Page
1
of
5
DRAFT
February
22,
2005
FIFRA
SCIENTIFIC
ADVISORY
PANEL
(
SAP)
OPEN
MEETING
MARCH
1­
2,
2005
FIFRA
SAP
WEB
SITE
http://
www.
epa.
gov/
scipoly/
sap/
OPP
Docket
Telephone:
(
703)
305­
5805
Docket
Number:
OPP­
2004­
0395
SCIENTIFIC
ISSUES
ASSOCIATED
WITH
THE
HUMAN
HEALTH
ASSESSMENT
OF
THE
CRY
34Ab1
PROTEIN
TUESDAY,
MARCH
1,
2005
Holiday
Inn
­
National
Airport
2650
Jefferson
Davis
Highway
Arlington,
VA
22202
Telephone:
(
703)
684­
7200

8:
30
AM
Introduction
and
Identification
of
Panel
Members
 
Stephen
Roberts,
PhD.
(
FIFRA
SAP
Session
Chair)


8:
45
AM
Administrative
Procedures
by
Designated
Federal
Official
 
Paul
Lewis,
PhD.


8:
50
AM
Welcome
 
Clifford
Gabriel,
PhD.
(
Director,
Office
of
Science
Coordination
and
Policy,
EPA)


8:
55
AM
Opening
Remarks
 
Janet
Andersen,
PhD.
(
Director,
Biopesticides
and
Pollution
Prevention
Division,
Office
of
Pesticide
Programs,
EPA)


9:
00
AM
Scientific
Issues
Associated
with
the
Human
Health
Assessment
of
the
Cry34Ab1
Protein
 
Rebecca
Edelstein,
PhD.
(
Office
of
Pesticide
Programs,
EPA)


10:
15
AM
BREAK

10:
30
AM
Public
Comments

12:
30
PM
LUNCH

1:
30
PM
Panel
Discussion
Protocols
for
Digestibility
Assays
1)
Dow
has
stated
that
enzyme
kinetic
theory
predicts
first
order
kinetics
for
pepsin
hydrolysis
under
conditions
of
high
enzyme
and
low
substrate
concentrations
and
has
demonstrated
that
the
rate
of
substrate
disappearance
under
these
conditions
follows
firstorder
kinetics
for
a
number
of
proteins.
However,
for
several
proteins,
initial
time
points
were
omitted
to
achieve
a
good
fit
to
the
model.
Dow
states
that
the
data
were
not
Page
2
of
5
included
"
based
on
theoretical
considerations,
which
include:
potential
zero­
order
or
mixed
order
kinetics
due
to
high
substrate
concentration,
possible
presence
of
denatured
and
highly
digestible
protein
contaminating
the
native
protein
preparation,
or
the
possibility
of
an
initial
burst
phase
or
transient
phase
preceding
the
first­
order
phase
of
digestion
(
Schnell
and
Maini,
2000;
Milgrom
et
al.,
1998)."

The
Panel
is
requested
to
comment
on
whether
the
explanation
justifies
omitting
early
time
points
or
whether
the
poor
fit
of
early
time
points
indicates
a
problem
with
the
model.

2)
Dow
has
asserted
that
first­
order
decay
is
predicted
based
on
enzyme
theory
as
long
as
the
pepsin
concentration
is
high
and
the
substrate
concentration
is
low
(<<
Km)
and
that
the
first­
order
rate
constant
determined
under
these
conditions
is
equal
to
Vmax/
Km.
Dow
has
also
stated
that
as
long
as
first­
order
conditions
are
met,
first­
order
rate
constants
and
half­
lives
are
unaffected
by
changes
in
substrate
protein
concentration
and
that
first­
order
rate
constants
can
be
used
to
predict
relative
digestion
efficiencies
for
proteins
even
if
the
protein
concentration
is
varied
among
experiments.
In
addition,
Dow
has
stated
that
at
the
USP
concentration
for
pepsin
of
0.32%,
the
enzyme
concentration
is
saturating
and
can
also
be
varied
between
experiments
without
affecting
the
first­
order
rate
constant.

The
Panel
is
asked
to
comment
on
these
statements.
How
much
can
the
pepsin
or
protein
substrate
concentrations
vary
without
affecting
the
kinetics
of
pepsin
digestion
and
firstorder
rate
constants?


3:
00
PM
BREAK

3:
15
PM
Panel
Discussion
(
continued)

3)
Typically,
for
comparing
the
in
vitro
digestibility
of
different
proteins,
researchers
have
used
fixed
concentrations
of
pepsin
and
substrate
protein
on
a
weight
basis
(
mg/
mL)
rather
than
adjusting
for
molecular
weight
of
the
substrate
protein,
presumably
because
larger
proteins
likely
have
more
potential
pepsin
cleavage
sites.
However,
Dow
states
that
"
while
multiple
pepsin­
labile
sites
may
occur
within
a
protein,
a
single
site
is
often
responsible
for
limiting
digestion
rates,
and
thus
the
number
of
molecules,
rather
than
total
weight,
is
most
often
more
influential
in
determining
the
kinetics
that
describe
decay."

The
Panel
is
asked
to
comment
on
Dow's
statement.
To
compare
the
rate
of
pepsin
digestion
of
different
proteins,
is
it
more
appropriate
for
the
concentration
of
test
protein
to
be
constant
on
a
weight
basis
(
mg/
mL)
or
a
mole
basis
(
mol/
L)?

4)
Typically,
researchers
have
looked
at
the
effect
of
pepsin
to
substrate
ratio
rather
than
concentrations
on
digestion
(
Karamac,
et
al.,
2002).
How
do
varying
the
ratios
and/
or
concentrations
affect
the
rate
of
hydrolysis?


4:
30
PM
ADJOURNMENT
Page
3
of
5
FIFRA
SCIENTIFIC
ADVISORY
PANEL
(
SAP)
OPEN
MEETING
MARCH
1­
2,
2005
FIFRA
SAP
WEB
SITE
http://
www.
epa.
gov/
scipoly/
sap/
OPP
Docket
Telephone:
(
703)
305­
5805
Docket
Number:
OPP­
2004­
0395
SCIENTIFIC
ISSUES
ASSOCIATED
WITH
THE
HUMAN
HEALTH
ASSESSMENT
OF
THE
CRY
34Ab1
PROTEIN
WEDNESDAY,
MARCH
2,
2005
Holiday
Inn
­
National
Airport
2650
Jefferson
Davis
Highway
Arlington,
VA
22202
Telephone:
(
703)
684­
7200

8:
30
AM
Introduction
and
Identification
of
Panel
Members
 
Stephen
Roberts,
Ph.
D.
(
FIFRA
SAP
Session
Chair)


8:
35
AM
Administrative
procedures
by
Designated
Federal
Official
 
Paul
Lewis,
PhD.


8:
40
AM
Follow­
up
from
Previous
Day's
Discussion
 
Rebecca
Edelstein,
PhD.
(
Office
of
Pesticide
Programs,
EPA)


9:
00
AM
Panel
Discussion
(
continued)

5)
Different
assays
exist
for
determining
pepsin
activity.
A
pepsin
activity
assay
based
on
measuring
the
trichloracetic
acid­
soluble
products
of
pepsin
hydrolysis
of
hemoglobin
is
provided
in
USP,
2004
under
the
entry
for
pepsin.
However,
the
entry
in
USP,
2004
for
"
gastric
fluid,
simulated"
references
the
Food
Chemicals
Codex
for
pepsin
activity,
which
provides
an
assay
that
measures
pepsin
digestion
of
egg
albumen.

The
Panel
is
asked
to
comment
on
the
appropriateness
of
using
a
fixed
concentration
of
pepsin
versus
using
a
fixed
specific
activity
of
pepsin
in
digestibility
protocols.
How
would
the
use
of
different
pepsin
activity
assays
affect
the
measured
pepsin
activity
units?

6)
Typically,
scientists
have
used
SDS­
PAGE
with
staining
or
western
blot
analysis
for
monitoring
digestion
reactions.
HPLC
is
also
sometimes
used.

The
Panel
is
asked
to
comment
on
the
pros
and
cons
of
the
different
methods
that
could
be
used
for
monitoring
digestion
reactions.


10:
30
AM
BREAK

12:
00
PM
LUNCH

1:
00
PM
Panel
Discussion
(
continued)
Page
4
of
5
7)
Some
researchers
have
used
one
digestion
reaction
and
removed
aliquots
at
various
times
for
monitoring,
while
others
have
set
up
separate
reactions
for
each
of
the
time
points.

What
are
the
pros
and
cons
of
these
approaches?

8)
Under
the
current
protocol,
Dow's
kinetic
approach
is
only
applicable
to
moderately
digestible
proteins
(
i.
e.,
using
Dow's
protocol,
many
proteins
digest
too
quickly
and
some
too
slowly
to
obtain
an
adequate
number
of
data
points
for
quantitative
kinetic
analysis).

Please
comment
on
the
usefulness
of
the
kinetic
approach
for
proteins
that
are
not
rapidly
degraded.


3:
00
AM
BREAK

3:
15
PM
Panel
Discussion
(
continued)

Allergenicity
Assessment
9)
The
2001
FAO/
WHO
report
and
2003
Codex
guidelines
both
recommend
using
in
vitro
digestibility
in
assessing
the
allergenicity
potential
of
a
protein.
The
FAO/
WHO
report
provides
a
"
decision
tree"
approach,
while
the
Codex
guidelines
suggest
a
weight
of
evidence
approach.
Codex
guidelines
state
"
resistance
of
a
protein
to
degradation
in
the
presence
of
pepsin
under
appropriate
conditions
indicates
that
further
analysis
should
be
conducted
to
determine
the
likelihood
of
the
newly
expressed
protein
being
allergenic,"
and
"
it
should
be
taken
into
account
that
a
lack
of
resistance
to
pepsin
does
not
exclude
that
the
newly
expressed
protein
can
be
a
relevant
allergen."
The
Codex
guidelines,
however,
don't
specify
how
a
protein
should
be
further
evaluated
if
it
is
"
resistant"
to
degradation,
and
"
resistant"
is
not
defined.

a)
What
weight
should
in
vitro
digestibility
studies
be
given
in
the
overall
assessment
compared
with
other
criteria
such
as
sequence
homology?

b)
The
Panel
is
asked
to
comment
on
the
appropriateness
of
setting
acceptable/
unacceptable
limits
for
digestibility
in
assessing
the
safety
of
a
protein.

10)
Stable
digestion
fragments
are
often
formed
during
pepsin
digestion
of
proteins,
and
Dow
has
used
the
kinetic
approach
to
estimate
the
half­
lives
of
several
digestion
fragments.

Please
comment
on
the
significance
of
the
rate
of
digestion
of
protein
fragments
for
allergenicity
assessments.

Cry34Ab1
and
Cry35Ab1
Assessment
11)
Cry34Ab1
appears
to
be
moderately
digested
in
SGF,
rather
than
rapidly
digested.
Considering
all
of
the
available
information
 
Cry34Ab1
originates
from
a
non­
allergenic
Page
5
of
5
source,
has
no
sequence
similarity
with
known
allergens,
is
not
glycoslyated,
is
inactivated
by
heat,
is
moderately
digested
in
SGF,
and
will
only
be
present
at
low
levels
in
food
 
EPA
has
concluded
that
Cry34Ab1
is
unlikely
to
be
a
food
allergen.

Please
comment
on
the
Agency's
conclusions
regarding
the
allergenicity
of
Cry34Ab1.


5:
00
PM
ADJOURNMENT
Please
be
advised
that
agenda
times
are
approximate.
For
further
information,
please
contact
the
Designated
Federal
Official
for
this
meeting,
Paul
Lewis
via
telephone:
(
202)
564­
8450;
fax:
(
202)
564­
8382;
or
email:
lewis.
paul@
epa.
gov
