[Federal Register Volume 60, Number 179 (Friday, September 15, 1995)]
[Rules and Regulations]
[Pages 47871-47874]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 95-23077]



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ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 180

[PP 4F4331/R2170; FRL-4976-9]
RIN 2070-AB78


Plant Pesticide Bacillus Thuringiensis CryIA(c) Delta-Endotoxin 
and the Genetic Material Necessary for Its Production in Cotton; 
Tolerance Exemption

AGENCY: Environmental Protection Agency (EPA).

ACTION: Final rule.

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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 protected]. 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 

[[Page 47872]]
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 
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, 2800 
Crystal Drive, Arlington, VA 22202, telephone no.: 703-308-8128; e-
mail: [email protected].

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.

Product Analysis

    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.

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, 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.

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 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 

[[Page 47873]]
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.

Submitted Data

    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 
preparations.
Classification: Acceptable.
    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.
Classification: 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.
Classification: Acceptable.
    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.
Classification: Acceptable.
    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 
necropsy.
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.
Classification: Acceptable.

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 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 
forth below.
    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 

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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:
    opp-D[email protected]

    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 
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: August 31, 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. 180.1155, 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.

[FR Doc. 95-23077 Filed 9-13-95; 12:19 pm]

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