Issuing

Agency/Office:

Animal and Plant Health Inspection Service / Biotechnology Regulatory Services

Title of

Document:

Guidance for Preparing Proposals to Exempt Plants with Additional

Modifications from Regulation Pursuant to 7 CFR § 340.1(b)(4)

Document ID: BRS-GD-2021-0001

Date of

Issuance:

January 19, 2021

Replaces: N/A

Summary: This document provides guidance on preparing proposals to exempt plants 

with additional modifications from regulation, as described in 7 CFR § 

340.1(b)(4). APHIS protects and enhances U.S. agricultural and natural 

resources using a science-based and risk-based regulatory framework to 

ensure the safe movement – including importation, interstate movement, 

and confined environmental release – of organisms developed using genetic 

engineering. APHIS receives its regulatory authority from the Plant Protection 

Act of 2000, and oversees organisms developed using genetic engineering in 

accordance with its regulations under 7 CFR part 340 (Movement of 

Organisms Modified or Produced Through Genetic Engineering).

Disclaimer: The contents of this guidance document do not have the force and effect of 

law and are not meant to bind the public in any way. This document is 

intended only to provide clarity to the public regarding existing requirements 

under the law or agency regulations.
2

USDA-APHIS Biotechnology Regulatory Services

Guidance for Preparing Proposals to Exempt Plants with Additional Modifications from Regulation Pursuant 

to

7 CFR § 340.1(b)(4) 

v. 01/07/2021

Biotechnology Regulatory Services 

Animal and Plant Health Inspection Service 

United States Department of Agriculture

4700 River Road

Riverdale, MD 20737

Pursuant to the Congressional Review Act (5 U.S.C. § 801 et seq.), the Office of Information and 

Regulatory Affairs designated this document as a non-major rule, as defined by 5 U.S.C. § 804(2).

The information contained in this document is intended solely as guidance. Except where noted, 

persons may choose to follow APHIS guidance or follow different procedures, practices, or protocols 

that meet applicable statutes and regulations.

Language implying that guidance is mandatory (e.g., “shall,” “must,” “required,” or “requirement”) 

should not be construed as binding unless the terms are used to refer to a statutory or regulatory 

requirement.

Following the guidance contained in this document should not be construed as a guarantee of 

compliance with applicable statutes and regulations.
3

Table of Contents

Introduction to Preparing Proposals to Exempt Plants with Additional Modifications Plants from

Regulation Pursuant to 7 CFR § 340.1(b)(4) ............................................................................................. 4

Submitting Proposals Under 7 CFR § 340.1(b)(4)...................................................................................... 5

Appendix 1 ............................................................................................................................................ 6

Example 1 based on § 340.1(b)(1)........................................................................................................... 6

Example 2 based on § 340.1(b)(2)........................................................................................................... 7

Guidance for Preparing Proposals to Exempt

Plants With Additional Modifications from 

Regulation Pursuant to 7 CFR § 340.1(b)(4) 
4

APHIS regulations at 7 CFR part 340 govern the movement of certain organisms that are modified or 

produced through genetic engineering. The regulations specify certain plants that are exempt from the 

regulations (7 CFR § 340.1(b), (c)). Among plants containing genetic modifications (i.e., changes to the 

plant genome), that could be achieved through conventional breeding, only plants containing a single 

genetic modification of one of the types listed in 7 CFR § 340.1(b)(1-3) are initially exempt from the 

regulations. Over time, APHIS expects new plant breeding innovations to evolve with advancements in 

science and technology, along with further development of scientific information related to conventional 

plant breeding. To ensure the regulations keep pace with advancements in science and technology, 

§ 340.1(b)(4) of the regulations establishes a process by which the Administrator can identify additional 

modifications that plants can contain and be exempt from the regulations, based on what could be 

achieved through conventional breeding. Through this process the Administrator could, for example, 

exempt plants that contain multiple genetic modifications on a species-specific basis, or the Administrator 

could list a new type of genetic modification that any plant could contain and be 

exempt from regulation. 

Proposals to exempt plants with additional modifications from the regulations may be APHIS-initiated or 

may be initiated by another party accompanied by adequate supporting information. In either case, 

assuming APHIS finds the proposal by the outside party to be scientifically credible, APHIS will publish a 

notice in the Federal Register of the proposal, along with the supporting information and will request 

public comments. After reviewing the comments, APHIS will publish a subsequent notice in the Federal 

Register announcing its action on the proposal. A list specifying modifications that plants can contain and 

be exempt pursuant to § 340.1(b) including § 340.1(b)(4) will be available on the APHIS website at: 

https://www.aphis.usda.gov/aphis/ourfocus/biotechnology. 

APHIS is providing the following guidance to help those who wish to prepare and submit proposals for 

exempting plants with additional modifications from the regulations pursuant to 7 CFR § 340.1(b)(4). We 

recommend discussing your proposal with APHIS prior to your first submission. 

Introduction to Preparing Proposals to Exempt Plants with Additional Modifications Plants from Regulation 

Pursuant to 7 CFR § 340.1(b)(4)
5

Submitting Proposals Under 7 CFR § 340.1(b)(4)

You must electronically submit your proposal via email (ExemptionProposals@usda.gov) to:

Bernadette Juarez

APHIS Deputy Administrator 

Biotechnology Regulatory Services

The proposal must include the following information:

• Proposer’s name and contact information, including email address.

• A description that clearly describes the additional modification(s) that a plant or plants can contain

and qualify for exemption (e.g., a number of changes achieved in all plants generally; a number of

changes in a particular plant species; a type of modification other than those already listed 7CFR

§ 340.1(b)(1-3)).

• The factual grounds demonstrating that the proposed genetic modification(s) could be

achieved through conventional plant breeding1

.

• Copies of scientific literature or publicly available information that support the proposal

and demonstrate the stated modification(s) can be achieved in conventional breeding.

• Any information known to the proposer that would be unfavorable to the proposal.

Examples of how to prepare a proposal for additional modifications plants can contain and qualify for 

exemption, based on existing exemptions (7 CFR § 340.1(b)(1) and (2)), are shown in Appendix 1.

After APHIS receives all the information required for a proposal, APHIS will complete its review 

and make a final determination within 12 months, except in circumstances that could not 

reasonably have been anticipated.

• If, after review of the proposal, APHIS determines there is insufficient publicly available

information and/or data or disagrees with the conclusion that plants containing the

modification(s) could be achieved through conventional breeding methods, APHIS will return

the proposal to the submitter and note the reasons for the return in writing.

• If, after review of the proposal, APHIS initially determines that plants containing the

modification(s) could be achieved through conventional breeding methods, APHIS will

publish a notice in the Federal Register proposing to exempt plants containing the additional

modification(s) from the regulations, including publicly available information and/or data

demonstrating that the modification(s) could be achieved through conventional breeding

methods, in accordance with the process set forth in § 340.1(b)(4)(i), requesting public

comment. After reviewing the comments, APHIS will publish a subsequent notice in the

Federal Register announcing its final determination.

A list specifying additional modifications that plants can contain and be exempt from regulation pursuant to 7 

CFR § 340.1(b)(4) will be available on the APHIS website at: 

https://www.aphis.usda.gov/aphis/ourfocus/biotechnology

1 As described in the Preamble to the revised 7 CFR part 340, our standard for “could be achieved through conventional 

breeding” is that the genetic modification could practically be expected to be pursued and achieved in a conventional 

breeding program. For example, evidence that multiple desired traits or genetic modifications can be introduced in a 

plant in a single step on a practical basis is needed to meet this standard. We are unlikely to adopt an exemption for 

plants containing statistically improbable modifications.
6

Appendix 1

Example 1 based on § 340.1(b)(1)

Name: Dr. Professor Scientist, 111 Plant Breeding Road, Anycity, Anystate, 00000 

drprofessorscientist@any.edu

Description of the modification.

The genetic modification is a single change in any plant species resulting from cellular repair of a targeted 

DNA break in the absence of an externally provided repair template.

The factual grounds demonstrating that the proposed modification(s) could be achieved 

through conventional plant breeding.

Spontaneous mutation rates in higher plants are low, ranging from 10-5 to 10-8 (1). During the 

1920s and 1930s, scientists discovered that mutation rates could be increased using radiation 

and chemical treatments (2). Breeding programs based on efficient mutation techniques have 

been widely used by plant breeders and many of our food crops are derived either directly or 

indirectly from such programs (2, 3). The molecular basis of spontaneous mutation, chemical 

mutagenesis, and radiation mutagenesis is known to result from damage to DNA( 2-4). Plants 

have evolved a number of DNA repair pathways to repair damaged DNA. One of the common 

types of DNA damage is the double strand break (3, 4). When double strand breaks are 

repaired, the frequent outcome is a deletion or insertion (also known as an indel) (5). Genome 

editing, just like conventional breeding, can be used to generate indels. Whole genome 

sequencing of individuals within a breeding population reveals the wide extent of 

polymorphisms created from DNA repair events (6, 7). As cellular repair of a targeted DNA 

break in the absence of an externally provided repair template is a common occurrence in 

conventional breeding, a plant containing a modification involving DNA breaks induced 

through genome editing should be eligible for exemption.

Copies of scientific literature, unpublished studies, or other data that support the 

proposal demonstrating that the stated modification(s) can routinely be achieved in 

conventional breeding.

1. S.-Y. Jiang, S. Ramachandran, Assigning biological functions to rice genes by genome annotation,

expression analysis and mutagenesis. Biotechnology Letters 32, 1753-1763 (2010).

2. M. C. Kharkwal, "A brief history of plant mutagenesis" in Plant Mutation Breeding and Biotechnology,

Q. Y. Shu, B.P. Forster, and H. Nakagawa, Ed. (CABI, 2012), 10.1079/9781780640853.0000 chap. 2, pp.

21-30.

3. Y. Oladosu et al., Principle and application of plant mutagenesis in crop improvement: a review.

Biotechnology & Biotechnological Equipment 30, 1-16 (2016).

4. Q. Que et al., Plant DNA Repair Pathways and Their Applications in Genome Engineering. Methods Mol

Biol 1917, 3-24 (2019).

5. H. Puchta, The repair of double-strand breaks in plants: mechanisms and consequences for genome

evolution. Journal of Experimental Botany 56, 1-14 (2004).

6. S. Li et al., Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole

genome sequencing. J Zhejiang Univ Sci B 17, 905-915 (2016).

7. S. Sun et al., Extensive intraspecific gene order and gene structural variations between Mo17 and

other maize genomes. Nat Genet 50, 1289-1295 (2018).

Information unfavorable to the proposal

None
7

Example 2 based on § 340.1(b)(2) 

Name: Dr. Professor Scientist, 111 Plant Breeding Road, Anycity, Anystate, 00000 

drprofessorscientist@anycompany.com

Description of the modification.

The genetic modification is a single targeted single base pair substitution in the genome of any plant 

species. 

The factual grounds demonstrating that the proposed modification(s) could be achieved 

through conventional plant breeding.

Chemical and radiation mutagenesis has been widely used in plant breeding (1-4). The Food and Agriculture 

Organization of the United Nations/International Atomic Energy Agency – Mutant Variety Database 

(FAO/IAEA-MVD) data (2019) reports on the developed and officially released mutants, a total of 3,275 

accessions from 225 species (4). The types of chemical mutagens most widely used in plants include ethylmethane sulfonate (EMS) (1, 3, 5, 6), methyl-methansulfonate (MMS) (3), sodium azide (7), N-methyl-Nnitrosourea (MNU) (8) and hydroxylamine (4, 9).

From (2).

EMS, MMS, and MNU are strong alkylating agents (2). These chemicals modify nucleotides by attaching an 

ethyl (EMS) or methyl (MMS and MNU) to the N or O moieties of the DNA bases.(10). The most frequently 

alkylated sites are shown in Figure 12.4 from reference (2). Modifications that occur on Guanine-O6, cytosine- 
8

N3, cytosine O2, Adenine-N1, Adenine-N3, Thymine-O4, Thymine-N3, and Thymine-O2 can influence base 

pairing (2, 11-14). In some cases, the modification causes mispairing that leads to mutation. The most well 

documented case of mispairing results from ethylation of Guanine-O6, after treatment with ethyl methane 

sulfonate (EMS), diethyl sulfate, or ethyl nitrosourea (ENU) (9). When Guanine-O6 is ethylated, guanine can 

pair with thymine instead of its usual cytosine (2, 9). During DNA replication, a DNA repair process removes 

the substituted guanine and replaces it with an adenine, the usual base pair for thymine (2, 9). This mutation 

results in a G to A transition (2, 9). Transitions are substitutions for purines with purines (A for G and G for A) 

and pyrimidines with pyrimidines (C for T and T for C). Transversions are substitutions between purines and 

pyrimidines (A or G for C or T and vice versa). The predominant mutation resulting from EMS mutagenesis is 

the G to A transition (1, 2). At a low frequency, EMS generates G to C or G to T transversions by 7-ethylguanine 

hydrolysis or A to G transition by 3-ethyladenine pairing errors (1, 13). Minoia et al. (2010) also observed A to 

C transversions in EMS treated tomato (15).

Some of the alkylation sites on DNA bases do not influence base pairing. The most heavily alkylated site by 

methyl methanesulfonate (MMS) is Guanine-N7 representing 83% of the alkylation sites (2, 11) and this 

modification does not influence base pairing (14). It is thought that when alkylation of the base occurs on a 

moiety not involved in base pairing, mutagenesis can still occur. The modified base is likely to be removed 

from the DNA strand (11-14) and when depurination occurs, faulty excision repair may lead to mutagenesis 

(16). MMS frequently leads to the formation of T to G transversions (2) and G to A and A to G transitions (1, 2).

MNU alkylates Guanine-O6 to a much greater extent than MMS showing a bias for G to A transitions (2). It can 

also alkylate Guanine-N7 and Adenine at N1, N3, and N7 (2). Suzuki et al. (2008) sequenced mutants isolated 

after MNU mutagenesis and also detected C to T transitions at high frequency, as well as T to A and A to T 

transversions (8).

Non-alkylating mutagenesis.

Hydroxylamine and sodium azide are non-alkylating mutagens frequently used in plant mutagenesis (2, 3). 

Hydroxylamine reacts with N substituents that are not alkylated by alkylating agents forming either N4 

hydroxycytosine or N6 hydroxyadenosine (9, 17). As depicted in Figure 12.4, both target sites play a role in 

base pairing. Brown (1968) speculated that the Cytosine derivative can base pair with T leading to a C to A 

transition (17).

Sodium azide is the principle chemical mutagen used in barley breeding (7). Sodium azide reacts with the 

endogenous metabolite O-acetylserine to form a reactive intermediate, beta-azidoalanine; presumably the 

beta azidoalanine reacts directly with DNA or through another intermediate to modify the DNA(7). It is not 

clear how this DNA modification results in base pairing substitutions (7). Olsen et al. (7) isolated several 

mutant lines from a screen for pigment mutants and sequenced candidate genes. They identified a number of 

single base pair substitutions in azide mutagenized barley including A to G; G to A, and T to C transitions and A 

to T and T to A transversions (7).

Summary of reviewed mutagenesis outcomes.

Chemical mutagenesis results in single base pair mutations as a result of DNA modification and repair. The 

agents used in mutagenesis modify DNA in various ways. In some cases, the modification leads to mispairing 

and repair. In other cases, excision of the modified base followed by faulty repair is thought to lead to the 

mutation. In most cases, the mutation is a G to A transition. However, all 12 possible combinations of single 

base pair substitution have been reported in the course of breeding experiments. These data are summarized 

in the table below:
9

Original

base SBS

1 A G Transition MMS (1); NA (7); EMS ((1)

2 A C Transversion EMS (15)

3 A T Transversion MMS (1); MNU (8); NA (13)

4 G A Transition EMS (1); MNU (2, 8); MMS (2); NA (7)

5 G C Transversion EMS (1)

6 G T Transversion EMS (1)

7 C G Transversion MNU (2); MMS(2)

8 C T Transition MNU (8); EMS (6))

9 C A Transversion HA (17)

10 T A Transversion MNU (8); NA (7); EMS (6)

11 T C Transition NA (7)

12 T G Transversion MMS (1); EMS (6)

MMS-methyl methane sulfonate 

NA-sodium azide

EMS-ethane methyl sulfonate 

MNU-methyl nitrosourea

HA-hydroxylamine

SBS-single base pair substitution

Numbers in parentheses refer to references.

Conclusion

As chemical mutagenesis is widely used in plant breeding, as single base pair substitutions are a frequent 

outcome of chemical mutagenesis, and all possible single nucleotide substitutions have been observed by 

sequencing plant DNA derived from plants subjected to chemical mutagenesis, a modification that results in a 

single nucleotide substitution should be exempt from regulation.

Copies of scientific literature, unpublished studies, or other data that support the 

proposal demonstrating that the stated modification(s) can routinely be achieved in 

conventional breeding.

1. Y. Kim, K. S. Schumaker, J. K. Zhu, EMS mutagenesis of Arabidopsis. Methods Mol Biol 323, 101-103 

(2006).

2. J. M. Leitao, "Chemical Mutagenesis" in Plant Mutation Breeding and Biotechnology, Q. Y. Shu, Forster, 

B. P., Nakagawa, H., Ed. (CABI, 2012), http://doi.org/10.1079/9781780640853.0135 chap. 12, pp. 135-

158.

3. M. A. J. Parry et al., Mutation discovery for crop improvement. Journal of Experimental Botany 60, 

2817-2825 (2009).

4. V. E. Viana, C. Pegoraro, C. Busanello, A. Costa de Oliveira, Mutagenesis in Rice: The Basis for Breeding 

a New Super Plant. Frontiers in Plant Science 10 (2019).

5. J. Maple, S. G. Møller, Mutagenesis in Arabidopsis. Methods Mol Biol 362, 197-206 (2007).

6. X. Serrat et al., EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly 

obtaining TILLING mutant populations. Plant Methods 10, 5 (2014).

7. O. Olsen, X. Wang, D. von Wettstein, Sodium azide mutagenesis: preferential generation of A.T-->G.C 

transitions in the barley Ant18 gene. Proc Natl Acad Sci U S A 90, 8043-8047 (1993).

8. T. Suzuki et al., MNU-induced mutant pools and high performance TILLING enable finding of anygene
10

mutation in rice. Molecular Genetics and Genomics 279, 213-223 (2008).

9. B. Singer, J. T. Kusmierek, Chemical Mutagenesis. Annual Review of Biochemistry 51, 655-691(1982).

10. G. R. Hoffmann, Genetic effects of dimethyl sulfate, diethyl sulfate, and related compounds. Mutation 

Research/Reviews in Genetic Toxicology 75, 63-129 (1980).

11. D. T. Beranek, Distribution of methyl and ethyl adducts following alkylation with monofunctional 

alkylating agents. Mutation Research 231, 11-30 (1990).

12. J. G. Jansen et al., Molecular analysis of hprt gene mutations in skin fibroblasts of rats exposed in vivo 

to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea. Cancer Res 54, 2478-2485 (1994).

13. D. R. Krieg, Ethyl Methanesulfonate-induced reversion of bacteriophage T4rII mutants. Genetics48

(1963).

14. R. Saffhill, G. P. Margison, P. J. O'Connor, Mechanisms of carcinogenesis induced by alkylating agents.

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 823, 111-145 (1985).

15. S. Minoia et al., A new mutant genetic resource for tomato crop improvement by TILLINGtechnology.

BMC Res Notes 3, 69-69 (2010).

16. M. Ensminger et al., DNA breaks and chromosomal aberrations arise when replication meets base 

excision repair. Journal of Cell Biology 206, 29-43 (2014).

17. D. M. Brown, M. J. E. Hewlins, P. Schell, The tautomeric state of N(4)-hydroxy- and of N(4)-aminocytosine derivatives. Journal of the Chemical Society C: Organic 10.1039/J39680001925, 1925-1929 

(1968).

Information Unfavorable to the Proposal

None