1
UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
OFFICE
OF
PREVENTION,
PESTICIDES
AND
TOXIC
SUBSTANCES
MEMORANDUM
SUBJECT:
Review
of
Comments
by
Valent
BioSciences
on
the
Tolerance
Exemption
for
Cry3Bb1
(
Docket
ID
Number
OPP­
2003­
0317)

TO:
Mike
Mendelsohn
Regulatory
Action
Leader
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

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

ACTION
REQUESTED:
To
review
the
comments
received
from
Valent
BioSciences
on
the
shortcomings
of
the
EPA
assessment
for
dietary
safety
of
Cry3Bb1.

SUMMARY
OF
COMMENTS
BY
VALENT
BIOSCIENCES:
The
commenter
has
maintained
that
the
basis
of
the
safety
assessment
for
the
Cry3Bb1
protein
is
the
expression
of
Cry
proteins
in
Bacillus
thuringiensis
microbial
pesticides
that
have
been
safely
used
for
over
40
years.
The
commenter
contends
that
the
strain
of
B.
thuringiensis
subspecies
kumamotoensis
has
never
been
registered
as
a
microbial
pesticide
and
therefore
Cry3Bb1
does
not
deserve
to
benefit
from
the
implied
40
years
of
safe
use
argument.

The
commenter
also
states
that
any
new
strain
of
B.
thuringiensis,
such
as
B.
thuringiensis
subspecies
kumamotoensis,
would
be
required
to
demonstrate
safety
through
new
data
on
mammalina
toxicology
and
pathogenicity
for
non­
target
organisms.

There
are
questions
raised
by
the
commenter
on
the
fact
that
the
Cry3Bb1
protein
subject
to
the
tolerance
determination
is
a
variant
of
the
natural
Cry3Bb1
endotoxin
engineered
specifically
to
enhance
activity
against
the
corn
rootworm
larvae
suggesting
that
the
binding
characteristics
have
2
been
altered.

The
results
of
the
three
variants's
toxicity
tests
indicate
a
toxicity
categoryIII
designation
with
accompanying
label
language
would
be
required.
The
commenter
is
concerned
about
these
toxicity
results
reflecting
negatively
on
the
currently
registered
microbial
Bt
use.

Finally,
the
commenter
is
concerned
that
the
validated
ELISA
method
for
detecting
Cry3Bb1
protein
would
distinguish
the
variant
from
the
natural
toxins.

CONCLUSION:
The
basis
of
the
Cry3Bb1
tolerance
determination
is
toxicology
data
that
has
been
generated
separately
from
any
registered
microbial
B.
thuringiensis.
While
there
has
been
the
reference
to
the
safe
use
of
microbial
formulations
in
both
the
reassessment
of
the
B.
thuringiensisbased
PIPs
and
more
recently
registered
PIPs,
all
of
these
PIP
proteins
have
had
extensive
mammalian
safety
data
generated
for
the
expressed
protein
itself.
Therefore,
reference
to
the
safe
use
of
microbial
B.
thuringiensis,
while
cogent
to
the
safety
assessment
to
inform
on
previous
exposure
to
the
Cry
proteins,
is
not
the
sole
basis
of
the
safety
finding
to
support
a
tolerance
exemption.

The
commenter
is
correct
that
the
specific
strain
of
B.
thuringiensis
subspecies
kumamotoensis
has
not
been
through
the
extensive
toxicity
and
pathogenicity
testing
required
for
a
microbial
pest
control
agent.
However,
a
Cry3B
protein
was
considered
as
a
part
of
the
assessment
of
the
Raven
B.
thuringiensis
subspecies
kurstaki
product
registered
by
Ecogen.

The
fact
that
all
three
variants
of
Cry3Bb1
protein
have
been
tested
for
toxicity
and
allergenicity
indicate
that
the
safety
of
these
three
variants
at
least,
is
similar
for
mammalian
species.
The
indication
of
these
test
results
is
that
minor
modifications
due
to
protein
engineering
for
enhanced
target
activity
does
not
necessarily
alter
non­
target
toxicity
for
mammalian
species.
This
supposition
does
not
mean
that
all
protein
engineering
modifications
would
result
in
equally
benign
results
for
non­
target
species.
No
insecticidal
activity
was
seen
in
specific
non­
target
insect
species
which
included
six
species
of
coleopteran
and
two
species
of
lepidopteran
pests
with
the
variant
Cry3Bb1
protein
suggesting
that
the
host
range
activity
is
limited.
There
are
also
results
from
bioassays
with
two
of
the
variant
Cry3Bb1
proteins
against
two
sensitive
coleopteran
species
(
Leptinotarsa
decemlineata
and
Diabrotica
virgifera)
that
indicates
that
there
are
not
significant
changes
in
the
activity
between
the
two
variants.

The
category
III
designation
for
the
results
of
the
acute
oral
toxicity
test
using
purified
Cry3Bb1
toxin
do
not
represent
any
change
from
the
results
that
would
be
seen
with
microbial
preparations.
The
category
classification
is
due
to
the
limitation
of
dose
volume
for
the
test
animal
rather
than
any
sign
of
toxicity
in
the
test
or
concern
for
possible
exposure.
The
oral
tests
were
done
with
purified
protein
doses
that
are
orders
of
magnitude
higher
than
would
be
seen
in
any
microbial
B.
thuringiensis
products.
Actual
exposure
to
Cry
proteins
in
PIP
products
are
not
expected
to
represent
any
hazard
of
oral
toxicity
given
the
results
seen
in
these
tests.

The
results
of
the
ELISA
assay
for
detection
of
the
Cry3Bb1
variant
protein
expressed
in
corn
indicate
that
the
test
does
detect
the
Cry3Bb1
protein
in
corn
grain
with
acceptable
variation
and
3
sensitivity.
However,
the
acceptance
of
the
ELISA
test
as
a
valid
analytical
method
is
contingent
on
the
further
testing
of
the
method
by
an
independent
laboratory
as
well
as
confirmation
of
the
test
by
the
EPA
laboratory
at
Ft.
Meade.
During
this
further
validation
process,
the
issue
of
specificity
of
the
reagents
for
the
Cry3Bb1
variant
protein
compared
to
other
proteins
that
may
be
present
in
commerce
needs
to
be
addressed.
