[Federal Register Volume 60, Number 158 (Wednesday, August 16, 1995)]
[Rules and Regulations]
[Pages 42443-42446]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 95-20014]



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ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 180

[PP 4F4395/R2161; FRL-4971-3]

RIN 2070-AB78


Plant Pesticide Bacillus Thuringiensis CryIA(b) Delta-Endotoxin 
and the Genetic Material Necessary for its Production (Plasmid Vector 
pCIB4431) in Corn

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(b) delta-endotoxin and the genetic 
material necessary for its production (plasmid vector pCIB4431) in 
corn. A request for an exemption from the requirement of a tolerance 
was submitted by Ciba-Geigy Corp. (Ciba Seeds). This regulation 
eliminates the need to establish a maximum permissible level for 
residues of this plant pesticide in the raw agricultural commodities of 
field corn, sweet corn, and popcorn.

EFFECTIVE DATE: Effective on August 16, 1995.

ADDRESSES: Written objections and hearing requests, identified by the 
document control number, [PP 4F4395/R2161] and may be submitted to: 
Hearing Clerk (1900), Environmental Protection Agency, Rm. M3708, 401 M 
St., SW., Washington, DC 20460. 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 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 Resources 
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.
    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 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 4F4395/R2161]. 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: Michael L. Mendelsohn, 
Biopesticides and Pollution Prevention Division, Office of Pesticide 
Programs, Environmental Protection Agency, 401 M St., SW., Washington, 
DC 20460. 

[[Page 42444]]
Office location and telephone number: 5th Floor, CS #1, 2800 Crystal 
Drive, Arlington, VA 22202, Telephone No.: (703)-308-8715; e-mail: 
[email protected].
SUPPLEMENTARY INFORMATION: Ciba Seeds has genetically modified corn 
plants to produce a truncated version of the pesticidal CryIA(b) delta-
endotoxin protein (derived from the soil microbe Bacillus 
thuringiensis). EPA issued a notice, published in the Federal Register 
of February 1, 1995 (60 FR 6093), which announced that Ciba-Geigy 
Corp., P.O. Box 12257, Research Triangle Park, NC 27709-2257, had 
submitted a pesticide petition, PP 4F4395, 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 delta-endotoxin as produced in corn by a CryIA(b) gene 
and its controlling sequences as found on plasmid vector pCIB4431. EPA 
has assigned the active ingredient of this product the name Bacillus 
thuringiensis CryIA(b) delta-endotoxin and the genetic material 
necessary for its production (plasmid vector pCIB4431) in corn. 
``Genetic material necessary for its production'' means the genetic 
material which comprise (1) genetic material encoding the CryIA(b) 
delta-endotoxin and (2) its regulatory regions. ``Regulatory regions'' 
are the genetic materials that control the expression of the genetic 
material encoding the CryIA(b) delta-endotoxin, such as promoters, 
terminators, and enhancers.
    There were no adverse comments or requests for referral to an 
advisory committee received in response to the notice of filing of the 
pesticide petition, PP 4F4395.

Product Analysis

    Ciba Seeds submitted information which adequately described the 
truncated CryIA(b) delta-endotoxin as expressed in corn, along with 
data on the genetic material necessary for its production.
    Product analysis data were submitted to show that microbially 
expressed and purified CryIA(b) delta-endotoxin used for mammalian 
toxicological testing purposes is not significantly different than the 
delta-endotoxin expressed in the plant. The following assays were used 
to determine the similarity of the microbially expressed and purified 
CryIA(b) delta-endotoxin and that produced in corn: SDS-PAGE, western 
blots, amino acid sequencing, certain tests for post-translational 
modifications, and insect bioactivity. These assays have demonstrated 
the truncated CryIA(b) delta-endotoxin expressed in corn and the 
tryptic digested CryIA(b) delta-endotoxin to be similar. The N-terminal 
amino acid sequences of both delta-endotoxins were found to be 
identical except that the plant produced delta-endotoxin had portions 
at the N-terminus deleted, perhaps due to internal plant proteases and 
a higher bioactivity. These differences were not considered 
toxicologically significant since they are not expected to change the 
activity of the deltaendotoxin in mammalian systems.

Toxicology Assessment

    The toxicology data provided are sufficient to demonstrate that 
there are no foreseeable human health hazards likely to arise from the 
use of Bacillus thuringiensis CryIA(b) delta-endotoxin and the genetic 
material necessary for its production (plasmid vector pCIB4431) when 
used as a plant pesticide in any corn plant.
    The data Ciba Seeds submitted regarding potential health effects 
include information on the characterization of the expressed CryIA(b) 
delta-endotoxin in corn, the acute oral toxicity, and in vitro 
digestibility of the delta-endotoxin.

Toxicity

    The Agency expects that proteins with no significant amino acid 
homology to known protein toxins and which are readily inactivated by 
heat or mild acidic conditions would also be readily degraded in an in 
vitro digestibility assay and have little likelihood for displaying 
oral toxicity.
    The data submitted by Ciba Seeds support the prediction that the 
CryIA(b) protein would be nontoxic to humans. When proteins are toxic, 
they are known to act via acute mechanisms and at very low dose levels 
[Sjobald, Roy D., et al. ``Toxicological Considerations for Protein 
Components of Biological Pesticide Products,'' Regulatory Toxicology 
and Pharmacology, 15, 3-9 (1992)]. Therefore, since no significant 
acute effects were observed, even at relatively high-dose levels, the 
CryIA(b) delta-endotoxin is not considered acutely or chronicly toxic. 
Adequate information was submitted to show that the test materials 
derived from microbial cultures were biochemically and insecticidally 
similar to the delta-endotoxin as produced by corn. Production of 
microbial produced CryIA(b) delta-endotoxin was chosen in order to 
obtain sufficient material for mammalian testing. In addition, the in 
vitro digestibility studies indicate the delta-endotoxin would be 
rapidly degraded following ingestion.
    The genetic material necessary for the production of the Bacillus 
thuringiensis CryIA(b) delta endotoxin are the nucleic acids (DNA) 
which comprise (1) genetic material encoding the CryIA(b) delta-
endotoxin and (2) its regulatory regions. ``Regulatory regions'' are 
the genetic material that control the expression of the genetic 
material encoding the CryIA(b) deltaendotoxin, such as promoters, 
terminators, and enhancers. DNA is common to all forms of plant and 
animal life, and the Agency knows of no instance where these nucleic 
acids have been associated with toxic effects related to their 
consumption. These ubiquitous nucleic acids as they appear in the 
subject active ingredient have been adequately characterized by the 
applicant. Therefore, no mammalian toxicity is expected from dietary 
exposure to the genetic material necessary for the production of the 
Bacillus thuringiensis CryIA(b) delta endotoxin in corn.

Allergenicity

    Current scientific knowledge suggests that common food allergens 
tend to be resistant to degradation by heat, acid, and proteases and 
are glycosylated and present at high concentrations in the food. Ciba 
Seeds has submitted data to indicate that the CryIA(b) delta-endotoxin 
is rapidly degraded by gastric fluid in vitro, is not present as a 
major component of food (i.e., is not found in corn kernels and is not 
detectable in finished silage) and is apparently nonglycosylated or 
otherwise post-translationally modified when produced in plants.
    Studies submitted to EPA done in laboratory animals 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, unlike known food allergens that are 
resistant to degradation.
    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 to the 
delta-endotoxin itself despite significant oral, dermal, and inhalation 
exposure to the microbial product. Several reports under FIFRA section 
6(a)2 have been made for various Bacillus thuringiensis products with 
allergic reactions being reported. However, these reactions were 

[[Page 42445]]
determined not to be due to Bacillus thuringiensis itself or any of the 
cry toxins.

Submitted Data

    1. Acute Oral Toxicity of Bacterially Produced CryIA(b) Delta- 
endotoxin Five male and five female mice received a single dose of 
3,280 mg/kg of CryIA(b) delta-endotoxin by oral gavage. No animals 
died, nor were there significant clinical signs as a result of the 
exposure. One female failed to gain weight between day 7 and day 14. 
All animals gained weight by the end of the study. Males gained more 
weight over the study than females. The LD50 was therefore greater 
than 3,280 mg/kg, the highest dose tested.
    2. In-Vitro Digestibility of CryIA(b) Delta-endotoxin. The CryIA(b) 
delta-endotoxin from either corn or B.t.k. HD19 is rapidly degraded in 
the presence of pepsin. Using 1/1000 strength pepsin, a time course 
study shows that the introduced delta-endotoxin from either source 
degrades within 10 minutes to fragments that lack any immunorecognition 
in a western blot assay. While this study provides useful information 
demonstrating the digestibility of the CryIA(b) delta-endotoxin 
produced in corn, it is not yet a validated study for assessing protein 
toxicology. It is not clear whether lack of toxicity correlates with in 
vitro digestibility under the conditions of the assay. EPA was relying 
on this study to demonstrate rapid degradation of the delta-endotoxin.
    3. Acute Oral Toxicity of Corn Leaf Protein Extracted from Bt Corn. 
Application of this study to dietary risk assessment is not possible 
because of extremely low doses administered, small test populations, 
and unexplained deaths occurring in both control and treated groups. 
Therefore, EPA is not relying on this study to support the tolerance 
exemption.

Residue Chemistry Data

    Residue chemistry data were not required because of the lack of 
mammalian toxicity of this active ingredient. In the acute mouse oral 
toxicity study, the CryIA(b) delta-endotoxin was shown to have an 
LD50 greater than 3,280 mg/kg. When proteins are toxic, they are 
known to act via acute mechanisms and at very low dose levels [Sjobald, 
Roy D., et al. ``Toxicological Considerations for Protein Components of 
Biological Pesticide Products,'' Regulatory Toxicology and 
Pharmacology, 15, 3-9 (1992)]. Therefore, since no significant acute 
effects were observed, even at relatively high dose levels, the 
CryIA(b) delta-endotoxin is not considered acutely or chronicly toxic. 
This is similar to the Agency position regarding toxicity and the 
requirement 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, further toxicity testing to verify 
the observed effects and clarify the source of the effects (Tiers II 
and III) and residue data are triggered by significant acute effects in 
studies such as the mouse oral toxicity study.
    The genetic material necessary for the production of the Bacillus 
thuringiensis CryIA(b) delta endotoxin are the nucleic acids (DNA) 
which comprise: (1) genetic material encoding the CryIA(b) delta-
endotoxin and (2) its regulatory regions. ``Regulatory regions'' are 
the genetic materials that control the expression of the genetic 
material encoding the CryIA(b) deltaendotoxin, such as promoters, 
terminators, and enhancers. As stated above, no mammalian toxicity is 
expected from dietary exposure to the genetic material necessary for 
the production of the Bacillus thuringiensis CryIA(b) delta endotoxin 
in corn. Therefore, no residue data are required in order to grant an 
exemption from the requirements of a tolerance for the plant pesticide, 
Bacillus thuringiensis CryIA(b) delta-endotoxin and the genetic 
material necessary for its production (plasmid vector pCIB4431) in 
corn.

Conclusions

    Based on the information considered, the Agency concludes that 
establishment of a tolerance is not necessary to protect the public 
health. Therefore, the exemption from tolerance is established as set 
forth below.
    Any person adversely affected by this regulation may, within 30 
days after publication of this document in the Federal Register, file 
written objections to the regulation and may also request a hearing on 
those objections. Objections and hearing requests must be filed with 
the Hearing Clerk, at the address given above (40 CFR 178.20). A copy 
of the objections and/or hearing requests filed with the Hearing Clerk 
should be submitted to the OPP docket for this rule making. The 
objections submitted must specify the provisions of the regulation 
deemed objectionable and the grounds for the objections (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 such issues, and a summary of any evidence 
relied upon by the objector as well as the other materials required by 
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 genuine and substantial issue of fact; there is 
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 4F4395/R2161] (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 4F4395/R2161], 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. 

[[Page 42446]]

    Under Executive Order 12866 (58 FR 51735, October 4, 1993), the 
Agency must determine whether the regulatory action is ``significant'' 
and therefore subject to all the requirements of the Executive Order 
(i.e., Regulatory Impact Analysis, review by the Office of Management 
and Budget (OMB)). Under section 3(f), the order defines 
``significant'' as those actions likely to lead to 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 known as 
``economically significant''); (2) creating serious inconsistency or 
otherwise interfering with an action taken or planned by another 
agency; (3) materially altering the budgetary impacts of entitlement, 
grants, user fees, or loan programs; or (4) 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 this 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 exemptions from tolerance requirements 
do not have a significant economic impact on a substantial number of 
small entities. A certification statement to this effect was published 
in the Federal Register of May 4, 1981 (46 FR 24950).

List of Subjects in 40 CFR Part 180

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

    Dated: August 7, 1995.

Penelope A. Fenner-Crisp,
Acting Director, Office of Pesticide Programs.

PART 180--[AMENDED]

    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. 180.1152, to read as follows:


Sec. 180.1152   Bacillus thuringiensis CryIA(b) delta-endotoxin and the 
genetic material necessary for its production (plasmid vector pCIB4431) 
in corn; exemption from the requirement of a tolerance.

    Bacillus thuringiensis CryIA(b) delta-endotoxin and the genetic 
material necessary for its production (plasmid vector pCIB4431) in corn 
is exempt from the requirement of a tolerance when used as a plant 
pesticide in the raw agricultural commodities of field corn, sweet 
corn, and popcorn. ``Genetic material necessary for its production'' 
means the genetic materials which comprise genetic material encoding 
the CryIA(b) delta-endotoxin and its regulatory regions. ``Regulatory 
regions'' are the genetic materials that control the expression of the 
genetic material encoding the CryIA(b) delta-endotoxin, such as 
promoters, terminators, and enhancers.

[FR Doc. 95-20014 Filed 8-15-95; 8:45 am]
BILLING CODE 6560-50-F