1
PRODUCT
CHARACTERIZATION
&

HUMAN
HEALTH
SAFETY
ASSESSMENT
FOR
STACKED
PLANT­
INCORPORATED
PROTECTANTS
June
8,
2004
2
OUTLINE
OF
PRESENTATION
°
Overview
of
food
safety
regulation
°
Active
ingredient
versus
new
product
°
Traditional
breeding
versus
transformation
°
Oral
Toxicity
not
Allergenicity
Assessment
is
subject
of
discussion
3
FEDERAL
OVERSIGHT
OF
FEDERAL
OVERSIGHT
OF
FOOD
SAFETY
FOR
FOOD
SAFETY
FOR
GENETICALLY
GENETICALLY­
ENGINEERED
ENGINEERED
PLANTS
PLANTS
°
Environmental
Protection
Agency­
Office
of
Pesticide
Programs
°
Food
and
Drug
Administration­
Center
for
Food
Safety
and
Applied
Nutrition
4
PLANT
PLANT­
INCORPORATED
INCORPORATED
PROTECTANTS
(

PROTECTANTS
(
PIPs
PIPs)

°
A
PIP
is
defined
as
the
pesticidal
substance
and
the
genetic
material
necessary
for
its
production
in
a
living
plant.

°
Genetic
material
is
essential
for
a
plant
to
express
a
new
pesticidal
substance.

°
The
basis
of
the
risk
assessment
is
the
hazard
of,
and
exposure
to,
the
pesticidal
substance.

°
New
active
ingredient
versus
new
product.

°
Transformation
versus
traditional
breeding.
5
PLANT
PLANT­
INCORPORATED
INCORPORATED
PROTECTANTS
(

PROTECTANTS
(
PIPs
PIPs)

°
Most
PIPs
have
marker
genes
associated
with
them
as
inert
ingredients.

°
Several
PIPs
have
more
than
one
active
pesticidal
substance.

°
Potato­
Cry3A
&
PLRV
replicase.

°
Corn­
Cry1Ab
&
Cry3Bb1­
YieldGard
Plus.

°
Cotton­
Cry1Ac
&
Cry1F­
WideStrike.

°
Cotton­
Cry1Ac
&
Cry2Ab2­
BollGard
II.
6
DATA
CATEGORIES
RELATED
TO
DATA
CATEGORIES
RELATED
TO
TOXICITY
&
DIETARY
SAFETY
OF
TOXICITY
&
DIETARY
SAFETY
OF
PROTEIN
PIPS
PROTEIN
PIPS
°
CHARACTERIZATION
OF
THE
PROTEIN.

°
PESTICIDAL
MODE
OF
ACTION.

°
AMINO
ACID
SEQUENCE
HOMOLOGY.

°
IN
VITRO
DIGESTIBILITY/
PROCESSING.

°
ACUTE
ORAL
TOXICITY.

°
ALLERGENICITY
DETERMINATION.
7
PROTEIN
TOXICITY
ASSESSMENT
ORAL
TOXICITY
°
Dietary
Proteins
Paradigm
°
Known
Protein
Toxins/
Longer
Term
Effects
°
Limit
Dose
(
Maximum
Hazard)
Approach
°
June
7,
2000
SAP
comments
on
protein
fragments
and
safety
factors
approach
8
PROTEIN
TOXICITY
ASSESSMENT
ACUTE
ORAL
TOXICITY
LIMIT
DOSE
°
Pure
protein
test
substance
°
Alternate
expression
vehicle
°
Dose
of
2­
5
g/
kg
bodyweight
°
Concentration
of
test
substance
°
Dose
volume
limitation
2.0ml/
100gm
9
WIDESTRIKE
COTTON
CRY1F
&
CRY1Ac
PROTEIN
TOXICITY
ASSESSMENT
°
Cry1Ac
tested
at
700mg/
kg
(
14%).

°
Cry1F
tested
at
600
mg/
kg
(
30%).

°
Combination
of
1Ac
and
1F
tested
at
350
and
375
mg/
kg
respectively.

°
No
adverse
effects
in
any
study.

°
No
similarity
to
any
toxins.

°
No
receptor
interaction/
Synergy.
10
DNA
/
AMINO
ACID
SEQUENCE
°
June
2000
SAP­
"
novel"
proteins
needed
greater
scrutiny
°
Changes
to
DNA
sequence
for
codon
preference
/
Southern
blot/
plasmid
map
°
Chimeric
Cry
proteins
for
protoxin
°
Full
amino
acid
(
AA)
sequence
data
is
always
available
for
the
introduced
trait/
expressed
protein
°
Confirmation
of
AA
sequence
by
Nterminal
or
MALDI­
TOF
11
AGRONOMIC
AGRONOMIC
CHARACTERISTICS/

CHARACTERISTICS/

CONSTITUENT
ANALYSIS
CONSTITUENT
ANALYSIS
°
APHIS/
CFSAN
data
°
Analysis
of
crosses
with
expected
Mendelian
ratios
°
Tissue
expression
over
time
°
Efficacy
and
other
performance
parameters
°
Fatty
acid/
gossypol
levels
12
CONCLUSIONS
°
No
signs
of
toxicity
from
the
submitted
tests.

°
No
significant
direct
exposure
to
Cry
proteins
in
cottonseed.

°
Data
for
individual
proteins
sufficient
for
human
dietary
hazard
assessment
unless
mode
of
action
indicates
binary
toxin
or
synergy.

°
Traditional
breeding
to
introduce
or
combine
traits
less
problematic
than
sequential
transformations.
13
QUESTION
1
°
The
Agency
examines
the
safety
of
proteins
based
on
the
source
of
the
protein,
the
protein's
pesticidal
mode
of
action,
comparisons
of
the
amino
acid
sequence
to
toxins
and
allergens
and
the
results
of
acute
oral
toxicity
testing.
The
company
provided
numerous
mammalian
oral
toxicity
studies
to
demonstrate
the
safety
of
the
introduced
Cry1Ac
and
Cry1F
protein
insecticidal
toxins.
The
toxins
were
tested
both
separately
and
in
combination.
The
Agency
believes
tests
with
combinations
of
pure
proteins
may
address
possible
synergistic
interactions
between
introduced
proteins.
14
QUESTION
1
(
continued)

°
However,
the
Agency
believes
that
unless
there
is
an
indication
that
the
two
proteins
would
interact,
such
as
being
parts
of
a
binary
toxin
or
attaching
to
the
same
receptor,
there
is
little
to
justify
testing
the
two
protein
together
when
separate
oral
toxicity
tests
indicate
a
lack
of
toxicity
for
the
individual
proteins.

°
Does
the
panel
have
additional
comments
on
this
position
including
instances
where
it
would
be
justified
to
require
the
toxicity
testing
of
two
proteins
in
combination?
15
QUESTION
2
°
When
traits
are
introduced
into
crop
plants
using
transformation
techniques
of
modern
biotechnology
of
even
traditional
breeding,

one
of
the
areas
of
concern
is
the
possibility
of
unintentional
changes.
There
is
a
general
difficulty
in
screening
for
these
changes
since
it
is
a
conceptual
leap
to
anticipate
the
unexpected.
However,
in
general
the
approach
has
been
to
examine
general
performance
of
the
new
cultivars
like
agronomic
performance
and
compositional
analysis
to
detect
unintentional
effects.
16
QUESTION2
(
continued)

°
PIP
products
can
be
both
transformed
lines
and
the
result
of
traditional
breeding
of
two
transformed
lines
to
yield
a
new
product
like
WideStrike
cotton.
In
both
cases,
the
new
PIP
product
must
be
registered
just
as
other
combinations
of
pesticide
active
ingredients
must
be
registered.

°
For
PIP
products
resulting
from
traditionally
bred
transformed
lines,
under
what
circumstances,
if
any,

would
it
be
appropriate
to
examine
agronomic
performance
and
compositional
analysis
to
provide
a
screen
for
unintentional
changes
in
the
crop?
Please
describe
other
ways
EPA
might
consider
screening
for
potential
unintentional
changes
in
a
crop.
