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Bacillus thuringiensis CryIA(b) Delta-Endotoxin - Tolerance Exemption—Final Rule 5/95

ENVIRONMENTAL PROTECTION AGENCY
40 CFR Part 180
[PP 3F4273/R2132; FRL-4953-2]
RIN 2070-AB78
Plant Pesticide Bacillus Thuringiensis CryIIIA Delta-Endotoxin and the
Genetic Material Necessary for Its Production; Tolerance Exemption
AGENCY: Environmental Protection Agency (EPA).
ACTION: Final rule.
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SUMMARY: EPA is establishing an exemption from the requirement of a
tolerance for residues of the plant pesticide active ingredient
Bacillus thuringiensis CryIIIA delta-endotoxin and the genetic material
necessary for [[Page 21726]] its production in potatoes. The Monsanto
Co. requested this exemption from the requirement of a tolerance. This
regulation eliminates the need to establish a maximum permissible level
for residues of this plant pesticide in potatoes.

EFFECTIVE DATE: Effective on May 3, 1995.

ADDRESSES: Written objections and hearing requests, identified by the
document control number, [PP 3F4273/R2132], may be submitted to:
Hearing Clerk (1900), Environmental Protection Agency, Rm. M3708, 401 M
St., SW., Washington, DC 20460. A copy of any objections and hearing
requests filed with the Hearing Clerk should be identified by the
document control number and submitted to: Public Response and Program
Branch, Field Operations Division (7506C), Office of Pesticide
Programs, Environmental Protection Agency, 401 M St., SW., Washington,
DC 20460. In person, bring copy of objections and hearing requests to:
Rm. 1132, CM #2, 1921 Jefferson Davis Hwy., Arlington, VA 22202. Fees
accompanying objections shall be labeled "Tolerance Petition Fees"
and forwarded to: EPA Headquarters Accounting Operations Branch, OPP
(Tolerance Fees) P.O. Box 360277M, Pittsburgh, PA 15251.

A copy of objections and requests for hearings filed with the Hearing
Clerk may also be submitted electronically by sending electronic mail
(e-mail) to: opp-docket@epamail.epa.gov. Copies of objections and
requests for hearings must be submitted as an ASCII file avoiding the
use of special characters and any form of encryption. Copies of
objections and requests for hearings will also be accepted on disks in
WordPerfect in 5.1 file format or ASCII file format. All copies of
objections and requests for hearings in electronic form must be
identified by the docket number [PP 3F4273/R2132]. No Confidential
Business Information (CBI) should be submitted through e-mail.
Electronic copies of objections and requests for hearings on this rule
may be filed online at many Federal Depository Libraries. Additional
information on electronic submissions can be found below in this
document.

FOR FURTHER INFORMATION CONTACT: By mail: Willie H. Nelson,
Biopesticides and Pollution Prevention Division, Office of Pesticide
Programs, Environmental Protection Agency, 401 M St., SW., Washington,
DC 20460. Office location and telephone number: Rm. 51B6, CS #1, 2800
Crystal Drive, Arlington, VA 22202, (703)-308-8128; e-mail:
nelson.willie@epamail.epa.gov.

SUPPLEMENTARY INFORMATION: EPA issued a notice, published in the
Federal Register of December 8, 1993 (58 FR 64583), which announced
that the Monsanto Co., 700 Chesterfield Village Parkway, St. Louis, MO
63198, had submitted a pesticide petition, PP 3F4273, to EPA requesting
that the Administrator, pursuant to section 408(d) of the Federal Food,
Drug and Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), establish an
exemption from the requirement of a tolerance for the plant pesticide
Bacillus thuringiensis subsp. tenebrionis (B.t.t) Colorado potato
beetle (CPB) control protein (CryIIIA).

EPA has assigned the active ingredient of this product the name
Bacillus thuringiensis CryIIIA delta-endotoxin and the genetic material
necessary for its production. "Genetic material necessary for
production" means the CryIIIA gene and its regulatory regions.
"Regulatory regions" are the genetic material that control the
expression of the gene, such as promoters, terminators, and enhancers.
Monsanto has genetically modified potato plants to produce the
pesticidal protein derived from the common soil bacterium Bacillus
thuringiensis subsp. tenebrionis. The protein produced by CPB-resistant
potatoes is identical to that found in nature. Monsanto has genetically
engineered potatoes by using plant-expressed vectors that transferred
the CryIIIA and neomycin phosphotransferase II (nptII) marker gene into
the genomic DNA of the potato plants. In the Federal Register of
September 28, 1994 (59 FR 49353), EPA exempted nptII and the genetic
material necessary for its production in or on all raw agricultural
commodities when used as an inert. There were no adverse comments or
requests for referral to an advisory committee received in response to
the notice of filing of the petition, PP 3F4273 (58 FR 64582, Dec. 8,
1993).

Residue Chemistry Data

Residue chemistry data were not required because of the lack of
toxicity to this active ingredient. This is similar to the Agency
position regarding the submission of residue data for the microbial
Bacillus thuringiensis products from which this plant pesticide was
derived. (See 40 CFR 158.740(b).) For microbial products, residue data
are required only when Tier II or III toxicology data are required. The
kinds of studies submitted for this plant pesticide are like those in
Tier I, not Tiers II or III. Submitted data indicated that the product
is of low mammalian toxicity/pathogenicity and the kinds of studies
required in Tier II or III were not appropriate. Therefore, no residue
data are required in order to grant an exemption from the requirement
of a tolerance for Monsanto's plant pesticide, Bacillus thuringiensis
Cry IIIA delta-endotoxin protein, the CryIIIA gene and the genetic
material necessary for its production in potato.

Product Analysis

Monsanto submitted information which adequately described the CryIIIA
delta-endotoxin from B.t.t., as expressed in potato, along with the
genetic material necessary for its production. Because it would be
difficult, or impossible, to extract sufficient biologically active
toxin from the plants to perform toxicology tests, Monsanto used delta-
endotoxin produced in bacteria. Product analysis data were submitted to
show that the microbially expressed and purified CryIIIA delta-
endotoxin is sufficiently similar to that expressed in the plant to be
used for mammalian toxicological purposes.

1. Molecular characterization of CPB-resistant Russet Burbank Potatoes
equivalence of microbially produced B.t.t. protein. The relative size
and number of copies of the DNA inserted into potatoes was demonstrated
with endonuclease digested chromosomal DNA from field- grown potato
plants southern blotted with the entire introduced plasmid PV-STBT02 as
the probe. These southern blots provided information about the number
of copies of introduced DNA, the lack of significant amount of DNA
introduced outside the border regions, and integrity of the introduced
DNA near the endonuclease cut site. These results indicate only that
the DNA necessary to produce the CryIIIA delta endotoxin were
introduced into the plant, thus indicating that exposure would only be
to the CryIIIA delta-endotoxin and the nucleic acids found in the
genetic material necessary for its production. Such nucleic acids have
not, by themselves, been associated with toxic effects to animals or
humans and are regular constituents of the human diet.

2. Equivalence of microbially produced and plant-produced B.t.t.
protein also called Colorado potato beetle active protein from Bacillus
thuringiensis subsp. tenebrionis. Microbially produced delta endotoxin
from the CryIIIA gene as expressed in Escherichia coli and in potato
tubers were compared. The data consists of SDS-PAGE comigration,
Western blot analysis, staining for carbohydrate residues, N-terminal
amino acid sequence analysis, and biological [[Page 21727]] equivalence
against Leptinotarsa decemlineata. These data are adequate to support
the equivalence of the microbially produced and plant-produced protein
for use in the toxicology studies.

3. Characterization of the major tryptic fragment from Colorado potato
beetle active bacillus thuringiensis subsp. tenebrionis. The purity and
activity of a 55kD protein released with tryptic digestion of the
B.t.t. delta endotoxin purified from E. coli was shown to have a
similar size, immunoreactivity, and amino acid sequence to the 55kD
fragment found in potato tubers. The 55kD protein had somewhat higher
bioactivity than the 68kD full-length delta endotoxin from B.t.t. These
data support the contention that both the 55kD and 68kD forms of the
CryIIIA delta-endotoxin found in the plant were similar to those
occurring in B.t.t.

4. Characterization of Colorado potato beetle active bacillus
thuringiensis subsp. tenebrionis protein produced in escherichia coli.
The method of preparing by fermentation the delta endotoxin from B.t.t.
in E. coli was presented. The protein was characterized for purity and
stability after purification. These data indicate that normal
fermentation techniques were used to produce the plant equivalent,
microbial CryIIIA delta-endotoxin.

5. Compositional comparison of Colorado potato beetle (CPB) active
bacillus thuringiensis subsp. tenebrionis proteins produced in CPB-
resistant potato plants and commercial microbial products. The CryIIIA
delta-endotoxin as expressed in potato tissue or an E. coli alternative
gives a similar immunoreactivity and electrophoretic mobility to
registered microbial products producing the same delta-endotoxin.

Toxicology Assessment

Toxicity

The delta-endotoxin proteins of B. thuringinesis have been intensively
studied, and no indications of mammalian toxicity have been reported.
Furthermore, approximately 176 different B. thuringiensis products have
been registered since 1961, and the Agency has not received any reports
of dietary toxicity attributable to their use. This is especially
significant because FIFRA section 6(a)(2) requires registrants to
report any adverse effects to EPA. Therefore, EPA does not expect any
mammalian toxicity from this protein in plants based on the use history
of B. thuringiensis products.

The data submitted by Monsanto support the prediction that this protein
would be nontoxic to humans. Adequate information was submitted to show
that the test material derived from microbial cultures was essentially
identical to the protein as produced by the potatoes. Production of a
plant equivalent, microbial CryIIIA delta-endotoxin, was chosen to
obtain sufficient material for mammalian testing. In addition, the in
vitro digestibility studies indicate the protein would rapidly be
degraded following ingestion.

The genetic material necessary for the production of the Bacillus
thuringiensis CryIII(A) delta endotoxin are the nucleic acids (DNA and
RNA) which comprise the CryIII(A) gene and its controlling sequences.
DNA and RNA are common to all forms of life, including plants, and the
Agency knows of no instance where these nucleic acids have been
associated with toxic effects related to the consumption of food. These
ubiquitous nucleic acids as they appear in the subject active
ingredient have been adequately characterized by the applicant.
Therefore, no mammalian toxicity is anticipated from dietary exposure
to the genetic material necessary for the production of the Bacillus
thuringiensis CryIII(A) delta endotoxin in potatoes.

Allergenicity

Despite decades of widespread use of Bacillus thuringiensis as a
pesticide (it has been registered since 1961), there have been no
confirmed reports of immediate or delayed allergic reactions from
exposure. Such incidents, should they occur, are required to be
reported under FIFRA section 6(a)(2) and as a data requirement for
registration of microbial pesticides (40 CFR 158.740 and Subdivision M
of the FIFRA testing guidelines, NTIS # PB89-211676).

Studies done in laboratory animals or as reported in the literature
also have not indicated any potential for allergic reactions to B.
thuringiensis or its components, including the delta-endotoxin in the
crystal protein. Recent in vitro studies also confirm that the delta
endotoxin would be readily digestible in vivo.

Current scientific knowledge suggests that common food allergens tend
to be resistant to degradation by heat, acid, and proteases, are
glycosylated, and are present at high concentrations in the food. The
delta endotoxins are not present at high concentrations, are not
resistant to degradation by heat, acid and proteases, and are
apparently not glycosylated when produced in plants. The company has
submitted data to indicate that the CryIIIA delta endotoxin is rapidly
degraded by gastric fluid in vitro, is not present as a major component
of food, and is apparently nonglycosylated when produced in plants.

Submitted Data

1. Acute oral toxicity of B.t.t. protein. The B.t.t. proteins were
determined to be stable and the dosing concentrations were determined
to be 74.9 mg/mL, 14.62 mg/mL, and 7.4 mg/mL. B.t.t. protein was not
toxic by oral gavage when mice were dosed with up to 5220 mg/kg body
weight. These results placed this protein in Tox Category IV.

2. In-vitro digestibility of B.t.t. protein. The 68 kD and 55kD B.t.t.
proteins degraded within 30 seconds in simulated gastric fluid when
analyzed by western blot and were not active against Colorado potato
beetles after degradation. The 68kD B.t.t. protein degraded to 55kD
within 2 hours of incubation in simulated intestinal fluid. The 55 kD
form remained unchanged after 14 hours of incubation and retained its
bioactivity and western blot results. These results indicate that,
following ingestion by humans, the B.t.t. proteins will be degraded
like other proteins to amino acids and peptides similar to those
occurring in a normal human diet.

Scientific Advisory Panel Subpanel on Plant Pesticides

A Subpanel of the FIFRA Scientific Advisory Panel (SAP) met on March 1,
1995, to discuss the Agency's Preliminary Scientific Review for this
use and concluded that "The Monsanto B. t. potato presents little
potential for human dietary toxicity. At a dose of one million- fold
greater than that contained in a potato (a 150-gram potato contains
about 300 micorgrams B.t. protein, 70 kg person = 4.5 micrograms/kg),
no toxicity was observed. Moreover, several studies of B.t. potatoes
are indistinguishable from strains of wild-type potatoes in nutritive
content (total protein, total sugars, vitamin C, minerals, etc.).
Furthermore, the B.t. toxin is rapidly digested by pepsin and is
inactivated by heat encountered in cooking."

Conclusions

In summary, based upon the submitted studies and other available
information, the Agency does not foresee any human health hazards from
the use of the Bacillus thuringiensis CryIII(A) delta-endotoxin and the
genetic material necessary for its production.

Based upon submitted data and a review of its use, EPA has found that
when used in accordance with good [[Page 21728]] agricultural practice,
this ingredient is useful for the purpose for which the tolerance
exemption is sought. Based on the information considered, EPA concludes
that a tolerance is not necessary to protect the public health.
Therefore, the exemption from the requirement of a tolerance is
established as set forth below.

Acceptable daily intake (ADI) and maximum permissible intake (MPI)
considerations are not relevant to this petition because the data and
information submitted demonstrate that this active ingredient is not
toxic to mammalian species. No enforcement actions are expected, based
upon the toxicity for this plant pesticide. Therefore, the requirement
for an analytical method for enforcement purposes is not applicable to
this exemption request.

Any person adversely affected by this regulation may, within 30 days
after publication of this document in the Federal Register, file
written objections and/ or a request for a hearing with the Hearing
Clerk, at the address given above (40 CFR 178.20). A copy of the
objections and hearing requests filed with the Hearing Clerk should be
submitted to the OPP docket for this rulemaking. The objections
submitted must specify the provisions of the regulation deemed
objectionable and the grounds for the objections, and must conform to
the other requirements of 40 CFR 178.25. Each objection must be
accompanied by the fee prescribed by 40 CFR 180.33(i). If a hearing is
requested, the objections must include a statement of the factual
issue(s) on which a hearing is requested, the requestor's contentions
on each such issue, and a summary of any evidence relied upon by the
objector (40 CFR 178.27). A request for a hearing will be granted if
the Administrator determines that the material submitted shows the
following: There is a genuine and substantial issue of fact; there is a
reasonable possibility that available evidence identified by the
requestor would, if established, resolve one or more of such issues in
favor of the requestor, taking into account uncontested claims or facts
to the contrary; and resolution of the factual issue(s) in the manner
sought by the requestor would be adequate to justify the action
requested (40 CFR 178.32).

A record has been established for this rulemaking under docket number
[PP 3F4273/R2132] (including copies of any objections and requests for
hearings submitted electronically as described below). A public version
of this record, including printed, paper versions of electronic
comments, which does not include any information claimed as CBI, is
available for inspection from 8 a.m. to 4:30 p.m., Monday through
Friday, excluding legal holidays. The public record is located in Rm.
1132 of the Public Response and Program Resources Branch, Field
Operations Division (7506C), Office of Pesticide Programs,
Environmental Protection Agency, Crystal Mall #2, 1921 Jefferson Davis
Highway, Arlington, VA.

An electronic copy of objections and requests for hearings can be sent
directly to EPA at:
opp-Docket@epamail.epa.gov.

A copy of electronic objections and requests for hearings must be
submitted as an ASCII file avoiding the use of special characters and
any form of encryption.

The official record for this rulemaking, as well as the public version,
as described above will be kept in paper form. Accordingly, EPA will
transfer any copy of objections and requests for hearings received
electronically into printed, paper form as they are received and will
place the paper copies in the official rulemaking record which will
also include any objections and requests for hearings submitted
directly in writing. The official rulemaking record is the paper record
maintained at the address in "ADDRESSES" at the beginning of this
document.

Under Executive Order 12866 (58 FR 51735, Oct. 4, 1993), the Agency
must determine whether the regulatory action is "significant" and
therefore subject to review by the Office of Management and Budget
(OMB) and the requirements of the Executive Order. Under section 3(f),
the order defines a "significant" regulatory action" as an action
that is likely to result in a rule (1) having an annual effect on the
economy of $100 million or more, or adversely and materially affecting
a sector of the economy, productivity, competition, jobs, the
environment, public health or safety, or State, local or tribal
governments or communities (also referred to as "economically
significant"); (2) creating serious inconsistency or otherwise
materially altering the budgetary impacts of entitlement, grants, user
fees, or loan programs or the rights and obligations or recipients
thereof; or (3) raising novel legal or policy issues arising out of
legal mandates, the President's priorities, or the principles set forth
in this Executive Order.

Pursuant to the terms of the Executive Order, EPA has determined that
this rule is not "significant" and is therefore not subject to OMB
review.

Pursuant to the requirements of the Regulatory Flexibility Act (Pub. L.
96-354, 94 Stat. 1164, 5 U.S.C. 601-612), the Administrator has
determined that regulations establishing new tolerances or raising
tolerance levels or establishing exemption from tolerance requirements
do not have a significant economic effect on a substantial number of
small entities. A certification statement to this effect was published
in the Federal Register of May 4,1981 (49 FR 24950).

List of Subjects in 40 CFR Part 180

Environmental protection, Administrative practice and procedure,
Agricultural commodities, Pesticides and pests, Reporting and
recordkeeping requirements.

Dated: April 25, 1995.

Daniel M. Barolo,
Director, Office of Pesticide Programs.

Therefore, 40 CFR part 180 is amended as follows:

PART 180--[AMENDED]

1. The authority citation for part 180 continues to read as follows:

Authority: 21 U.S.C. 346a and 371.

2. In subpart D, by adding new Sec. 180.1147, to read as follows:

Sec. 180.1147 Bacillus thuringiensis CryIIIA delta-endotoxin and the
genetic material necessary for its production.

Bacillus thuringiensis CryIIIA delta-endotoxin and the genetic material
necessary for its production are exempted from the requirement of a
tolerance when used as a plant pesticide in potatoes. "Genetic
material necessary for its production" means the CryIIIA gene and its
regulatory regions. `Regulatory regions" are the genetic materials
that control the expression of the gene, such as promoters,
terminators, and enhancers.

[FR Doc. 95-10864 Filed 4-28-95; 12:21 pm] BILLING CODE 6560-50-F