Document ID: EPA-HQ-OPP-2005-0122-0012
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2005-07-26T04:00Z

­
1
­
UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
FEB
11
2005
OFFICE
OF
PREVENTION,
PESTICIDES
AND
TOXIC
SUBSTANCES
MEMORANDUM
SUBJECT:
Addendum
to
Product
Characterization
and
Expression
Analysis
for
Modified
Cry3A
(
mCry3A)
Bacillus
thuringiensis
insecticidal
protein
and
the
genetic
material
necessary
for
their
production
(
via
pZM26)
in
transgenic
maize
(
corn)
plants
derived
from
Syngenta
Seeds'
transformation
Event
MIR604,
EPA
Reg.
No.
67979­
EUP­
U
TO:
Mike
Mendelsohn,
Regulatory
Action
Leader
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

FROM:
Annabel
Fellman,
Environmental
Protection
Specialist
Microbial
Pesticides
Branch,
Biopesticides
and
[
signed]
Pollution
Prevention
Division
(
7511C)

THROUGH:
John
L.
Kough,
Ph.
D.,
Biologist
Microbial
Pesticides
Branch,
Biopesticides
and
[
signed]
Pollution
Prevention
Division
(
7511C)

ACTION
REQUESTED:
To
review
responses
to
data
deficiencies
noted
in
a
previous
data
submission
[
see
memorandum
from
A.
Fellman
through
J.
Kough
to
M.
Mendelsohn,
02/
11/
05]
in
support
for
an
Experimental
Use
Permit
and
a
temporary
exemption
from
tolerances
for
Modified
Cry3A
insect
control
protein
and
maize
(
Corn)
Plants
Derived
from
Event
MIR604,
submitted
by
Syngenta
Seeds,
Inc.­
Field
Crops
 
NAFTA.

CONCLUSION:
Based
on
the
review
of
the
data,
the
registrant
has
satisfactorily
addressed
the
agency's
concerns
and
the
responses
to
data
deficiencies
are
adequate
to
upgrade
the
previous
studies
classified
as
supplemental
to
acceptable.

*
THIS
REVIEW
DOES
NOT
CONTAIN
CONFIDENTIAL
BUSINESS
INFORMATION*
­
2
­
_______________________________________________________________________
DATA
REVIEW
RECORD:
Active
Ingredient:
Modified
Cry3A
(
mCry3A)
Bacillus
thuringiensis
insecticidal
protein
and
the
genetic
material
necessary
for
their
production
(
via
pZM26)
in
transgenic
maize
(
corn)
plants
derived
from
Syngenta
Seeds'
transformation
Event
MIR604.
Product
Name:
Event
MIR604
Maize
Plants
Expressing
Modified
Cry3A
Bacillus
thuringiensis
Protein
Company
Name:
Syngenta
Seeds,
Inc.
 
Field
Corps­
NAFTA
ID
No:
67979
Chemical
Number:
006509
Decision
Number:
________
DP
Barcode:
________

MRID
No:___________

Syngenta
Seeds'
Responses
to
Questions
Raised
Jan.
5,
2005
during
the
Review
of
Modified
Cry3A
protein
as
expressed
in
Event
MIR604
Maize,
EPA
Reg.
No.
67979­
EUP­
U
BACKGROUND:
During
the
course
of
the
agency's
review
of
the
studies
submitted
in
support
for
a
EUP
application
and
temporary
tolerance
exemption
for
Modified
Cry3A
protein
as
expressed
in
Event
MIR604
Maize,
several
data
deficiencies
were
identified
and
required
further
clarification,
justification,
and
corrections
for
them
to
be
considered
complete
and
acceptable.
These
deficiencies
were
presented
to
the
registrant
by
the
agency
on
January
5,
2005
via
conference
call.
As
per
the
agency,
the
registrant
has
submitted
a
compilation
of
each
data
clarification/
explanation
requested
in
a
single
MRID.

RECOMMENDATION:
ACCEPTABLE.
The
registrant
has
satisfactorily
addressed
the
agency's
concerns
and
the
responses
to
data
deficiencies
are
adequate
to
upgrade
the
previous
studies
classified
as
supplemental
to
acceptable.

SUMMARY
OF
DATA:

Several
questions/
concerns
were
presented
to
the
registrant
by
the
agency
on
January
5,
2005
via
conference
call.
These
questions
arose
during
the
course
of
the
agency's
reviews
of
the
following
studies,
which
the
registrant
submitted
in
support
of
an
EUP
application
and
temporary
tolerance
exemption
for
Modified
Cry3A
insect
control
protein
and
Maize
(
Corn)
Plants
Derived
from
Event
MIR604.
This
product
is
intended
to
provide
protection
against
western
and
northern
corn
rootworm
larvae.
As
per
the
agency,
the
registrant
has
submitted
a
compilation
of
each
data
clarification/
explanation
requested
in
a
single
MRID.
­
3
­
MRID
NO.
Syngenta
Response
to
Agency
Questions
MRID
No.
461556­
02
The
agency
had
concerns
regarding
similar
molecular
size
of
the
mcry3A
and
pmi
genes
in
the
Southern
analyses
of
the
pZM26
plasmid
using
probes
for
mcry3A,
pmi,
and
the
plasmid
backbones.
The
agency
requested
an
explanation
for
the
fortuitous
co
migration
of
the
two
genes
observed
on
the
gels.

The
registrant
claimed
that
the
resulting
hybridizing
bands
seen
in
the
mcry3A
and
pmi
Southern
analyses
of
Event
MIR604
actually
have
unique
molecular
weights.
This
conclusion
was
derived
from
closer
examination
of
the
resulting
hybridizing
bands
in
the
mcry3A
and
pmi
Southern
analyses,
which
were
<
5.5
to<
6.0
kb
and
>
5.0
to
<
5.5
kb,
respectively.

The
registrant
further
validated
this
conclusion
based
on
subsequent
analyses
of
the
Zea
mays
genomic
sequences
flanking
the
Event
MIR604
T­
DNA
insert.
It
revealed
the
presence
of
two
flanking
KpnI
recognition
sequences
as
well
as
a
single
KpnI
recognition
sequence
located
within
the
MIR604
T­
DNA
insert
(
see
Figure
1).
Based
on
this
observation,
the
registrant
reasoned
that
the
restriction
enzyme
digestion
of
Event
MIR604
with
KpnI
prior
to
Southern
analysis
should
predictably
result
in
a
band
of
5.6
kb
for
the
KpnI
restriction
fragment
containing
the
mcry3A
gene
and
a
band
of
5.2
kb
for
the
KpnI
restriction
fragment
containing
the
pmi
gene.
Since
the
respective
Southern
blots
of
Event
MIR604
with
mcry3A­
and
pmi­
specific
probes
did
result
in
hybridizing
bands
observed
to
match
the
molecular
weights
predicted,
the
registrant
has
provided
substantial
evidence
demonstrating
similar,
yet
unique,
molecular
weights
of
KpnI
restriction
enzyme
digests
using
mcry3A­
and
pmi­
specific
probes.

Figure
1.
Location
of
KpnI
restriction
sites
within
flanking
Zea
mays
genomic
sequences
and
within
MIR604
T­
DNA
insert
and
predicted
resultant
KpnI
restriction
digest
fragments.

mcry3A
(
1797
bp)
pmi
(
1176
bp)

5'
flanking
sequence
(
1452
bp)
3'
flanking
sequence
(
1766
bp)
ZmUbiInt
(
1993
bp)

MTL
(
2556
bp)
NOS
(
253
bp)

NOS
(
253
bp)

5.6
kb
KpnI
fragment
(
5611
bp)

5.2
kb
KpnI
fragment
(
5218
bp)
Kpn
I
(
681)
Kpn
I
(
6292)

Kpn
I
(
11510)
­
4
­
Reviewer's
Comment:
The
agency
recommends
the
registrant
provide
more
convincing
evidence
for
this
observation
by
staining
with
both
probes
and
showing
two
bands
occurring.
This
data
could
be
provided
in
support
for
Section
3
registration.

CLASSIFICATION:
ACCEPTABLE­
The
registrant
has
satisfactorily
addressed
the
agency's
questions.
In
addition,
MRID
No.
461556­
02
can
now
be
upgraded
to
acceptable.

MRID
No.
461556­
04
The
agency
had
concerns
with
the
absence
of
total
protein
determination
for
the
extracts
of
mCry3A
and
PMI
proteins
in
Event
604­
derived
corn.

The
registrant's
justification
for
this
report
was
that
the
total
protein
determination
was
superfluous
in
calculating
the
concentration
of
mCry3A
in
the
sample.
The
concentration
calculations
were
reported
as
µ
g
mCry3A/
g
fresh
wt
(
gfw),
which
are
converted
to
µ
g
mCry3A/
g
dry
wt
(
gdw)
by
using
the
%
dry
weight
(
calculated
by
weighing
the
sample
before
and
after
lyophilization).
The
registrant
further
explained
that
these
values
have
more
relevance
than
the
concentration
of
mCry3A
on
a
total
protein
basis,
and
are
consistent
with
previous
instructions
from
the
Agency
to
report
such
data
on
a
dry
weight
basis.

CLASSIFICATION:
ACCEPTABLE­
The
registrant
has
satisfactorily
addressed
the
agency's
questions.

MRID
No.
461556­
05
The
agency
had
several
questions
with
the
molecular
characterization
of
mCry3A
test
protein.
This
includes
the
absence
of
the
SDS­
PAGE
gel
data
and
Western
blot
data
confirming
the
molecular
size
of
the
long
and
short
forms
of
mCry3A
protein
as
well
as
the
unexpected
molecular
weight
of
the
test
protein
(~
60
kDa
instead
of
67.7
kDa)
discerned
from
the
SDSPAGE
blot.
Additional
concerns
were
presented
to
the
registrant
in
regards
to
the
control
mortality
data
for
the
insecticidal
bioassay.

The
registrant
claimed
that
the
difference
between
the
expected
molecular
weight
and
the
apparent
molecular
weight
of
mCry3A
using
SDS­
PAGE
is
due
to
the
inherent
lack
of
accuracy
in
the
technique.
The
lack
of
accuracy
materializes
because
proteins
of
widely
varying
amino
acid
compositions
may
complex
with
SDS
in
different
ratios,
leading
to
differential
mass­
to­
charge
ratios
and
anomalous
migration
behavior
upon
electrophoresis.
Moreover,
the
apparent
molecular
weight
of
the
target
protein
may
vary
depending
on
the
choice
of
molecular
weight
markers.
The
registrant
also
provided
a
reference
(
Dube
and
Flynn,
1998),
who
reviewed
the
limitations
of
SDS­
PAGE
for
molecular
weight
determinations,
and
indicated
that
the
apparent
molecular
weight
of
a
protein
by
this
method
is
typically
within
10%
of
its
true
molecular
weight.
Due
to
these
reasons,
the
registrant
utilized
mass
spectrometry
to
provide
much
higher
accuracy
for
molecular
weight
determination
than
is
possible
using
electrophoretic
techniques.

To
satisfy
the
requirement
of
visual
confirmation
of
the
molecular
weights
for
the
long
and
short
forms
of
mCry3A
protein,
the
registrant
provided
gels
from
the
SDS­
PAGE
and
­
5
­
western
blot
analysis
(
see
Figure
2).
These
gels
indicated
single
intense
bands
consistent
with
the
predicted
molecular
weights
of
ca.
69,500
Da
and
ca.
67,700
Da
for
the
mCry3A­
LF
and
mCry3A­
SF
samples,
respectively.

Figure
2.
SDS­
PAGE
and
Western
Blot
Analysis
of
the
Long
and
Short
Forms
of
mCry3A
Protein
(
samples
mCry3A­
LF
and
mCry3A­
SF)

A)
SDS­
PAGE
Lanes
1
and
3:
2.5
and
5
µ
g
mCry3A­
LF,
respectively
Lanes
2
and
4:
2.5
and
5
µ
g
mCry3A­
SF,
respectively
B)
Western
Blot
Lanes
1
and
3:
50
and
100
ng
mCry3A­
LF,
respectively
Lanes
2
and
4:
50
and
100
ng
mCry3A­
SF,
respectively
The
registrant
also
explained
that
in
MRID
461556­
06,
the
purpose
for
SDS­
PAGE
analysis
was
to
separate
the
two
different
forms
of
mCry3A
that
had
been
found
by
mass
spectrometry,
not
to
establish
their
molecular
weights.
Therefore,
the
mass
spectrometry
was
utilized
to
demonstrate
that
both
bands
represented
forms
of
mCry3A
and
more
importantly,
to
establish
the
molecular
weights
of
the
long
and
short
forms
more
accurately.

The
registrant
explained
the
observed
differences
in
the
estimated
LC50
values
were
due
to
the
inherent
variability
and
the
inability
to
full
optimize
the
western
corn
rootworm
(
WCRW)
bioassay.
Their
claim
of
quantitatively
inconsistent
results
from
day­
to­
day
were
substantiated
with
evidence
of
others
unable
to
establish
robust
WCRW
bioassays
(
Herman
et
al.,
2002)
or
descriptions
of
this
insect
as
delicate
and
difficult
to
work
with
148
98
64
50
36
22
1
2
3
4
1
2
3
4
A
B
­
6
­
(
English
et
al.,
2003).
Moreover,
despite
the
apparent
numerical
differences
in
the
estimated
LC50
values
reported
for
the
mCry3A
long
form
(
361
µ
g/
ml)
the
mCry3A
short
form
(
82
µ
g/
ml),
and
the
concurrent
bioassay
of
test
substance
MCRY3A­
0102
(
141
µ
g/
ml),
these
materials
were
considered
to
have
equivalent
bioactivity
because
of
overlapping
95%
confidence
intervals.
Thus,
the
confidence
intervals
reflect
some
of
the
variability
inherent
in
the
WCRW
bioassay.
Therefore,
due
to
the
particular
difficulties
and
variables
in
culturing
WCRW
larvae,
the
registrant
has
adequately
explained
the
observed
differences
noted.

CLASSIFICATION:
ACCEPTABLE.
The
registrant
has
satisfactorily
addressed
the
agency's
questions.
In
addition,
MRID
No.
461556­
05
can
now
be
upgraded
to
acceptable.

MRID
No.
461556­
06
The
agency
requested
further
documentation
for
the
presence
of
16
additional
amino
acids
at
the
beginning
of
the
N­
terminal
end
of
the
long
form
component
of
MCRY3A­
0102
test
substance.
The
identities
of
which
component
depicted
in
the
UV­
Absorbance
Spectra
was
also
requested.

In
response
to
the
agency'
concerns,
the
registrant
conducted
an
in­
depth
examination
of
the
nucleotide
sequence
of
the
cry1Ac
promoter
utilized
for
expression
of
the
mCry3A
protein
in
the
MCRY3A­
0102
test
substance.
An
"
ATG"
initiation
codon
sequence
was
identified
within
the
cry1Ac
promoter
indicating
the
presence
of
an
additional
putative
open
reading
frame
(
ORF)
(
see
Figure
3).
This
demonstrated
that
the
resultant
putative
ORF
would
be
in­
frame
with
the
mcry3A
gene
and
would
result
in
an
additional
16
amino
acids
on
the
N­
terminus
of
the
intended
mCry3A
protein
(
see
Figure
4).

Figure
3.
Graphical
representation
of
the
sequence
data
in
Figure
4
that
depicts
location
of
two
ATG
initiation
codons
in
the
E.
coli
over­
expression
plasmid
and
the
corresponding
N­
termini
of
the
long
and
short
forms
of
mCry3A
present
in
test
substance
MCRY3A­
0102.

Figure
4.
Nucleotide
sequence
of
cry1Ac
promoter
and
mcry3A
gene
in
E.
coli
over­
expression
plasmid
used
to
produce
test
substance
MCRY3A­
0102.
The
locations
of
two
"
ATG"
initiation
codons
in
the
E.
coli
plasmid
and
the
deduced
amino
acid
sequences
of
the
long
and
short
forms
of
short
form
mCry3A
long
form
mCry3A
Cry1Ac
promoter
ATG
ATG
­
7
­
mCry3A
present
in
test
substance
MCRY3A­
0102
are
shown.
ATG
codons
and
the
additional
16
amino
acids
found
in
the
long
form
of
mCry3A
are
indicated
with
the
following
text
format:
ATG.

cry1Ac
promoter
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
121
TGCATTTTTT
CATAAGATGA
GTCATATGTT
TTAAATTGTA
GTAATGAAAA
ACAGTATTAT
ACGTAAAAAA
GTATTCTACT
CAGTATACAA
AATTTAACAT
CATTACTTTT
TGTCATAATA
long
form
mcry3A
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
cry1Ac
promoter
short
form
mcry3A
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~
M
N
W
Y
L
N
K
R
D
G
G
L
N
G
S
T
M
T
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
181
ATCATAATGA
ATTGGTATCT
TAATAAAAGA
GATGGAGGTT
TAAACGGATC
CACCATGACG
TAGTATTACT
TAACCATAGA
ATTATTTTCT
CTACCTCCAA
ATTTGCCTAG
GTGGTACTGC
long
form
mcry3A
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
short
form
mcry3A
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
A
D
N
N
T
E
A
L
D
S
S
T
T
K
D
V
I
Q
K
G
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
241
GCCGACAACA
ACACCGAGGC
CCTGGACAGC
AGCACCACCA
AGGACGTGAT
CCAGAAGGGC
CGGCTGTTGT
TGTGGCTCCG
GGACCTGTCG
TCGTGGTGGT
TCCTGCACTA
GGTCTTCCCG
The
registrant
also
explained
that
the
identities
of
the
peaks
in
the
HPLC­
chromatogram
were
not
further
investigated
because
the
HPLC
analysis
was
utilized
as
an
additional
technique
to
demonstrate
that
the
test
substance
MCRY3A­
0102
contained
two
mCry3A
components.
However,
they
inferred
that
the
first
peak
(
11.868
min)
on
the
reversedphase
HPLC
chromatogram
is
the
long
form
of
mCry3A
because
it
was
the
most
abundant
form
identified
by
mass
spectrometry.

CLASSIFICATION:
ACCEPTABLE­
The
registrant
has
satisfactorily
addressed
the
agency's
questions.
In
addition,
MRID
No.
461556­
06
can
now
be
upgraded
to
acceptable.

MRID
No.
461556­
07
The
agency
had
questioned
the
mCry3A
protein
bands
showing
a
molecular
weight
around
~
90
kDa
instead
of
the
expected
molecular
weight
of
67.7kDa
in
the
Western
blot
analysis
in
Figure
3.
The
registrant
credited
this
inconsistent
finding
to
mislabeled
molecular
weight
standards.
It
should
have
been
labeled
210,
105,
78,
55,
45,
34,
17,
16,
7,
and
4
instead
of
250,
148,
98,
64,
50,
36,
22,
16,
6
and
4
kDa.
A
corrected
Figure
3
was
submitted
and
the
mCry3A
protein
occurred
between
the
standards
of
78
and
55
kDa
(
see
Figure
5).
Component
Basepair
Location
of
Component
cry1Ac
Promoter
33­
218
long
form
mCry3A
187­
2028
short
from
mCry3A
235­
2028
­
8
­
This
demonstrated
the
predicted
molecular
weight
of
ca.
67.7
kDa
for
the
mCry3A
proteins;
therefore,
the
registrant
has
adequately
addressed
the
agency
concerns.

Figure
5.
In
vitro
digestibility
of
plant
expressed
mCry3A
(
sample
IAPMIR604­
0103)
and
microbially
expressed
mCry3A
(
sample
MCRY3A­
0102).

Western
blot
analysis.
Lane
1:
Molecular
weight
standard
(
SeeBlue
®
Plus2,
Invitrogen);
Lanes
2
to
5:
IAPMIR604­
0103
in
SGF,
0,
2,
5,
10,
min,
respectively;
Lanes
6
and
11:
MCRY3A­
0102
in
G­
con,
0
and
60
min,
respectively;
Lanes
7
to
10:
MCRY3A­
0102
in
SGF,
0,
2,
5,
10
min,
respectively.
SGF
=
Simulated
Gastric
Fluid,
G­
con
=
SGF
without
pepsin.

CLASSIFICATION:
ACCEPTABLE­
The
registrant
has
satisfactorily
addressed
the
agency's
questions.
In
addition,
MRID
No.
461556­
07
can
now
be
upgraded
to
acceptable.

MRID
No.
461556­
09
The
agency
had
previous
concerns
with
the
lack
of
extraction
efficiencies
conducted
as
well
as
extraction
values
calculated
on
a
dry
weight
basis
in
the
analysis
for
the
presence
of
mCry3A
protein
in
wet
and
dry
milled
fractions,
corn
oil
and
corn
chips
from
processing
Event
MIR604
maize.

The
registrant
reported
that
the
mCry3A
values
would
not
have
been
significantly
different
if
reported
on
a
dry
weight
basis
because
all
the
processing
samples
in
solid
form
had
very
low
moisture
content
upon
receipt
(
ranging
from
3%
to
7%
moisture,
see
Table
1).
In
a
separate
study,
the
mCry3A
extraction
efficiency
for
most
corn
plant
tissues
(
kernels,
roots,
leaves,
whole
plants)
was
determined
to
be
70
to
77%
(
see
MRID
461556­
04).
The
registrant
reasoned
that
the
extraction
efficiencies
were
similar
for
these
samples,
because
1
2
3
4
5
6
7
8
9
10
11
210
105
78
55
45
34
17
16
7
4
mCry3A
mCry3A
breakdown
product
mol
wt
[
kDa]
­
9
­
the
same
extraction
procedure
(
SOP
2.58)
was
used
in
MRID
No.
461556­
09
for
the
analyses
of
mCry3A
in
all
samples
(
except
steep
water).

Table
1:
Percent
Moisture
Values
for
Wet­
and
Dry­
Milling
Fractions
of
Event
MIR604
Corn
CLASSIFICATION:
ACCEPTABLE­
The
registrant
has
satisfactorily
addressed
the
agency's
questions.

MRID
No.
464252­
01
The
agency
requested
a
more
detailed
explanation/
rationale
for
the
presence
of
Bovine
Serum
albumin
demonstrating
cross­
reactivity
with
the
 

­
pavalbumin
in
the
assessment
of
amino
acid
homology
of
PMI
with
known
allergens.

The
registrant
provided
three
possible
reasons
to
account
for
the
IgE
reactivity
between
the
BSA
and
the
serum
drawn
from
the
patient
with
confirmed
allergy
to
Rana
species
CH2001
 ­
parvalbumin
(
as
judged
by
Western
blotting),
which
are
as
follows:

1.
An
artefact.
This
is
very
unlikely
because
an
immunoblot
of
the
same
proteins
conducted
with
a
negative
control
serum
failed
to
display
any
reactivity
for
any
protein,
including
the
preparation
of
BSA.
Note:
the
commercial
sample
of
BSA
was
reported
by
the
supplier
to
have
98%
purity
(
Sigma­
Aldrich
Cat.
No.
A4503,
lot
no.
062K1064).

1
Values
are
from
raw
data
generated
by
the
processing
facility,
Iowa
State
University,
on
June
20,
2003.
2
Values
are
from
raw
data
generated
by
the
processing
facility,
Iowa
State
University,
on
April
15,
2003.
Dry­
Milled
Fractions1
%
Moisture
Flaking
Grits
7.09
Coarse
Grits
8.07
Fine
Grits
7.56
Corn
Meal
7.96
Corn
Cones
8.24
Corn
Flour
8.68
Hulls
7.05
Wet­
Milled
Fractions2
%
Moisture
Starch
7.38
Gluten
Meal
10.53
Germ
Meal
3.01
Coarse
Fiber
3.36
Medium
Fiber
3.43
Fine
Fiber
2.49
­
10
­
2.
Cross­
reactivity.
It
is
also
very
unlikely
that
there
exists
any
serological
reactivity
between
Rana
species
CH2001
 ­
parvalbumin
and
BSA.
There
is
very
little
homology
between
these
proteins
and
furthermore,
no
level
of
homology
that
would
signal
potential
cross­
reactivity.
An
amino
acid
homology
search
was
conducted
on
all
of
the
BSA
proteins
identified
in
the
NCBI
database3,
which
indicated
that
all
BSA
entries
are
highly
homologous
(>
99.5%
identity).
One
representative
BSA
sequence
(
Accession
No.
CAA41735)
was
arbitrarily
selected
for
an
amino
acid
sequence
comparison
with
Rana
species
CH2001
 ­
parvalbumin
(
Accession
No.
CAC83047)
by
FASTA
search.
The
global
alignment
of
the
two
proteins
indicated
2.1%
overall
sequence
identity
(
see
Figure
7A).
A
local
alignment
of
the
portion
of
the
BSA
sequence
(
residues
255­
364)
having
the
greatest
similarity
to
Rana
species
CH2001
 ­
parvalbumin
was
also
provided
and
indicated
less
than
12%
sequence
identity
(
see
Figure
7B).

3.
Co­
sensitization
The
most
likely
explanation
is
that
the
patient
with
confirmed
allergy
to
Rana
species
CH2001
 ­
parvalbumin,
and
whose
serum
was
used
for
these
investigations,
had
concomitant
sensitization
to,
and
IgE
antibodies
reactive
with,
BSA.
There
is
not
firm
evidence
for
this,
but
the
subject
from
which
the
serum
was
drawn
appears
to
have
been
atopic
because,
in
addition
to
his
reactivity
to
 ­
parvalbumin
from
Rana
species
CH2001,
there
was
evidence
(
from
specific
skin
prick
testing)
that
he
was
reactive
with
grass
pollen,
birch
pollen
and
hazelnuts
(
Hilger
et
al.,
2002).
Note:
BSA
protein
is
an
allergen
that
has
sometimes
been
associated
with
sensitization
to
beef
and
milk
(
Restani
et
al.,
2004;
Hilger
et
al.,
2001).

Dr.
Hilger,
who
conducted
this
study,
was
asked
by
Syngenta
to
comment
further
on
the
weak
reactivity
observed
between
the
frog­
allergic
patient's
serum
and
the
BSA
sample.
Dr.
Hilger
concurred
that
co­
sensitization
was
the
most
likely
explanation
for
the
reactivity.
She
has
additionally
tested
the
patient
for
beef
sensitivity,
and
observed
some
reactive
bands
upon
serum
testing
(
personal
communication;
Jan.
19,
2005).

Figure
6A.
FASTA
global
alignment
of
 ­
parvalbumin
from
Rana
species
CH2001
to
a
representative
bovine
serum
albumin
(
Accession
No.
CAA41735)

Result:
4.0%
positives
(
highlighted
in
green);
2.1%
identity
(
highlighted
in
yellow)

1
60
CAA41735­
BSA
(
1)
MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPF
CAC83047­
parvalbumin
(
1)
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
61
120
CAA41735­
BSA
(
61)
DEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEP
CAC83047­
parvalbumin
(
1)
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
121
180
CAA41735­
BSA
(
121)
ERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYY
CAC83047­
parvalbumin
(
1)
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
181
240
CAA41735­
BSA
(
181)
ANKYNGVFQECCQAEDKGACLLPKIETMREKVLTSSARQRLRCASIQKFGERALKAWSVA
3
Entrez
database
of
protein
sequences
maintained
by
the
(
US)
National
Center
for
Biotechnology
Information.
(
http://
www.
ncbi.
nlm.
nih.
gov/
entrez/
query.
fcgi?
db=
Protein)
­
11
­
CAC83047­
parvalbumin
(
1)
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
241
300
CAA41735­
BSA
(
241)
RLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKE
CAC83047­
parvalbumin
(
1)
­­­­­­­­­­­­­­
MPMTDVLAACDISKAMAAFPAAEPFNHKKFFELCGLKGKSQDDMKK
301
360
CAA41735­
BSA
(
301)
CCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRR
CAC83047­
parvalbumin
(
47)
VFHMLDKDQSGFIEKDELALILKGFTPEGRDLSDKETTALLAAGDKDGDGKIGVDEFVKL
361
420
CAA41735­
BSA
(
361)
HPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEK
CAC83047­
parvalbumin
(
107)
VSEC­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
421
480
CAA41735­
BSA
(
421)
LGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLIL
CAC83047­
parvalbumin
(
111)
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
481
540
CAA41735­
BSA
(
481)
NRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLP
CAC83047­
parvalbumin
(
111)
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
541
600
CAA41735­
BSA
(
541)
DTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVV
CAC83047­
parvalbumin
(
111)
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
601
CAA41735­
BSA
(
601)
STQTALA
CAC83047­
parvalbumin
(
111)
­­­­­­­

Figure
6B.
FASTA
local
alignment
of
region
of
greatest
similarity
between
 ­
parvalbumin
from
Rana
species
CH2001
and
residues
255
 
364
of
a
representative
bovine
serum
albumin
(
Accession
No.
CAA41735)

Results:
21.8%
positives
(
highlighted
in
green);
11.8%
identity
(
highlighted
in
yellow)

1
60
CAA76847­
BSA
(
255)
TKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKSHCIA
CAC83047­
parvalbumin
(
1)
MPMTDVLAACDISKAMAAFPAAEPFNHKKFFELCGLKGKSQDDMKKVFHMLDKDQSGFIE
61
110
CAA76847­
BSA
(
315)
EVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEY
CAC83047­
parvalbumin
(
61)
KDELALILKGFTPEGRDLSDKETTALLAAGDKDGDGKIGVDEFVKLVSEC
The
conclusion
drawn
is
that,
irrespective
of
likely
cross­
sensitization
of
the
serum
for
the
BSA
preparation,
the
evidence
indicated
that
there
is
no
serological
cross­
reactivity
between
Rana
species
CH2001
 ­
parvalbumin
and
PMI.

CLASSIFICATION:
ACCEPTABLE­
The
registrant
has
satisfactorily
addressed
the
agency's
questions.
In
addition,
MRID
No.
464252­
01
can
now
be
upgraded
to
acceptable.

CONCLUSION:

Based
on
the
review
of
the
data,
the
registrant
has
satisfactorily
addressed
the
agency's
concerns
and
the
responses
to
data
deficiencies
are
adequate
to
upgrade
the
previous
studies
classified
as
supplemental
to
acceptable.
­
12
­
REFERENCES
MRID
No.
461556­
05
Dube,
S.
and
E.
Flynn
(
1998)
Estimating
protein
molecular
weights
using
SDS­
PAGE.
Focus
20:
24­
25.
(
http://
www.
invitrogen.
co.
jp/
focus/
201024.
pdf)

English,
L.
H.,
S.
M.
Brussock,
T.
M.
Malvar,
J.
W.
Bryson,
C.
A.
Kulesza,
F.
S.
Walters,
S.
L.
Slatin,
M.
A.
Von
Tersch
and
C.
Romano
(
2003)
Nucleic
acid
compositions
encoding
modified
Bacillus
thuringiensis
coleopteran­
toxic
crystal
proteins.
U.
S.
Patent
No.
6,642,030
(
http://
www.
uspto.
gov).

Herman,
R.,
P.
N.
Scherer,
D.
L.
Young,
C.
A.
Mihaliak,
T.
Meade,
A.
T.
Woodsworth,
B.
A.
Stockhoff
and
K.
E.
Narva
(
2002)
Binary
insecticidal
crystal
protein
from
Bacillus
thuringiensis,
Strain
PS149B1:
Effects
of
individual
protein
components
and
mixtures
in
laboratory
bioassays.
J.
Econ.
Entomol.
95(
3):
635­
639.

MRID
No.
464252­
02
Hilger,
C.,
F.
Grigioni,
C.
De
Beaufort,
G.
Michel,
J.
Freilinger
and
F.
Hentges
(
2001)
Differential
binding
of
IgG
and
IgA
antibodies
to
antigenic
determinants
of
bovine
serum
albumin.
Clin.
Exp.
Immunol.
2001;
123:
387­
394
Hilger,
C.,
F.
Grigioni,
L.
Thill,
L.
Mertens
and
F.
Hentges
(
2002)
Severe
IgE­
mediated
anaphylaxis
following
consumption
of
fried
frog
legs:
definition
of
 ­
parvalbumin
as
the
allergen
in
cause.
Allergy
57:
1053­
1058.
Note:
A
copy
of
this
reference
was
provided
in
the
original
Syngenta
homology
reports
for
PMI
(
EPA
MRID
No.
46425201)
and
MIR604
PMI
(
EPA
MRID
No.
46425202).

Restani,
P.,
C.
Ballabio,
A.
Cattaneo,
P.
Isoardi,
L.
Terracciano
and
A.
Fiocchi
(
2004)
Characterization
of
bovine
serum
albumin
epitopes
and
their
role
in
allergic
reactions.
Allergy
59
(
Suppl.
78):
21
 
24