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Bacillus thuringiensis var. tenebrionis - Experimental Use Permit Issuance and Amendment 6/93

Issuance of an Experimental Use Permit for Four Transgenic Plant Pesticides
AGENCY: Environmental Protection Agency (EPA).
ACTION: Issuance and amendment.
SUMMARY: On April 29, 1993, EPA issued an Experimental Use Permit
(EUP) to Monsanto Company to conduct field testing of four transgenic
plant pesticides. EPA has determined that this permit may be
of regional and national significance because it is the second
EUP approved under the Federal Insecticide, Fungicide, and Rodenticide
Act, for field testing altered plants having pesticidal properties.
The Office of Pesticide Programs (OPP) within EPA is responsible
for scientific review, risk assessment and issuance or denial
of EUPs. OPP has evaluated the data submitted by Monsanto and,
based on these data and other available data, can foresee no
significant risks to humans or to nontarget organisms from this
group of field tests as proposed by Monsanto. EPA's assessment,
however, is based solely on the EUP; eventual commercialization
of Monsanto's four transgenic potato pesticides may raise other
issues not addressed with this EUP. The permit was assigned
EUP number 524-EUP-79 and issued for 1 year, beginning April
29, 1993 and ending April 29, 1994; in accordance with 40 CFR
172.11(a), the Agency is soliciting public comments. On April
23, 1993, just prior to issuance of the EUP, Monsanto applied
to EPA for an amendment. This amendment was for the addition
of two sites thereby increasing the acreage for this EUP an
additional 0.03 acres; EPA has granted this amendment.

DATES: Written comments must be received on or before July 21,

ADDRESSES: Comments, in triplicate, should bear the docket control
number OPP-50753B and be submitted to: Public Response and Program
Resources Branch, Field Operations Division (H7506C), Office
of Pesticide Programs, Environmental Protection Agency, 401
M St., SW., Washington, DC 20460. In person bring comments to:
Rm. 1128, Crystal Mall #2, 1921 Jefferson Davis Highway, Crystal
City, VA 22202.

   Information submitted in any comment concerning this notice
may be claimed confidential by marking any part or all of that
information as "Confidential Business Information" (CBI).
Information so marked will not be disclosed except in accordance
with procedures set forth in 40 CFR part 2. A copy of the comment
that does not contain CBI must be submitted for inclusion in
the public record. Information not marked confidential may be
disclosed publicly by EPA without prior notice to the submitter.
Written comments will be available for public inspection in
Rm. 1128 at the address given above, from 8 a.m. to 4:30 p.m.,
Monday through Friday, excluding legal holidays.

Product Manager (PM) 18, Registration Division (H7505C), Office
of Pesticide Programs, Environmental Protection Agency, 401
M St., SW., Washington, DC 20460. Office location and telephone
number: Rm. 213, Crystal Mall #2, 1921 Jefferson Davis Highway,
Crystal City, VA, (703) 305-7690.

SUPPLEMENTARY INFORMATION: The permit was issued to Monsanto
Agricultural Company, 700 Chesterfield Village Parkway, St.
Louis, Missouri 63198. Monsanto is testing the Colorado Potato
Beetle (CPB) control protein, delta-endotoxin, derived from
the soil microbe Bacillus thuringiensis subspecies tenebrionis
(B.t.t.), as expressed in plants and tubers of several lines
of potato cultivars. According to the application, CPB control
protein, B.t.t. delta-endotoxin, will be present at no more
than .2 percent of the total weight of the potato plants or

   Some of the potato cultivar lines will contain only the B.t.t.
ë-endotoxin gene or an expression-enhancer fusion product of
B.t.t. for mediating Colorado Potato Beetle resistance. Other
potato cultivar lines have been modified to contain the B.t.t.
gene mediating Colorado Potato Beetle resistance and genes expressing
viral coat proteins mediating Potato Virus Y (PVY) or Potato
Leaf Roll Virus (PLRV) resistance.
   The ë-endotoxin as produced by the eight lines of genetically-
engineered potato plants will be evaluated for effectiveness
against the Colorado Potato Beetle (CPB), and its impact, if
any, upon nontarget insect species. The experimental program
is designed to evaluate the expressed CPB control proteins,
from the B.t.t. CryIIIA gene or the expression enhancer-CryIIIA
fusion product, for efficacy, agronomic evaluations, performance
confirmation, host plant resistance and population dynamics.
These experiments are designed to further evaluate the performance
of the expressed ë-endotoxin proteins against the Colorado Potato
Beetle in the various geographical areas in which potato is
commercially grown.
   In January of 1993, Monsanto amended their EUP application
with the elimination of Hawaii as a test site. A FR Notice announcing
this amendment was published on February 17, 1993. On April
23, 1993, Monsanto submitted a request to amend the EUP after
its issuance, to include additional sites in Idaho and Maryland.
Regarding Idaho, Monsanto is requesting to split the original
acreage to make two separate sites in order to reduce the potential
risk of frost damage and the possible spread of disease throughout
the site if it were to occur. The addition of the Maryland site
will increase the overall acreage of the EUP from 86.87 to 86.90
acres. According to the application, not more than 80 potato
plants of Russet Burbank variety expressing vector PV-STBT02,
which contains the B.t.t. gene only, will be planted at the
Maryland site.
   Monsanto's test sites are located in the following 13 States:
Colorado, Idaho, Maine, Maryland, Michigan, Montana, New York,
North Dakota, Ohio, Oregon, Pennsylvania, Washington and Wisconsin.
A maximum of 15,000 plants or tubers will be planted per acre,
each weighing approximately 5.6 grams per plant and 60 grams
per tuber. Some of the potato cultivar lines will contain only
the B.t.t. gene for mediating CPB resistance. Other potato cultivar
lines have been modified to contain genes mediating both CPB
and PYV or PLRV resistance. The total plant material, at planting,
will contain 129.31 grams B.t.t. protein, with levels rising
to a maximum of 39.4 kilograms of B.t.t. protein at harvest.
Likewise, the amount of viral coat protein, at planting, will
be approximately 0.005 grams of PLRV coat protein and 0.10 grams
of PVY coat protein; how much would be present at harvest is
not known, however, for the viral coat proteins.
   Upon completion of testing, some potato plants and tubers
will be collected and saved for future research, analyses or
plantings. All other plant material will be destroyed. Because
no plants or tubers will be used for food or feed, no tolerances
for this EUP are requested.
   The labeling states the following:

   The package contains Colorado potato beetle resistant potato
plants containing a Bacillus thuringiensis subspecies tenebrionis
protein. Contains potato variety _____ containing vector PV-
ST-TO-. For use only at an application site of a cooperator
and in accordance with the terms and conditions of the Experimental
Use Permit. This labeling must be in the possession of the user
at the time of planting of the potato plants or tubers. Not
for sale to any person other than a participant or cooperator
of the EPA-approved Experimental Use Program.

   1. Product Label One. Active Ingredient: IR-22, Bacillus
thuringiensis subspecies tenebrionis ë- endotoxin as produced
in potato by Cry IIIA gene and its controlling sequences and
found in the following constructs:
   PV-STBT02 ..........0.01 - 0.2 %*
   PV-STBT04 ............01 - 0.2 %*
   PV-STMT01 ..........0.01 - 0.2 %*

   2. Product Label Two. Active Ingredient: IR-23, Bacillus
thuringiensis subspecies tenebrionis ë-endotoxin as produced
in potato by an expression enhancer-CryIIIA fusion product and
its controlling sequences and found in the following construct:

   PV-STBT05............0.01 - 0.2 %*

   3. Product Label Three. Active Ingredients: IR-22, Bacillus
thuringiensis subspecies tenebrionis ë-endotoxin as produced
in potato by Cry IIIA gene and its controlling sequences and
found in the following constructs:
   PV-STMT02............0.01 - 0.2 %*

   PV-STMT04............0.01 - 0.2 %*
   PV-STMT10............0.01 - 0.2 %*
   PV-STMT11............0.01 - 0.2 %*
   PV-STMT12............0.01 - 0.2 %*
   PV-STMT13............0.01 - 0.2 %*
   PV-STMT14............0.01 - 0.2 %*

   Potato Leaf Roll Virus (PLRV) coat protein as produced in
potato by PLRV modCP gene and its controlling sequences and
found in the following constructs:
   PV-STMT02.............0.001 - 0.01 %*
   PV-STMT04. ...........0.001 - 0.01 %*
   PV-STMT10 ............0.001 - 0.01 %*
   PV-STMT11 ............0.001 - 0.01 %*
   PV-STMT12 ............0.001 - 0.01 %*
   PV-STMT13 ............0.001 - 0.01 %*
   PV-STMT14 ............0.001 - 0.01 %*

   4. Product Label Four. Active Ingredients: IR-22, Bacillus
thuringiensis subspecies tenebrionis ë-endotoxin as produced
in potato by Cry IIIA gene and its controlling sequences and
found in the following constructs:

   PV-STMT15............ 0.01 - 0.2 %*
   PV-STBT02 in combination with PV-STPY01....0.01 - 0.2 %*

   Potato Y Virus (PYV) coat protein as produced in potato by
PYV gene and its controlling sequences and found in the following

   PV-STMT15 ........... 0.006 - 0.1 %*
   PV-STPY01 in combination with PV-STBT02 .....0.006 - 0.1 %*

   [The active ingredient percentages are each asterisked (*)
to indicate that the values are percentages of total protein
on a dry weight basis.] It is a violation of Federal law to
use these plants or tubers in any manner inconsistent with this
labeling. This plant material contains Bacillus thuringiensis
subspecies tenebrionis insecticidal protein and may only be
used according to the protocols as included in the approved
EUP program for evaluation of the control of the following insect:
Colorado Potato Beetle/Leptinotarsa decemlineata

   Cooperators must have a copy of each applicable protocol
prior to initiating any research with these plants or tubers.
Potatoes should be planted at a maximum of 15,000 plants or
tubers per acre depending on the site variety. Do not contaminate
water, food, or feed by storage and/or disposal. Store in cool
dry place inaccessible to children. Any plants or tubers not
used in these experiments must be returned to Monsanto or disposed
of as specified in the field protocols. All plant material that
is not saved for further research analyses or future plantings
must be destroyed as specified in the field protocols. None
of the plants or plant material may be sold or allowed to enter
into commerce. Do not reuse bag. Discard in trash. Ensure that
the bag is completely empty of plants before disposing in the

EUP Program

   The EUP program will include the following five experiments
designed to evaluate the performance of the expressed protein
against the Colorado Potato Beetle: Efficacy and Agronomic Evaluations;
Performance Confirmation; Population Dynamics and Resistance
Management. In addition, seed increase trials will be conducted
in order to produce seed for future plantings. In keeping with
acceptable agronomic practices for each region, fertilizer,
herbicides, and fungicides will be used, if needed, to improve
soil nutrient levels, and to control weeds and diseases. If
CPB populations exceed economic threshold levels, additional
insecticides will be applied in accordance with local Integrated
Pest Management (IPM) practices to meet the objectives of the
experiment. Any conventional pesticides used in this EUP program
will be applied according to each pesticide's application rate
as specified on its label.
   The Agency has evaluated the potential for adverse effects
on nontarget species and the environment as a result of this
EUP. The Agency believes that the containment procedures as
described by Monsanto in their EUP application, and subsequently
modified by EPA are adequate to prevent any significant pesticide
production outside of the test site.
   The various toxins produced by Bacillus thuringiensis have
been studied extensively. Pending further testing to fully evaluate
any effect of the pesticidal toxin on human health and nontarget
environmental species, OPP has evaluated the exposure potential
to humans and nontargets only for this particular EUP. The amount
of toxin produced on these field test sites is not sufficient
to cause concern.
   Monsanto's use of viral coat proteins potentially raises
some issues; however, PVY viral coat protein requires an additional
protein called Helper Factor for transmission of the virus particles
by aphid vectors, and PLRV viral coat protein is believed to
require the presence of a readthrough product of the coat protein
gene for transmission by aphids; the additional protein and
gene were not transferred during the transformation process.
In addition, viral coat proteins are highly specific to plant
viruses, and have no apparent effect on nonviral organisms.
For these reasons, EPA believes that the PLRV and PVY coat proteins
that will be expressed by the various lines of transgenic potato,
for this EUP, present low potential risks. Because of the low
exposure, due to the limited acreage and duration of the EUP,
EPA believes that there will not be a situation warranting a
formal review under the Endangered Species Act for any endangered
mammals, birds, invertebrates, plants or aquatic species.
   Based upon EPA's scientific review of Monsanto's proposed
protocol and the scientific Peer Review recommendations, the
Agency is requiring the following protocol modifications to
Monsanto's Experimental Program.
   1. Fields planted in 1994 with transgenic tubers remaining
from the 1993 harvest, and/or fields which serve as a 1994 disposal
area for 1993 tubers, be monitored for volunteers during the
1995 growing season and any such volunteers destroyed if containment
of the potatoes to test sites is still required beyond the 1994
growing season.
   2. Monitoring for volunteer potato plants during the 1995
growing season will require that the fields in which the tests
were conducted during 1993 and the disposal areas for the 1993
tubers not be planted to nontransgenic potatoes in both 1994
and 1995.
   3. Disposal of potatoes by burying should be at a depth of
one foot or more to be effective.

Written Scientific Peer Review

   The Monsanto EUP application and OPP's Preliminary Scientific
Document were sent to four individuals in the scientific community
having specific expertise in biotechnology to obtain a "peer
review" of OPP's Scientific Position. For this EUP application,
the Agency asked these individuals to address specific risk
issues, containment provisions, and the protocol modifications
recommended by OPP. The following individuals provided a written
scientific peer review: George G. Kennedy, Ph.D., Department
of Entomology, North Carolina State University; Kathleen H.
Keeler, Ph.D., School of Biological Sciences, University of
Nebraska, Lincoln; Dr. Peter Palukaitis, Ph.D., Department of
Plant Pathology, Cornell University; Richard E. Wetzler, Ph.D.,
Center for Environmental Management, Tufts University.
   Dr. Kennedy concurred "with finding of negligible environmental
and health risks." Although Dr. Kennedy agreed with OPP's protocol
modifications, he recommended that it be explicitly stated that
the fields should not be planted to nontransgenic potatoes during
the 1994 and 1995 growing seasons because of the difficulty
in monitoring for transgenic volunteers.  Dr. Kennedy's recommendations
have been incorporated into EPA's protocol modifications.
   Dr. Keeler stated that she doubted "there are meaningful
problems associated with these field tests"; however, she did
recommend that disposal of potatoes by burying should be at
a depth of 1 foot or more to be effective. Dr. Keeler also recommended
that additional data be collected to facilitate an environmental
assessment; her recommendations have been incorporated into
EPA's protocol modifications. Dr. Keeler's recommendations for
additional data also have been forwarded to Monsanto.
   Regarding plant viruses and their transgenically expressed
coat proteins for this EUP-Dr. Palukaitis foresees "no problem
with the application as described." In addition, Dr. Palukaitis
described the inability of PLRV and PVY coat protein from being
transferred to other plants in the ecosystem by aphid vectors
due to the absence, in PVY, of an additional protein called
Helper Factor and, in PLRV, of the readthrough product of the
coat protein gene. Dr. Wetzler agreed with OPP's scientific
position that "risks associated with proposed releases or potential
accidental releases of the transgenic Solanum tuberosum (Potato)
are minor." Dr. Wetzler concurred with Dr. Kennedy's recommendations
to explicitly state that nontransgenic potatoes cannot be grown
in the fields where testing will be conducted during the 1994
and 1995 growing seasons. Lastly, Dr. Wetzler made some suggestions
regarding insect resistance testing and efficacy evaluation
which have been forwarded to Monsanto.

Interagency Coordination

   As per an August 11, 1987, Letter of Agreement, the Animal
and Plant Health Inspection Service (APHIS) of the United States
Department of Agriculture (USDA) and EPA have shared preliminary
assessments of Monsanto's application for field testing the
effectiveness and environmental impact of B.t.t. ë-endotoxin
when produced by potato. APHIS assessed the potential for plant
material to escape into the environment and the possible effects
an escape would have on other plant species; the APHIS assessment
concurs with EPA's on these issues.
   In addition, many States have passed biotechnology laws which
require Monsanto to submit a state application for a permit
prior to experimental use testing in the State. Monsanto has
been advised to consult with the appropriate regulatory agency
of each of the 13 States to determine if a State permit is necessary
prior to the onset of field testing.

Public Involvement

   Notice of receipt of Monsanto's EUP application was published
in the Federal Register on December 14, 1992, and provided a
30-day public comment period. Concurrently, Monsanto's application,
deleted of all "Confidential Business Information," was assigned
public docket number OPP-50753 and was made available for public
inspection in OPP's Public Docket Room. A second Federal Register
Notice, which extended the public comment period for another
30 days, was published on February 17, 1993 (58 FR 8758), announcing
Monsanto's amendment of their EUP application eliminating Hawaii
as a site. An amendment Notice creates a secondary docket, and
therefore, a companion docket was created using the Public Docket
number OPP-50753A. As a consequence, the second amendment, which
icreases the number of sites in Maryland and Idaho, creates
the Public Docket number OPP-50753B.
   Both of Monsanto's amendment requests, EPA's "Peer Review"
comments, and EPA's Final Scientific Position document can be
retrieved by the public using the docket numbers OPP-50753A
and OPP-50753B. Because most of the aforementioned documents
were not available for public review until after issuance of
the EUP, the comment period has been extended an additional
30 days.
   To date, only one comment was received by this Agency; the
commenter, an independent research advocate living in the State
of Washington, voiced concerns about applying "classical toxicology"
to bioengineered organisms to ensure their safety. Regarding
consumption of bioengineered foods containing pesticides, the
commenter voiced concern that EPA is not taking into consideration
in its risk assessment sensitive subpopulations, who may be
allergic or suffer from digestive tract disorder whereby this
technology could be life-threatening. The Tolerance Support
and Science Analysis Branches of the Heatlh Effects Division
of OPP have received copies of the commenter's letter and are
evaluating the concerns. Moreover, the commenter's comments
will be forwarded for consideration to FDA, USDA, and the Biotechnology
Risk Assessment Research Planning Group for the EPA Office of
Research and Development. Because this EUP will be conducted
in a crop destruct fashion, the commenter's concerns regarding
the consumption of bioengineered foods are not relevant to this
   In addition to the above comment, APHIS provided EPA with
a copy of comments they had received from the Director of the
Division of Plant Industry of the Maine Department of Agriculture.
The Director indicated that a subcommittee of the State of Maine's
Commission of Biotechnology and Genetic Engineering had a number
of reservations about the impact of [future] large-scale trials
on the potential for the Colorado Potato Beetle developing resistance
to the B.t.t. ë-endotoxin. They suggested that a risk assessment
be performed before these products approach commercialization.
The EPA has recently formed an OPP workgroup to examine the
issue of resistance induction and to determine the best regulatory
approach for EPA to adopt.

   Dated: June 10, 1993.

Lawrence E. Culleen,
Acting Director, Registration Division, Office of Pesticide

[FR Doc. 93-14561 Filed 6-18-93; 8:45 am]