
(
since
1996)
for
resistance
to
Cry1Ac
has
already
occurred.
Field
resistance
to
Cry1Ac
places
additional
selection
pressure
on
the
Cry2Ab2
component
of
Bollgard
II
cotton.

Given
the
assumptions
and
uncertainties
in
Monsanto's
CBW
modeling
efforts,
the
Agency
asks
the
SAP
to
comment
on
the
utility
of
the
modeling
to
predict
the
effectiveness
of
natural
(
non­
cotton
C3
crops
+
non­
Bt
corn)
vs.
current
effective
refuge
(
non­
Bt
cotton
+
non­
Bt
corn
+
non­
cotton
C3
crops)
to
manage
CBW
resistance
to
the
toxins
expressed
in
Bollgard
II.
Discuss
the
impact
of
preselection
for
Cry1Ac
resistance
on
the
modeling
output.

b)
TBW.
The
intrinsic
durability
of
all
three
Bt
cotton
products
is
much
greater
for
TBW
than
for
CBW
because
of
the
"
high
dose"
of
Cry1Ac
for
TBW
expressed
in
all
three
products.
In
virtually
all
cases,
all
three
products
retained
their
efficacy
(
i.
e.,
no
resistance)
for
more
than
30
years
(
maximum
time
for
the
simulation)
even
if
all
cotton
in
a
region
is
planted
to
that
product
and
no
structured
refuge
is
required
(
i.
e.,
all
natural
refuge).
The
only
exceptions
occur
for
Bollgard
cotton
in
Tennessee
and
Mississippi.
Given
the
assumptions
of
the
three­
gene
model
and
its
limitations,
there
is
likely
enough
effective
natural
refuge
to
be
sufficient
to
delay
the
evolution
of
resistance
to
Bollgard
II
cotton
for
more
than
30
years
(
i.
e.,
the
time
horizon
of
the
model,
not
to
be
interpreted
as
a
precise
number
of
years)
under
all
plausible
scenarios
in
all
four
regions.
This
is
due
to
the
extremely
high
efficacy
of
Cry1Ac
against
TBW,
and
the
fact
that
Cry1Ac
is
present
in
all
three
Bt
cotton
products.
In
the
state
with
the
lowest
natural
refuge
for
TBW,
Mississippi
(
see
Table
13,
Appendix
2),
resistance
to
Cry1Ac
and
Cry1F
evolved
after
21
years
in
scenario
1­
N
if
the
structured
refuge
requirements
for
Bollgard
and
WideStrike
cotton
were
removed.
Uncertainties
in
the
pheromone
captures,
gossypol
analyses,
spatial
analysis,
estimation
of
effective
refuge
calculation,
degree
of
shared
binding
affinity
of
Cry1Ac
to
receptor
A
and
B,
genetics
of
resistance,
resistance
mechanism,
initial
resistance
allele
frequency,
and
other
modeling
assumptions
affect
the
precision
and
accuracy
of
the
modeling
predictions.
Monsanto's
modeling
also
does
not
consider
pre­
selection
for
Cry1Ac
resistance.
Ten
years
of
selection
pressure
(
since
1996)
for
resistance
to
Cry1Ac
has
already
occurred.
Field
resistance
to
Cry1Ac
places
additional
selection
pressure
on
the
Cry2Ab2
component
of
Bollgard
II
cotton.

Given
the
assumptions
and
uncertainties
in
Monsanto's
TBW
modeling
efforts,
the
Agency
asks
the
SAP
to
comment
on
the
utility
of
the
modeling
to
predict
the
effectiveness
of
natural
(
non­
cotton
hosts)
vs.
current
effective
refuge
(
non­
Bt
cotton
+
non­
cotton
hosts)
to
manage
TBW
resistance
to
the
Bt
toxins
expressed
6
in
Bollgard
II
cotton.
Discuss
the
impact
of
pre­
selection
for
Cry1Ac
resistance
on
the
modeling
output.

8)
Modeling
suggests
that
the
overall
durability
of
Bollgard
II
cotton
can
be
enhanced
if
Bollgard
cotton
is
removed
from
the
marketplace.
This
conclusion
is
supported
by
other
researchers
who
examined
the
benefit
of
managing
resistance
evolution
to
two
toxins
with
dissimilar
modes
of
action
using
a
pyramided
approach
(
Zhao
et
al.,
2005;
Roush,
1998;
Livingston
et
al.,
2004;
Hurley,
2000;
Caprio,
2005).
On
the
other
hand,
the
concurrent
use
of
single­
and
two­
gene
Bt
plants
can
offer
exposed
populations
a
"
stepping
stone"
to
develop
resistance
to
both
proteins.
Bollgard,
Widestrike,
and
Bollgard
II
cotton
exist
in
a
mosaic
in
southeastern
cotton
growing
regions,
with
Bollgard
dominating
the
total
acreage.
In
2004,
Bollgard
cotton
acreage
accounted
for
>
95%
of
all
Bt
cotton
acreage
in
the
U.
S.
(
see
Head
et
al.,
2005,
MRID#
467172­
03).
Encouraging
the
adoption
of
Bollgard
II
will
increase
the
overall
durability
of
all
three
Bt
cotton
products.
From
an
insect
management
point
of
view,
removal
of
Bollgard
cotton
from
the
marketplace
would
benefit
the
two­
gene
products,
Bollgard
II
and
WideStrike.

The
Panel
is
asked
to
address
the
implications
for
selection
for
CBW
and
TBW
resistance
if
the
mosaic
of
single
gene
and
dual
gene
products
remains
in
the
marketplace
for
a
number
of
years.
How
would
selection
pressure
be
reduced
if
the
single
gene
product
is
removed
from
the
marketplace
gradually
(
e.
g.,
>
3
years)
or
rapidly
(
e.
g.,
 
3
years)
over
a
period
of
years?

Overall
Data/
Results
Interpretation
9)
There
are
three
major
variables
to
evaluating
structured
refuge
for
Bt
crops:
a)
production
of
a
sufficient
number
of
susceptible
insects
relative
to
any
resistant
survivors
of
the
Bt
crop,
b)
proximity
of
the
refuge
to
the
transgenic
crop
to
facilitate
random
mating
between
susceptible
(
from
the
refuge)
and
resistant
(
from
the
Bt
crop)
insects,
and
c)
developmental
synchrony
of
the
refuge
with
the
transgenic
crop
to
promote
random
mating.

Given
Monsanto's
sampling,
gossypol
analysis,
spatial
and
temporal
analyses,
and
modeling
evaluation,
the
Agency
asks
the
panel
to
comment
on
whether
Monsanto's
analysis
scientifically
supports
the
conclusion
that
natural
refuge
will
be
comparable
to
the
effectiveness
of
structured
refuge
for
management
of
TBW
resistance
to
the
Bt
proteins
expressed
in
Bollgard
II
cotton
for
each
of
the
four
regions:
the
Carolinas,
Georgia,
Mississippi
Delta,
and
Texas.
