Rule2025-22927
SpCas9 Protein; Exemption From the Requirement of a Tolerance
Primary source
Metadata and text below are from the Federal Register, a public-domain U.S. government work. Always verify the official published version before relying on it for any legal matter.
Published
December 16, 2025
Effective
December 16, 2025
Issuing agencies
Environmental Protection Agency
Abstract
This regulation establishes an exemption from the requirement of a tolerance for residues of the SpCas9 protein in or on the food and feed commodities of citrus when used as a plant-incorporated protectant (PIP) in citrus. Soil Culture Solutions LLC (d/b/a Soilcea) submitted a petition to EPA under the Federal Food, Drug, and Cosmetic Act (FFDCA), requesting an exemption from the requirement of a tolerance. This regulation eliminates the need to establish a maximum permissible level for residues of SpCas9 protein.
Full Text
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<title>Federal Register, Volume 90 Issue 239 (Tuesday, December 16, 2025)</title>
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[Federal Register Volume 90, Number 239 (Tuesday, December 16, 2025)]
[Rules and Regulations]
[Pages 58167-58170]
From the Federal Register Online via the Government Publishing Office [<a href="http://www.gpo.gov">www.gpo.gov</a>]
[FR Doc No: 2025-22927]
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ENVIRONMENTAL PROTECTION AGENCY
40 CFR Part 174
[EPA-HQ-OPP-2025-0212; FRL-13100-01-OCSPP]
SpCas9 Protein; Exemption From the Requirement of a Tolerance
AGENCY: Environmental Protection Agency (EPA).
ACTION: Final rule.
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SUMMARY: This regulation establishes an exemption from the requirement
of a tolerance for residues of the SpCas9 protein in or on the food and
feed commodities of citrus when used as a plant-incorporated protectant
(PIP) in citrus. Soil Culture Solutions LLC (d/b/a Soilcea) submitted a
petition to EPA under the Federal Food, Drug, and Cosmetic Act (FFDCA),
requesting an exemption from the requirement of a tolerance. This
regulation eliminates the need to establish a maximum permissible level
for residues of SpCas9 protein.
DATES: This regulation is effective December 16, 2025. Objections and
requests for hearings must be received on or before February 17, 2026,
and must be filed in accordance with the instructions provided in 40
CFR part 178 (see also Unit I.C. of the SUPPLEMENTARY INFORMATION).
ADDRESSES: The docket for this action, identified by docket
identification (ID) number EPA-HQ-OPP-2025-0212, is available at
<a href="https://www.regulations.gov">https://www.regulations.gov</a>. Additional information about the docket
generally, along with instructions for visiting the docket in-person,
is available at <a href="https://www.epa.gov/dockets">https://www.epa.gov/dockets</a>.
FOR FURTHER INFORMATION CONTACT: Shannon Borges, Biopesticides and
Pollution Prevention Division (7511P), Office of Pesticide Programs,
Environmental Protection Agency, 1200 Pennsylvania Ave. NW, Washington,
DC 20460-0001; main telephone number: (202) 566-1400; email address:
<a href="/cdn-cgi/l/email-protection#4d0f1d1d090b1f032239242e283e0d283d2c632a223b"><span class="__cf_email__" data-cfemail="713321213537233f1e0518121402311401105f161e07">[email protected]</span></a>.
SUPPLEMENTARY INFORMATION:
I. Executive Summary
A. Does this action apply to me?
You may be potentially affected by this action if you are an
agricultural producer, food manufacturer, or pesticide manufacturer.
The following list of North American Industrial Classification System
(NAICS) codes is not intended to be exhaustive, but rather provides a
guide to help readers determine whether this document applies to them:
<bullet> Crop production (NAICS code 111).
<bullet> Animal production (NAICS code 112).
<bullet> Food manufacturing (NAICS code 311).
<bullet> Pesticide manufacturing (NAICS code 32532).
If you have any questions regarding the applicability of this
action to a particular entity, consult the person listed under FOR
FURTHER INFORMATION CONTACT.
B. What is EPA's authority for taking this action?
EPA is issuing this rulemaking under section 408 of the Federal
Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a. FFDCA section
408(c)(2)(A)(i) allows EPA to establish an exemption from the
requirement for a tolerance (the legal limit for a pesticide chemical
residue in or on a food) only if EPA determines that the exemption is
``safe.'' FFDCA section 408(c)(2)(A)(ii) defines ``safe'' to mean that
``there is a reasonable certainty that no harm will result from
aggregate exposure to the pesticide chemical residue, including all
anticipated dietary exposures and all other exposures for which there
is reliable information.'' This includes exposure through drinking
water and in residential settings but does not include occupational
exposure. Pursuant to FFDCA section 408(c)(2)(B), in establishing or
maintaining in effect an exemption from the requirement of a tolerance,
EPA must take into account the factors set forth in FFDCA section
408(b)(2)(C), which require EPA to give special consideration to
exposure of infants and children to the pesticide chemical residue in
establishing a tolerance and to ``ensure that there is a reasonable
certainty that no harm will result to infants and children from
aggregate exposure to the pesticide chemical residue. . . .''
Additionally, FFDCA section 408(b)(2)(D) requires that the Agency
consider, among other things, ``available information concerning the
cumulative effects of a particular pesticide's residues'' and ``other
substances that have a common mechanism of toxicity.''
C. How can I file an objection or hearing request?
Under FFDCA section 408(g), 21 U.S.C. 346a, any person may file an
objection to any aspect of this regulation and may also request a
hearing on those objections. If you fail to file an objection to the
final rule within the time period specified in the final rule, you will
have waived the right to raise any issues
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resolved in the final rule. You must file your objection or request a
hearing on this regulation in accordance with the instructions provided
in 40 CFR part 178. To ensure proper receipt by EPA, you must identify
docket ID number EPA-HQ-OPP-2025-0212 in the subject line on the first
page of your submission. All objections and requests for a hearing must
be in writing and must be received by the Hearing Clerk on or before
February 17, 2026.
EPA's Office of Administrative Law Judges (OALJ), in which the
Hearing Clerk is housed, urges parties to file and serve documents by
electronic means only, notwithstanding any other particular
requirements set forth in other procedural rules governing those
proceedings. See ``Revised Order Urging Electronic Filing and
Service,'' dated June 22, 2023, which can be found at <a href="https://www.epa.gov/system/files/documents/2023-06/2023-06-22%20-%20revised%20order%20urging%20electronic%20">https://www.epa.gov/system/files/documents/2023-06/2023-06-22%20-%20revised%20order%20urging%20electronic%20</a> filing%20and%20service.pdf.
Although EPA's regulations require submission via U.S. Mail or hand
delivery, EPA intends to treat submissions filed via electronic means
as properly filed submissions; therefore, EPA believes the preference
for submission via electronic means will not be prejudicial. When
submitting documents to the OALJ electronically, a person should
utilize the OALJ e-filing system at <a href="https://yosemite.epa.gov/OA/EAB/EAB-ALJ_upload.nsf">https://yosemite.epa.gov/OA/EAB/EAB-ALJ_upload.nsf</a>.
In addition to filing an objection or hearing request with the
Hearing Clerk as described in 40 CFR part 178, please submit a copy of
the filing (excluding any Confidential Business Information (CBI)) for
inclusion in the public docket at <a href="https://www.regulations.gov">https://www.regulations.gov</a>. Follow
the online instructions for submitting comments. Do not submit
electronically any information you consider to be CBI or other
information whose disclosure is restricted by statute. If you wish to
include CBI in your request, please follow the applicable instructions
at <a href="https://www.epa.gov/dockets/commenting-epa-dockets#rules">https://www.epa.gov/dockets/commenting-epa-dockets#rules</a> and clearly
mark the information that you claim to be CBI. Information not marked
confidential pursuant to 40 CFR part 2 may be disclosed publicly by EPA
without prior notice.
II. Petitioned for Exemption
In the Federal Register of July 3, 2025 (90 FR 29515) (FRL-12474-
05-OSCPP), EPA issued a document pursuant to FFDCA section 408(d)(3),
21 U.S.C. 346a(d)(3), announcing the filing of a pesticide tolerance
petition (PP 4E9159) by Soil Culture Solutions LLC (d/b/a Soilcea),
3802 Spectrum Blvd., Suite 157, Tampa, FL 33612. The petition requested
that 40 CFR part 174 be amended by establishing an exemption from the
requirement of a tolerance for residues of the PIP Cas9 protein in
citrus. That document referenced a summary of the petition prepared by
the petitioner Soilcea, which is available in the docket.
Thirty-six comments were received in response to the notice of
filing. All supported the proposed tolerance exemption for SpCas9
protein and urged the Agency to approve the tolerance exemption and
associated PIP product registration. No substantive issues were raised
in these comments that would affect this tolerance exemption action.
III. Final Tolerance Actions
A. EPA's Safety Determination
EPA evaluated the available toxicological and exposure data on Cas9
protein and considered their validity, completeness, and reliability,
as well as the relationship of this information to human risk. A full
explanation of the data upon which the EPA relied and its risk
assessment based on those data can be found within the document
entitled ``Product Characterization Review and Human Health Risk
Assessment of the Three Plant-Incorporated Protectants for the Loss-Of-
Function Edits in Accelerated Cell Death 2 (ACD2); Lethal Leaf Spot 1
(LLS1); and Papain-Like Cysteine Protease (PLCP) and the Genetic
Material Necessary for their Production (gRNAs g5, g213, g86) along
with the Cas9 Protein, as Expressed in Citrus Rootstock Containing
Event CarriCea T1'' (Human Health Risk Assessment). This document, as
well as other relevant information, are available in the docket for
this action as described under ADDRESSES.
The Cas9 protein is an enzyme (endonuclease) that is part of the
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
precision genome editing system. CRISPR is an adaptive immune system in
procaryotes and archaea that defends the organism from viral infection
by cleaving viral DNA. Specifically, CRISPR is a complex consisting of
the Cas9 enzyme and a guide RNA (gRNA) molecule. The gRNA binds to DNA
in a sequence-specific manner, while the Cas9 protein makes a double-
stranded break at that position. Cas9 alone does not have catalytic
activity; instead, the incorrect repair of the DNA cut caused by Cas9
results in the loss of function of the targeted gene.
In citrus, SpCas9 protein (derived from the bacterium Streptococcus
pyogenes), along with three gRNAs have been genetically engineered into
the Carrizo cultivar (``CarriCea T1'' rootstock). Each gRNA is
complementary to an endogenous citrus gene and the CRISPR system causes
loss of function to those genes. The proteins that would be otherwise
produced from the targeted genes are involved in pathways exploited by
the bacterium Candidatus Liberibacter asiaticus (CLas), the known
causal agent of Huanglongbing disease (also known as citrus greening).
The loss of function modifications in these genes disrupt the
interactions between the plant proteins and CLas effector proteins,
thus interfering with the bacterium's ability to suppress the plant's
immune response and establish infection. As a result, the CarriCea T1
rootstock exhibits resistance to bacterial infection by CLas.
The CRISPR/Cas9 system is ubiquitous in bacteria and archaea where
it functions by recognizing and removing foreign DNA, serving as a key
component of bacterial immune defense. Cas9 proteins are prevalent
across a wide range of bacterial groups, highlighting their ubiquitous
presence in the environment. Humans are expected to be exposed to
natural Cas9 proteins through their interactions with the natural
environment. For example, Cas9 is also found in bacteria used in food
production, such as Streptococcus thermophilus, and in commensal
bacteria like Lactobacillus plantarum which is found in the human
gastrointestinal tract. This widespread presence suggests regular human
exposure to Cas9 proteins through environmental and dietary sources.
While the amino acid sequences of the various Cas9 proteins vary
between species, the endonuclease function remains conserved. This
consistent mechanism across bacteria suggests that the function of Cas9
remains the same in pathogenic and non-pathogenic bacteria and
therefore indicates a history of safe exposure to Cas9.
In citrus, exposure to SpCas9 protein through the dietary route is
expected to be negligible as the SpCas9 protein is expected to remain
localized to the rootstock, with no presence identified in the leaves
of grafted scions and therefore no expectation of the protein to be
present in the fruit of grafted scions. Similarly, exposure via
drinking water is considered unlikely due to SpCas9's containment in
the rootstock, as well as the protein's susceptibility to degradation
by environmental conditions and microbial activity. If exposure were to
occur, the risk is expected to be negligible due to (1) the history of
safe exposure of humans to
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the Cas9 protein due to the ubiquitous presence of Cas9 proteins in the
environment, and the SpCas9 protein in CarriCea T1 sharing nearly
identical amino acid sequence homology with the naturally occurring
SpCas9; (2) a pesticidal mode of action that is not toxic; (3)
bioinformatic analyses demonstrating the absence of significant
homology between SpCas9 protein and known toxins or allergens; and (4)
data showing the rapid digestibility of SpCas9 protein in gastric
enzymes and heat lability at 100 [deg]C, which limits the likelihood
for allergenic effects.
As a PIP, the SpCas9 protein is contained within the plant cells
and as such, non-occupational and residential exposure is considered to
be negligible. Further, there are no proposed residential uses for the
CarriCea T1 product containing SpCas9 protein; therefore, a residential
handler and post-application exposure and risk assessment has not been
conducted.
Although FFDCA section 408(b)(2)(C) provides for an additional
tenfold margin of safety for infants and children in the case of
threshold effects, EPA has determined that there are no such effects
due to the lack of toxicity and allergenicity of SpCas9 protein. As a
result, an additional margin of safety for the protection of infants
and children is unnecessary.
B. Analytical Enforcement Methodology
EPA has determined that an analytical method is not required for
enforcement purposes since the Agency is establishing an exemption from
the requirement of a tolerance without any numerical limitation.
Nonetheless, a Western blot method using a monoclonal anti-FLAG
antibody was submitted to determine the levels of SpCas9 protein in
plant samples. The antibody used to detect SpCas9 protein through its
FLAG-tag could also be used to determine SpCas9 presence in citrus,
provided no other FLAG-tagged proteins are present at the same time.
C. Conclusion
Based upon its evaluation in the Human Health Risk Assessment,
which concluded that SpCas9 protein residues in or on citrus are not
toxic to mammals, the EPA concludes that there is a reasonable
certainty that no harm will result to the U.S. population, including
infants and children, from aggregate exposure to residues of SpCas9
protein. Therefore, an exemption from the requirement of a tolerance is
established for residues of SpCas9 protein in or on citrus when used
according to the label and good agricultural practices.
IV. Statutory and Executive Order Reviews
Additional information about these statutes and Executive Orders
can be found at <a href="https://www.epa.gov/laws-regulations/and-executive-orders">https://www.epa.gov/laws-regulations/and-executive-orders</a>.
A. Executive Order 12866: Regulatory Planning and Review
This action is exempt from review under Executive Order 12866 (58
FR 51735, October 4, 1993), because it establishes or modifies a
pesticide tolerance or a tolerance exemption under FFDCA section 408 in
response to a petition submitted to the Agency. The Office of
Management and Budget (OMB) has exempted these types of actions from
review under Executive Order 12866.
B. Executive Order 14192: Unleashing Prosperity Through Deregulation
Executive Order 14192 (90 FR 9065, February 6, 2025) does not apply
because actions that establish a tolerance under FFDCA section 408 are
exempted from review under Executive Order 12866.
C. Paperwork Reduction Act (PRA)
This action does not impose an information collection burden under
the PRA, 44 U.S.C. 3501 et seq., because it does not contain any
information collection activities.
D. Regulatory Flexibility Act (RFA)
This action is not subject to the RFA, 5 U.S.C. 601 et seq. The RFA
applies only to rules subject to notice and comment rulemaking
requirements under the Administrative Procedure Act (APA), 5 U.S.C.
553, or any other statute. This rule is not subject to the APA but is
subject to FFDCA section 408(d), which does not require notice and
comment rulemaking to take this action in response to a petition.
E. Unfunded Mandates Reform Act (UMRA)
This action does not contain an unfunded mandate of $100 million or
more (in 1995 dollars and adjusted annually for inflation) as described
in UMRA, 2 U.S.C. 1531-1538, and does not significantly or uniquely
affect small governments. The action imposes no enforceable duty on any
State, local or Tribal governments or the private sector.
F. Executive Order 13132: Federalism
This action does not have federalism implications as specified in
Executive Order 13132 (64 FR 43255, August 10, 1999), because it will
not have substantial direct effects on the States, on the relationship
between the National Government and the States, or on the distribution
of power and responsibilities among the various levels of government.
G. Executive Order 13175: Consultation and Coordination With Indian
Tribal Governments
This action does not have Tribal implications as specified in
Executive Order 13175 (65 FR 67249, November 9, 2000), because it will
not have substantial direct effects on Tribal governments, on the
relationship between the Federal Government and the Indian Tribes, or
on the distribution of power and responsibilities between the Federal
Government and Indian Tribes.
H. Executive Order 13045: Protection of Children From Environmental
Health Risks and Safety Risks
This action is not subject to Executive Order 13045 (62 FR 19885,
April 23, 1997) because it is not a significant regulatory action under
section 3(f)(1) of Executive Order 12866 (See Unit IV.A.), and because
EPA does not believe the environmental health or safety risks addressed
by this action present a disproportionate risk to children.
However, EPA's 2021 Policy on Children's Health applies to this
action. This rule finalizes an exemption from the requirement of a
tolerance under the FFDCA, which requires EPA to give special
consideration to exposure of infants and children to the pesticide
chemical residue in establishing a tolerance and to ``ensure that there
is a reasonable certainty that no harm will result to infants and
children from aggregate exposure to the pesticide chemical residue . .
.'' (FFDCA 408(b)(2)(C)). The Agency's consideration is documented in
Unit III.A.
I. Executive Order 13211: Actions Concerning Regulations That
Significantly Affect Energy Supply, Distribution or Use
This action is not subject to Executive Order 13211 (66 FR 28355)
(May 22, 2001) because it is not a significant regulatory action under
Executive Order 12866.
J. National Technology Transfer Advancement Act (NTTAA)
This action does not involve technical standards that would require
Agency consideration under NTTAA section 12(d), 15 U.S.C. 272.
K. Congressional Review Act (CRA)
This action is subject to the CRA, 5 U.S.C. 801 et seq., and EPA
will submit
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a rule report to each House of Congress and to the Comptroller General
of the United States. This action is not a ``major rule'' as defined by
5 U.S.C. 804(2).
List of Subjects in 40 CFR Part 174
Environmental protection, Administrative practice and procedure,
Agricultural commodities, Pesticides and pests, Reporting and
recordkeeping requirements.
Dated: December 4, 2025.
Edward Messina,
Director, Office of Pesticide Programs.
For the reasons set forth in the preamble, EPA is amending 40 CFR
chapter I as follows:
PART 174--PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED
PROTECTANTS
0
1. The authority citation for part 174 continues to read as follows:
Authority: 7 U.S.C. 136-136y; 21 U.S.C. 321(q), 346a and 371.
0
2. Add Sec. 174.556 to subpart W to read as follows:
Sec. 174.556 SpCas9 Protein; exemption from the requirement of a
tolerance.
An exemption from the requirement of a tolerance for residues of
SpCas9 protein in or on the food and feed commodities of Citrus when
used as a Plant-Incorporated Protectant in accordance with label
directions and good agricultural practices.
[FR Doc. 2025-22927 Filed 12-15-25; 8:45 am]
BILLING CODE 6560-50-P
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