[Federal Register Volume 60, Number 85 (Wednesday, May 3, 1995)]
[Rules and Regulations]
[Pages 21725-21728]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 95-10864]



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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: [email protected]. 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: 
[email protected].

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-D[email protected].


    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