Document ID: EPA-HQ-OPP-2004-0395-0044
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2005-03-02T05:00Z

Scientific
Issues
Associated
with
the
Human
Health
Assessment
of
the
Cry34Ab1
Protein
Rebecca
Edelstein,
Ph.
D,
Chemist
EPA
Office
of
Pesticide
Programs
Biopesticides
and
Pollution
Prevention
Division
2
Background
°
Cry34Ab1
(
14
kDa),
Cry35Ab1
(
44
kDa)

°
Occur
naturally
in
the
soil
bacterium
Bacillus
thuringiensis
(
Bt)

°
Active
against
western
and
northern
corn
rootworm
larvae
3
Background
(
cont.)

°
Exact
mode
of
action
is
not
known,
but
appears
to
be
similar
to
other
Bt
Cry
toxins
 
Pore­
forming
toxins,
increase
the
permeability
of
the
plasma
membrane
of
midgut
epithelial
cells
in
susceptible
larvae
leading
to
larval
death
4
Regulatory
Status
°
Dow
AgroSciences
and
Pioneer
submitted
applications
to
commercialize
the
plantincorporated
protectants
(
PIPs)
Bt
Cry34Ab1
and
Cry35Ab1
and
the
genetic
material
necessary
for
their
production
in
corn
°
Permits
issued
for
evaluation
in
the
field
prior
to
commercialization
°
Temporary
tolerance
exemption
issued
°
Commercial
registration
application
still
under
review
5
Human
Health
Assessment:

Submitted
Data
°
Acute
oral
toxicity
studies
in
mice
using
high
doses
of
purified
proteins
 
No
treatment­
related
effects
observed
°
Heat
stability
studies
°
Amino
acid
sequence
similarity
comparison
°
Protein
characterization:
not
glycoslyated
°
Digestibility
studies
6
Issues
°
Cry34Ab1
appears
to
degrade
slower
in
simulated
gastric
fluid
(
SGF)
than
other
proteins
previously
registered
for
food
use
°
Applicants
have
asserted
that
Cry34Ab1
is
digested
rapidly
but
appears
to
digest
slower
than
other
proteins
because
of
differences
in
sensitivities
in
digestion
assays
°
Used
a
kinetic
approach
to
assess
the
degradation
of
Cry34Ab1
in
SGF
7
Charge
to
the
Panel
°
Comment
on
the
kinetic
approach
for
assessing
in
vitro
digestion
°
Advise
EPA
on
appropriate
protocols
for
in
vitro
digestibility
assays
°
Discuss
how
in
vitro
digestibility
assays
should
be
used
in
allergenicity
assessments
°
Comment
on
EPA's
allergenicity
assessment
of
Cry34Ab1
8
Outline
°
EPA's
approach
to
assessing
allergenicity
potential
of
proteins
°
In
vitro
digestibility
assays
 
Protocols
 
Studies
submitted
with
Dow
and
Pioneer's
applications
°
Allergenicity
assessment
of
Cry34Ab1
°
Questions
to
the
Panel
9
Allergenicity
Assessment
°
1994
Allergenicity
Conference
 
Methods
are
available
to
assess
allergenic
potential
for
proteins
derived
from
allergenic
sources
where
serum
from
sensitive
individuals
is
available
 
No
direct
methods
for
proteins
from
sources
that
are
not
known
to
produce
allergy
 
Assurance
that
a
protein
is
unlikely
to
cause
an
allergic
reaction
by
evaluating
its
similarity
to
known
allergens
10
Allergenicity
Assessment
 
1994
Conference
(
cont.)

°
No
single
factor
is
fully
predictive
°
Assessment
should
be
based
on
the
totality
of
available
information
11
Allergenicity
Assessment:
EPA's
Approach
°
Weight­
of­
the­
evidence
approach:

 
Source
of
the
trait
 
Sequence
comparison
to
known
allergens
 
Heat
stability
 
Post­
translational
modification
 
Digestibility
 
Prevalence
in
food
°
No
single
factor
fully
predictive
12
Allergenicity
Assessment:

Weight­
of­
Evidence
Approach
°
Approach
has
been
presented
at
previous
SAP
meetings
 
Approach
supported
by
the
Panels
°
2003
Codex
Alimentarius
Commission
guidelines:
Assessment
of
Possible
Allergenicity,
Annex
to
Guideline
for
the
Conduct
of
Food
Safety
Assessment
of
Foods
Derived
from
Recombinant­
DNA
Plants
 
Weight
of
evidence
approach
13
Digestibility
and
Allergenicity
°
Many
allergenic
proteins
or
their
digestion
fragments
stable
in
simulated
gastric
fluid
(
SGF)
for
>
60
minutes
°
Many
non­
allergens
degrade
in
SGF
within
seconds
°
Correlation
is
not
absolute
 
Some
allergens
digest
quickly
 
Some
non­
allergens
stable
14
Biological
Significance
of
Digestibility
and
Allergenicity
°
Rapid
digestion
minimizes
the
likelihood
that
a
protein
will
be
absorbed
and
elicit
an
allergic
response
°
Small
peptides
likely
incapable
of
causing
sensitization
°
Digested
proteins
capable
of
eliciting
allergic
response
in
sensitized
individuals
°
Pepsin
stability
may
reflect
resistance
to
cleavage
by
intracellular
proteases
 
Inherent
stability
to
digestion
may
influence
nature
of
immune
response
(
Dearman,
et
al.,
2002)
15
Typical
Digestibility
Assay
°
Incubate
protein
in
simulated
gastric
fluid
(
SGF):
acidic
solution
containing
pepsin
°
Time
for
protein
or
digestion
fragment
to
become
undetectable
monitored
°
Two
approaches:

 
Separate
reactions
for
each
time
point,
quenched
by
addition
of
base
at
appropriate
time
 
Single
reaction,
samples
removed
and
quenched
at
appropriate
time
°
Disappearance
of
protein
or
digestion
fragment
monitored
by
gel
electrophoresis
(
SDS­
PAGE)
16
Digestibility
Protocols
°
Protein
can
appear
to
be
resistant
to
digestion
or
rapidly
digested
depending
on
 
pH,
temperature,
stirring
 
Pepsin
and
substrate/
test
protein
concentrations
 
Purity
of
the
proteins
 
Specific
activity
of
pepsin
 
Amount
of
protein
loaded
on
gel
 
Sensitivity
of
detection
method
17
Digestibility
Protocols:
U.
S.

Pharmacopeia
(
USP)

°
Standardized
SGF
preparation
 
pH
approximately
1.2
 
Pepsin
(
derived
from
porcine
stomach
mucosa)
concentration:
3.2
mg/
mL
 
Test
protein
concentration
not
specified
 
Pepsin
specific
activity:
800
to
2500
units
per
mg
protein
°
Pepsin
activity
unit:
quantity
of
enzyme
that
digests
3000
times
its
weight
of
coagulated
egg
albumen
18
Digestibility
Protocols:
Astwood,

et
al.,
1996
°
SGF:
pH
1.2,
pepsin
concentration
0.32%
(
w/
v)

°
Test
protein:
170
ng/
µ
L,
pepsin
to
test
protein
ratio
=
20:
1,
w/
w
°
Separate
reactions
for
each
time
point
°
37
°
C,
shaking
water
bath
°
Pepsin
activity:
approx.
20,000
units
(
method:

Ryle,
1984;
TCA­
soluble
products
using
hemoglobin
as
substrate)

°
Tested
allergens
and
non­
allergens
 
large
database
19
Digestibility
Protocols:
ILSI
Study
(
Thomas,
et
al.,
2004)

°
SGF:
pH
1.2
and
2.0,
pepsin
concentration:
0.7
mg/
mL
°
10
activity
units
per
microgram
test
protein
(
activity
tested
by
enzyme
supplier,
Ryle
method)

°
Ratio:
pepsin
to
test
protein,
3:
1,
w/
w
°
Single
reaction
for
each
protein
tested
°
37
°
C,
no
shaking
°
Tested
on
10
proteins,
allergens
and
non­
allergens
°
Protocol
reproducible
and
gave
consistent
results
in
different
labs
20
Digestibility
Protocols:
2001
FAO/
WHO
Report
°
SGF:
pH
2.0,
pepsin
concentration
3.2
mg/
mL
°
Test
protein
concentration
2.5
mg/
mL
(
pepsin
to
test
protein
ratio:
1.3:
1,
w/
w)

°
Separate
reactions
for
each
time
point
°
37
°
C,
shaking
water
bath
°
Pepsin
activity
should
be
assessed
(
Ryle)

°
Untested
 
no
database
21
Digestibility
Protocols:
Previously
Registered
Products
°
SGF
prepared
according
to
USP
 
pH
1.2
 
Pepsin
concentration:
3.2
%
(
w/
v)

 
Pepsin
activity
often
not
specified
 
Different
test
protein
concentrations
(
pepsin
to
test
protein
ratio
range:
1600:
1
to
0.007:
1,

w/
w)
22
Cry34Ab1/
Cry35Ab1
Digestibility
Study
(
MRID
452422­
12)

°
Conditions:

 
pH
1.2
 
Pepsin:
0.3
%
(
w/
v)

 
Pepsin
to
Cry34Ab1
ratio:
60:
1
w/
w,
20:
1
mol/
mol
 
Pepsin
to
Cry35Ab1
ratio:
17:
1
w/
w,
20:
1
mol/
mol
 
Single
reaction
for
each
protein
tested
 
37
°
C,
no
shaking
°
Samples
taken
at
1,
5,
7,
15,
20,
30,
and
60
min
23
Cry34Ab1/
Cry35Ab1
Digestibility
Study
(
MRID
452422­
12)

°
Results:

°
Cry35Ab1:
SDS­
PAGE
and
western
blot:

undetectable
at
5
minute
time
point
°
Cry34Ab1:
SDS­
PAGE:
undetectable
at
20
minute
time
point;
western
blot:

undetectable
at
30
minute
time
point
24
Cry34Ab1
Digestion
 
Kinetic
Study
(
MRID
455845­
02)

°
Conditions:
same
as
previous
study
except
reaction
mixtures
shaken
during
incubation
°
Samples
taken
at
1,
2,
3,
5,
7.5,
10,
15,

and
20
minutes
°
SDS­
PAGE
analyzed
by
densitometry
°
Band
volume
directly
proportional
to
amount
of
protein
°
Decay
fit
to
first­
order
model
 
Linear
regression
used
to
calculate
DT50
and
DT90
25
Cry34Ab1
Digestion
 
SDS­
PAGE
Cry34Ab1
Figure
4,
MRID
455845­
02
26
Cry34Ab1
Digestion
 
Western
Blot
8.5
18
31
42.6
85
kDa
M
0
20
0
1
2
3
5
7.5
10
15
20
SGF
SGF
Figure
5,
MRID
455845­
02
27
Cry34Ab1
Degradation
Cry34Ab1
Digestion
y
=
­
0.3719x
+
4.3602
R
2
=
0.9783
1
1.5
2
2.5
3
3.5
4
4.5
5
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Minutes
ln(%

remaining)

DT
50:
2
minutes
DT
90:
6
minutes
Figure
1,
MRID
455845­
02
28
Comparison
Studies
 
Digestion
of
Allergens
and
Non­
Allergens
 
Kinetic
Approach
°
First
study
(
MRID
461239­
20)
used
results
from
ILSI
study
(
Thomas,
et
al.,
2004)

 
Conditions
different
from
those
used
for
Cry34Ab1
study
 
Tested
5
allergens
and
4
non­
allergens
 
Most
proteins
in
the
study
digested
either
too
quickly
or
too
slowly
for
determination
of
kinetic
rates
 
Of
the
rates
that
were
analyzed
kinetically,
some
had
good
fit
to
first­
order
decay,
while
others
had
poor
fit
29
Comparison
Study
°
Second
study
(
MRID
463886­
01)

 
Conditions
same
as
those
used
for
Cry34Ab1
 
SGF:
pH
1.2,
pepsin
concentration:
0.3%
(
w/
v),
test
protein
concentration:
0.002
mM
(
and
0.01
mM),

pepsin
to
test
protein
ratio:
20:
1
mol/
mol
 
Single
reaction
for
each
replicate,
37
°
C,
constant
shaking
 
Samples
taken
at
0.5,
1,
2,
5,
10,
15,
20,
30,
45,
and
60
min
 
Tested
7
allergens
and
8
non­
allergens
30
Kinetic
Approach
 
data
plot
Kidney
Bean
Lectin
0
50000
100000
150000
200000
0
10
20
30
40
50
60
minutes
net
band
volume
kidney
bean
lectin
fragment
Appendix
D,
MRID
463886­
01,
p.
45
31
Half
Life
of
Most
Persistent
Fragment
0
10
20
30
40
50
60
70
HRP
RUBISCO
soy
lipoxygenase
pea
lectin
ConA
kidney
bean
lectin
collagen
lima
bean
trypsin
inhibitor
lysozyme
Ova
OVM
Ara
h
2
BSA
BLG
STI
minutes
Ranges
reflect
digestions
at
0.002
and
0.01
mM
substrate
concentrations
allergens
non­
allergens
>
60
minutes
<
30
seconds
Appendix
F,
MRID
463886­
01,
p.
64
32
Comparison
Study
 
Results
°
Strong
correlation
between
digestion
rate
and
allergenicity
not
observed
°
Data
fit
well
to
first­
order
decay
model
except
for
early
time
points
°
Half­
lives
calculated
using
initial
substrate
concentrations
that
differed
by
5­
fold
fairly
consistent
°
Half­
lives
of
two
allergens
and
two
nonallergens
similar
to
half­
life
estimated
for
Cry34Ab1
33
Cry34Ab1
In
vitro
Digestibility
°
Digestion
appears
to
be
moderate:

 
Slower
than
other
proteins
previously
registered
for
food
use
(<
30
seconds
 
5
minutes)

 
Faster
than
most
tested
food
allergens
(>
60
minutes)

 
Slower
than
most
tested
non­
allergens
(<
30
seconds
 
5
minutes)
34
Kinetic
Approach:
Dow's
Assertions
°
First­
order
decay
predicted
as
long
as
substrate
concentration
low
(<<
Km)
and
pepsin
concentration
high
°
First­
order
rate
constant
equal
to
Vmax/
Km
°
Measures
inherent
digestion
efficiency
of
pepsin
for
a
particular
protein
35
Kinetic
Approach:
Dow's
Assertions
(
cont.)

°
As
long
as
first­
order
conditions
are
met,

firstorder
rate
constants
and
half­
lives
are
unaffected
by
changes
in
test
protein
concentration
(
and
pepsin
concentration
when
saturating)

 
First­
order
rate
constants
can
be
used
to
predict
relative
digestion
efficiencies
for
proteins
even
if
protein
and
pepsin
concentrations
are
both
varied
among
experiments
°
Should
improve
accuracy
of
determining
pepsin
digestibility
36
EPA's
Assessment
of
Kinetic
Approach
°
Less
dependent
on
sensitivity
of
assay
°
Results
still
dependent
on
reaction
conditions
(
pH,
temperature,

concentrations
and
purity
of
proteins)

°
Digestion
rates
depend
on
fit
to
first­
order
kinetics
 
Pepsin
hydrolysis:
multi­
step/
multi­
reaction
process
37
Pepsin
°
Endopeptidase
found
in
gastric
juice
 
Breaks
down
ingested
proteins
°
Aspartic
protease
 
two
active
site
aspartic
acid
residues
°
Substrate
binding
cleft
can
accommodate
7­
8
residues
°
Broad
substrate
and
peptide
bond
specificity
 
Preferentially
cleaves
at
aromatic
and
hydrophobic
residues
38
Pepsin
Hydrolysis:

Mechanisms
One­
by­
One/
Processive
Mechanism
39
Pepsin
Hydrolysis:

Mechanisms
Zipper
Mechanism
40
EPA's
Assessment
of
Kinetic
Approach
(
cont.)

°
At
pH
4.5,
pepsin
hydrolyzed
native
hemoglobin
using
one­
by­
one
mechanism
and
denatured
hemoglobin
by
zipper
mechanism
(
Choisnard
et
al.,
2002)

°
When
digestion
fragments
are
formed,
they
may
compete
with
the
full­
length
protein
and
inhibit
the
rate
of
decay
and
affect
fit
to
firstorder
kinetics
°
Comparison
studies
show
fairly
good
fit
to
firstorder
kinetics,
though
early
time
points
were
omitted
for
several
proteins
41
EPA's
Assessment
of
Kinetic
Approach
(
cont.)

°
Strong
correlation
between
half­
lives
and
allergenicity
not
observed
°
Current
protocol
for
kinetic
approach
only
applicable
to
proteins
with
moderate
digestibility
42
EPA's
Assessment
of
Kinetic
Approach
(
cont.)

°
Dow
has
stated
that
the
first­
order
rate
constant
=
Vmax/
Km
°
When
[
S]
<<
Km
and
catalytic
quantities
of
E
are
used,
first­
order
rate
constant
=
Vmax/
Km
°
Unclear
whether
the
relationship
is
the
same
under
high
[
E]
conditions
 
Standard
assumptions
to
derive
rate
equation
do
not
apply
°
Km
values
for
Cry34Ab1
and
proteins
in
comparison
studies
not
determined
43
Cry34Ab1:
Allergenicity
Assessment
°
Originates
from
a
non­
allergenic
source
°
No
sequence
similarity
with
known
allergens
°
Inactivated
when
heated
for
30
minutes
at
90
°
C
(
bioactivity
assay)

°
Not
glycosylated
°
Moderately
digested
in
SGF
°
Expressed
at
low
levels
44
Cry34Ab1
Allergenicity
Assessment
Conclusion
°
Using
the
weight­
of­
evidence
approach,

EPA
has
concluded
that
Cry34Ab1
is
unlikely
to
be
an
allergen
45
Summary
°
Scientists
at
Dow
have
developed
a
kinetic
approach
for
assessing
pepsin
catalyzed
protein
digestion
°
EPA
is
asking
the
Panel
to
comment
on
the
kinetic
approach
and
discuss
how
digestion
assays
should
be
conducted
and
used
in
allergenicity
assessments
Panel
Questions
47
Protocols
for
Digestibility
Assays:
Question
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
first­
order
kinetics
for
a
number
of
proteins.
However,
for
several
proteins,

initial
time
points
were
omitted
to
achieve
a
good
fit
to
the
model.
Dow
has
stated
that
the
data
were
not
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
firstorder
phase
of
digestion
(
Schnell
and
Maini,
2000;

Milgrom
et
al.,
1998)."
48
Question
1
°
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.
49
Question
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
first­
order
rate
constants?
50
Question
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
has
stated
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)?
51
Question
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?
52
Question
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?
53
Question
6
°
Typically,
scientists
have
used
SDSPAGE
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.
54
Question
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?
55
Question
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.
56
Question
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.
57
Question
9a
°
What
weight
should
in
vitro
digestibility
studies
be
given
in
the
overall
assessment
compared
with
other
criteria
such
as
sequence
homology?
58
Question
9b
°
The
Panel
is
asked
to
comment
on
the
appropriateness
of
setting
acceptable/
unacceptable
limits
for
digestibility
in
assessing
the
safety
of
a
protein.
59
Question
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.
60
Question
11
°
Cry34Ab1
appears
to
be
moderately
digested
in
SGF,

rather
than
rapidly
digested.
Considering
all
of
the
available
information
 
Cry34Ab1
originates
from
a
nonallergenic
source,
has
no
sequence
similarity
with
known
allergens,
is
not
glycosylated,
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.