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Bacillus thuringiensis CryIA(b) Delta-Endotoxin - Tolerance Exemption in Corn—Final Rule 8/95

[Federal Register: August 16, 1995 (Volume 60, Number 158)] [Rules and
Regulations]
[Page 42443-42446]
From the Federal Register Online via GPO Access [wais.access.gpo.gov]
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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:
opp-docket@epamail.epa.gov. 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:
mendelsohn.michael@epamail.epa.gov.
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-Docket@epamail.epa.gov

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