Bacillus thuringiensis CryIA(b) Delta-Endotoxin - Tolerance
Exemption in Cotton—Final Rule 8/95
[Federal Register: September 15, 1995 (Volume 60, Number 179)]
ENVIRONMENTAL PROTECTION AGENCY
40 CFR Part 180
[PP 4F4331/R2170; FRL-4976-9]
Plant Pesticide Bacillus Thuringiensis CryIA(c) Delta-Endotoxin and the
Genetic Material Necessary for Its Production in Cotton; Tolerance
AGENCY: Environmental Protection Agency (EPA).
ACTION: Final rule.
SUMMARY: This rule establishes an exemption from the requirement of a
tolerance for residues of the plant pesticide active ingredient
Bacillus thuringiensis CryIA(c) delta-endotoxin and the genetic
material necessary for its production in cotton. The Monsanto Co.
requested the exemption from the requirement of a tolerance under the
Federal Food, Drug and Cosmetic Act. The rule eliminates the need to
establish a maximum permissible level for residues of this plant
pesticide in cotton.
EFFECTIVE DATE: September 15, 1995.
ADDRESSES: Written objections and hearing requests, identified by the
document control number [PP 4F4331/R2170] 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 hearing requests filed with the Hearing
Clerk may also be submitted electronically by sending electronic mail
(e-mail) to: email@example.com. Copies of objections and
hearing requests must be submitted as an ASCII file avoiding the use of
special characters and any form of encryption. Copies of objections and
hearing requests will also be accepted on disks in WordPerfect in 5.1 file
format or ASCII file format. All copies of objections and hearing requests in
electronic form must be identified by the docket number [PP 4F4331/R2170]. No
Confidential Business Information (CBI) should be submitted through e-mail.
Electronic copies of objections and hearing requests on this rule may
be filed online at many Federal Depository Libraries. Additional
information on electronic submissions can be found below in this
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, 2800
Crystal Drive, Arlington, VA 22202, telephone no.: 703-308-8128;
SUPPLEMENTARY INFORMATION: EPA issued a notice, published in the
Federal Register of September 14, 1994 (59 FR 47137), which announced
that Monsanto Co., 700 Chesterfield Village Parkway, St. Louis, MO
63198, had submitted pesticide petition (PP) 4F4331 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), exempt from the
requirement of a tolerance the plant pesticide Bacillus thuringiensis
var. kurstaki delta-endotoxin protein as produced by the CryIA(c) gene
and its controlling sequences. EPA has assigned the active ingredient
of this product the name Bacillus thuringiensis CryIA(c) delta-
endotoxin and the genetic material necessary for its production.
"Genetic material necessary for production" means the CryIA(c) gene
and its regulatory regions. "Regulatory regions" are the genetic
materials that control the expression of the gene, such as promoters,
terminators, and enhancers. Monsanto has genetically modified cotton
plants to produce the pesticidal protein derived from the common soil
bacterium Bacillus thuringiensis. The protein produced by these cotton
plants is identical to that found in nature.
There were no adverse comments or requests for referral to an
advisory committee received in response to the notice of filing.
Residue Chemistry Data
Residue chemistry data were not required because of the lack of
toxicity to this active ingredient. This position is similar to that
the Agency has taken 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 Tier 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 to grant an exemption from the requirements
of a tolerance for Monsanto's plant pesticide, Bacillus thuringiensis
CryIA(c) delta-endotoxin protein, the CryIA(c) gene and the genetic
material necessary for its production in cotton.
Monsanto submitted information which adequately described the
CryIA(c) delta-endotoxin from B.t., as expressed in cotton, 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 CryIA(c) delta-
endotoxin is sufficiently similar to that expressed in the plant to be
used for mammalian toxicological purposes. Plant and microbially
produced CryIA(c) delta-endotoxin were shown by these studies to have
similar molecular weights and immunoreactivity (SDS-PAGE and Western
blots), to lack detectable post-translational modication (glycosylation
tests), to have identical amino acid sequences in the N-terminal region
and to have similar results in bioassays against Heliothis virescens
and Helicoverpa zea. While it is difficult to prove that two proteins
are identical, the combined results of the above studies indicate a
high probability that these two sources produce proteins that are
essentially identical by available protein analytical assays.
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, the Agency
does not anticipate any mammalian toxicity from this protein in plants
based on the use history of B. thuringiensis products. The in vitro
digestibility assay provides useful information to predict the
metabolic fate of the CryIA(c) protein and its potential as a food
allergen. However, it is not clear how this assay's results relate to
protein toxicity. Therefore, the Agency requested that an acute oral
toxicity study be done to confirm the expected lack of toxicity
indicated by the in vitro digestibility results.
Monsanto's submitted oral toxicity data support the prediction that
this protein would be nontoxic to humans. CryIA(c) delta-endotoxin was
chosen in order to obtain sufficient material for mammalian testing if
any exposure were anticipated in food or feed. The in vitro
digestibility studies indicate that the protein would rapidly be
degraded following ingestion.
The genetic material necessary for the production of the Bacillus
thuringiensis CryIA(c) delta endotoxin are the nucleic acids (DNA and
RNA) that constitute the CryIA(c) 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 CryIA(c) delta-endotoxin in cotton.
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 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
(Conference on Scientific Issues, Related to Potential Allergenicity in
Transgenic Food Crops, April 18 and 19, Annapolis, MD, sponsored by
FDA, EPA, and USDA). 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 CryIA(c) delta-
endotoxin is rapidly degraded by gastric fluid in vitro, that it is not
present as a major component of food, and that it is apparently
nonglycosylated when produced in plants.
1. Product characterization (431452-01). Southern blot analysis
restriction digests of DNA extracts from cotton line 531 and the
parental Coker 312 showed that there is probably only one insert of the
cryIA(c) gene cassette present in the transformed line. The introduced
gene appears to be genetically stable in the cotton according to the
results of progeny selfing and backcrosses with elite lines. The amino
acid sequence is homologous to the cryIA(b) gene from HD-1 for
positions 1-466 and homologous to cryIA(c) for positions 467-1178 with
a single exception of a leucine-serine 766 in the crystal portion of
the protein cleaved prior to toxin activation. Western blot analysis of
purified toxin, leaf tissue from cotton line 531 and the parental Coker
312 shows that trypsinized extracts have comigrating bands similar to
that found in B.t.k HD-73 protein reference material and commercial
2. Product characterization (431452-02). B.t.k. HD-73 toxin
isolated from either cotton line 531 or 931 were compared to the same
toxin expressed in E. coli by SDS-PAGE, western blot, glycosylation and
bioactivity (Conference on Scientific Issues Related to Potential
Allergenicity in Transgenic Food Crops, April 18 and 19, 1994,
Annapolis, MD, sponsored by FDA, EPA, andUSDA). The data presented
suggest the bacterially produced protein and that found in cotton are
equivalent and suggest the bacterially produced B.t.k. HD-73 toxin can
serve as a surrogate test substance for the toxicological tests to
support the registration of transgenic cotton. This initial submission
was classified as supplementary because of the absence of sufficient
description of how the B.t.k. HD-73 protein was isolated and purified
from the cotton plant. A cursory description is found in "Assessment
of Equivalence Between E. coli-Produced and Cotton-Produced Btk HD73
Protein * * *." (MRID 431452-02, p.13). Monsanto has since provided
complete details regarding isolation and purification. With the
clarification of the extraction procedure described above, the product
characterization study (MRID 431452-02) has been upgraded from
supplementary to acceptable.
3. Product characterization (431452-03). The delta-endotoxin from
B.t.k. HD-73 (lot 5025385) produced in E. coli containing the plasmid
pMON10569 was purified, lyophilized and found to have the following
characteristics: 4.5% moisture, 75.6% protein (amino acid analysis),
70% protein (BCA), 88% HD-73 specific protein (ELISA), 80% HD-73
specific protein (Coomassie blue PAGE), 1.6 ug gram negative endotoxin/
mg and no significant trace metals except for sodium, potassium, and
phosphate. The molecular weight of the B.t.k. HD-73 toxin was estimated
to be 134.8 kD for the full length species and 77.1 kD for the tryptic.
The functional activity was found to be an LC50 of 0.28 ppm
against Heliothis virescens.
4. Product characterization (431452-04). Ten insect pest species
from 5 families were tested for their sensitivity to B.t.k. HD-73
protein. Only in the lepidopteran species was there significant
mortality. The green peach aphid showed marginal effects from treatment
with a tryptic digest of the CryIA(c) toxin from B.t.k. which was not
reproducible in a repeat test. The tryptic digest preparation positive
control also showed higher mortality in the TBW test.
5. Acute oral toxicity (431452-13). Ten male and female CD-1 mice
per dose level were exposed by oral gavage to 500, 1,000 and 4,200 mg/
kg body weight of E. coli produced B.t.k. HD-73 toxin. Controls were
given the protein equivalent of 6,340 mg/kg of BSA. No mortalities or
treatment related adverse effects were seen in either the treated or
control mice. There were no observable dose-related effects seen upon
Classification: Acceptable. Tox category IV.
6. In vitro digestibility (431452-14). The B.t.k. HD-73 protein was
rapidly degraded to fragments not recognized in a western blot after 7
minutes incubation in simulated gastric fluid (SGF) and was not active
in a TBW bioassay after SGF incubation. The in vitro digestibility
assay provides useful information to predict the metabolic fate of the
CryIA(c) protein and its potential as a food allergen.
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 CryIA(c) 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 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
Acceptable daily intake (ADI) and maximum permissible intake (MPI)
considerations are not relevant to this petition because the data/
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 prescribedby 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 4F4331/R2170] (including objections and hearing requests
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 Room 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.
Written objections and hearing requests, identified by the document
control number [PP 4F4331/R2170], may be submitted to the Hearing Clerk
(1900), Environmental Protection Agency, Rm. 3708, 401 M St., SW.,
Washington, DC 20460.
A copy of electronic objections and hearing requests filed with the
Hearing Clerk can be sent directly to EPA at:
A copy of electronic objections and hearing requests filed with the
Hearing Clerk 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 objections and hearing requests 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 all objections and hearing requests 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
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
Dated: August 31, 1995.
Daniel M. Barolo,
Director, Office of Pesticide Programs.
Therefore, 40 CFR part 180 is amended as follows:
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. 180.1155, to read as follows:
Sec. 180.1155 Bacillus thuringiensis CryIA(c) delta-endotoxin and the
genetic material necessary for its production; exemption from the
requirement of a tolerance.
Bacillus thuringiensis CryIA(c) 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 cotton. "Genetic
material necessary for its production" means the CryIA(c) gene and its
regulatory regions. "Regulatory regions" are the genetic materials
that control the expression of the gene, such as promoters,
terminators, and enhancers.