**NIH** Guidelines for Research Involving Recombinant **DNA Molecules
MSU/1998**

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## SECTION III. EXPERIMENTS COVERED BY THE NIH GUIDELINES

This section describes six categories of experiments involving
recombinant DNA: (i) those that require Institutional Biosafety
Committee (IBC) approval, RAC review, and NIH Director approval before
initiation (see Section III-A), (ii) those that require NIH/ORDA and
Institutional Biosafety Committee approval before initiation (see
Section III-B), (iii) those that require Institutional Biosafety
Committee and Institutional Review Board approvals and NIH/ORDA
registration before initiation (see Section III-C), (iv) those that
require Institutional Biosafety Committee approval before initiation
(see Section III-D), (v) those that require Institutional Biosafety
Committee notification simultaneous with initiation (see Section III-E),
and (vi) those that are exempt from the NIH Guidelines (see Section
III-F).

**Note:** If an experiment falls into Sections III-A, III-B, or III-C
and one of the other sections, the rules pertaining to Sections III-A,
III-B, or III-C shall be followed. If an experiment falls into Section
III-F and into either Sections III-D or III-E as well, the experiment is
considered exempt from the NIH Guidelines.

Any change in containment level, which is different from those specified
in the NIH Guidelines, may not be initiated without the express approval
of NIH/ORDA (see Section IV-C-l-b- (2) and its subsections, Minor
Actions).

Section III-A. Experiments that Require Institutional Biosafety
Committee Approval, RAC Review, and NIH Director Approval Before
Initiation (See Section IV-C-1-b-(2), Major Actions).

## Section III-A-l. Major Actions under the NIH Guidelines 

Experiments considered as Major Actions under the NIH Guidelines cannot
be initiated without submission of relevant information on the proposed
experiment to the Office of Recombinant DNA Activities, National
Institutes of Health/MSC 7010,6000 Executive Boulevard, Suite 302,
Bethesda, Maryland 20892-7010, (301) 496-9838, the publication of the
proposal in the Federal Register for 15 days of comment, review by RAC,
and specific approval by NIH. The containment conditions or stipulation
requirements for such experiments will be recommended by RAC and set by
NIH at the time of approval. Such experiments require Institutional
Biosafety Committee approval before initiation. Specific experiments
already approved are included in Appendix D**,** Major Actions Taken
under the NIH Guidelines, which may be obtained from the Office of
Recombinant DNA Activities, National Institutes of Health/MSC 7010, 6000
Executive Boulevard, Suite 302, Bethesda, Maryland 20892-7010 (301)
496-9838.

**Section III-A-l-a.** The deliberate transfer of a drug resistance
trait to microorganisms that are not known to acquire the trait
naturally (see Section V-B, *Footnotes and References of Sections
I-IV*), if such acquisition could compromise the use of the drug to
control disease agents in humans, veterinary medicine, or agriculture,
will be reviewed by RAC.

Section III-B. Experiments That Require NIH/ORDA and Institutional
Biosafety Committee Approval Before Initiation

Experiments in this category cannot be initiated without submission of
relevant information on the proposed experiment to NIH/ORDA. The
containment conditions for such experiments will be determined by
NIH/ORDA in consultation with ad hoc experts. Such experiments require
Institutional Biosafety Committee approval before initiation (see
Section IV -B- 2-b-( I ),

Institutional Biosafety Committee).

**Section III-B-I. Experiments Involving the Cloning of Toxin Molecules
with LD~5o~ of Less than 100 Nanograms per Kilogram Body Weight**

Deliberate formation of recombinant DNA containing genes for the
biosynthesis of toxin molecules lethal for vertebrates at an LD~5o~ of
less than 100 nanograms per kilogram body weight (e.g., microbial toxins
such as the botulinum toxins, tetanus toxin, diphtheria toxin, and
*Shigella dysenteriae* neurotoxin). Specific approval has been given for
the cloning in *Escherichia coli K-* 12 of DNA containing genes coding
for the biosynthesis of toxic molecules, which are lethal to vertebrates
at 100 nanograms to 10O micrograms per kilogram body weight. Specific
experiments already approved under this section may be obtained from the
Office of Recombinant DNA Activities, National Institutes of Health/MSC
7010, 6000 Executive Boulevard, Suite 302, Bethesda, Maryland
20892-7010, (301) 496-9838.

Section III-C. Experiments that Require Institutional Biosafety
Committee and Institutional Review Board Approvals and NIH/ORDA
Registration Before Initiation

Section III-C-l. Experiments Involving the Deliberate Transfer of
Recombinant DNA or DNA or RNA Derived from Recombinant DNA into One or
More Human Subjects

Research proposals involving the deliberate transfer of recombinant DNA,
or DNA or RNA derived from recombinant DNA, into human subjects (human
gene transfer) will be considered through a review process involving
both NIH/ORDA and RAC. Investigators shall submit relevant information
on the proposed human gene transfer experiments to NIH/ORDA. Submission
of human gene transfer protocols to NIH will be in the format described
in Appendix M-I, Submission Requirements \--Human Gene Transfer
Experiments. Submission to NIH/ORDA shall be for registration purposes
and will ensure continued public access to relevant human gene transfer
information in compliance with the NIH Guidelines. Investigational New
Drug (IND) applications should be submitted to FDA in the format
described in 21 CFR, Chapter I, Subchapter D, Part 312, Subpart B,
Section 23, IND Content and Format.

Institutional Biosafety Committee approval must be obtained from each
institution at which recombinant DNA material will be administered to
human subjects (as opposed to each institution involved in the
production of vectors for human application and each institution at
which there is ex vivo transduction of recombinant DNA material into
target cells for human application).

RAC prefers that submission to NIH/ORDA in accordance with Appendix M-I,
*Submission* *Requirements \--Human Gene Transfer Experiments*, contain
no proprietary data or trade secrets, enabling all aspects of the review
to be open to the public. Following receipt by NIH/ORDA, relevant
information shall be entered into the NIH human gene transfer database
for registration purposes. Summary information pertaining to the human
gene transfer protocol will be forwarded to RAC members. NIH/ORDA
summary information shall include comparisons to previously registered
protocols. Specific items of similarity to previous experiments include
(but are not limited to): (i) gene delivery vehicle, (ii) functional
gene, (iii) marker gene, (iv) packaging cell (if applicable), (v)
disease application, (vi) route of administration, and (vii) patient
selection criteria.

RAC members shall notify NIH/ORDA within 15 working days if the protocol
has been determined to represent novel characteristics requiring further
public discussion.

Full RAC review of an individual human gene transfer experiment can be
initiated by the NIH Director or recommended to the NIH Director by: (i)
three or more RAC members, or (ii) other Federal agencies. An individual
human gene transfer experiment that is recommended for full RAC review
should represent novel characteristics deserving of public discussion.
RAC recommendations on a specific human gene transfer experiment shall
be forwarded to the NIH Director, the Principal Investigator, the
sponsoring institution, and other DHHS components, as appropriate.

**Note:** For specific directives concerning the use of retroviral
vectors for gene delivery, consult Appendix B- V -I, *Murine Retroviral
Vectors.*

**Section III-D. Experiments that Require Institutional Biosafety
Committee Approval Before Initiation**

Prior to the initiation of an experiment that falls into this category,
the Principal Investigator must submit a registration document to the
Institutional Biosafety Committee which contains the following
information: (i) the source(s) of DNA; (ii) the nature of the inserted
DNA sequences; (iii) the host(s) and vector(s) to be used; (iv) if an
attempt will be made to obtain expression of a foreign gene, and if so,
indicate the protein that will be produced; and (v) the containment
conditions that will be implemented as specified in the *NIH
Guidelines.* For experiments in this category, the registration document
shall be dated, signed by the Principal Investigator, and filed with the
Institutional Biosafety Committee. The Institutional Biosafety Committee
shall review and approve all experiments in this category prior to their
initiation. Requests to decrease the level of containment specified for
experiments in this category will be considered by NIH (see Section
N-C-I-b-(2)-(c), *Minor Actions).*

**Section III-D-I. Experiments Using Risk Group 2, Risk Group 3, Risk
Group 4, or Restricted Agents as Host-Vector Systems** (See Section
II-A, *Risk Assessment)*

**Section III-D-l-a.** Experiments involving the introduction of
recombinant DNA into Risk Group 2 agents will usually be conducted at
Biosafety Level (BL) 2 containment. Experiments with such agents will
usually be conducted with whole animals at BL2 or BL2-N (Animals)
containment.

**Section III-D-l-b.** Experiments involving the introduction of
recombinant DNA into Risk Group 3 agents will usually be conducted at
BL3 containment. Experiments with such agents will usually be conducted
with whole animals at BL3 or BL3-N containment.

**Section III-D-l-c.** Experiments involving the introduction of
recombinant DNA into Risk Group 4 agents shall be conducted at BL4
containment. Experiments with such agents shall be conducted with whole
animals at BL4 or BL4-N containment.

**Section III-D-l-d.** Containment conditions for experiments involving
the introduction of recombinant DNA into restricted agents shall be set
on a case-by-case basis following NIWORDA review. A U.S. Department of
Agriculture permit is required for work with plant or animal pathogens
(see Section V-G and V-L, *Footnotes and References of Sections I-IV).*
Experiments with such agents shall be conducted with whole animals at BU
or BL4-N containment.

**Section III-D-2.** **Experiments in Which DNA From Risk Group 2, Risk
Group 3, Risk Group 4, or Restricted Agents is Cloned into Nonpathogenic
Prokaryotic or Lower Eukaryotic Host-Vector Systems**

**Section III-D-2-a**. Experiments in which DNA from Risk Group 2 or
Risk Group 3 agents (see Section II-A, *Risk Assessment)* is transferred
into nonpathogenic prokaryotes or lower eukaryotes may be performed
under BL2 containment. Experiments in which DNA from Risk Group 4 agents
is transferred into nonpathogenic prokaryotes or lower eukaryotes may be
performed under BL2 containment after demonstration that only a totally
and irreversibly defective fraction of the agent\'s genome is present in
a given recombinant. In the absence of such a demonstration, BU
containment shall be used. The Institutional Biosafety Committee may
approve the specific lowering of containment for particular experiments
to BL 1. Many experiments in this category are exempt from the *NIH
Guidelines* (see Section III-F, *Exempt Experiments).* Experiments
involving the formation of recombinant DNA for certain genes coding for
molecules toxic for vertebrates require NIH/ORDA approval (see Section
III-B-l, *Experiments Involving the Cloning of Toxin Molecules with
LD~5o~ of Less than 100 Nanograms Per Kilogram Body Weight)* or shall be
conducted under NIH specified conditions as described in Appendix F,
*Containment Conditions for Cloning of Genes Coding for the Biosynthesis
of Molecules Toxic for Vertebrates.*

**Section III-D-2-b.** Containment conditions for experiments in which
DNA from restricted agents is transferred into nonpathogenic prokaryotes
or lower eukaryotes shall be determined by NIH/ORDA following a
case-by-case review (see Section V-L, *Footnotes and References of
Sections I-IV).* A U.S. Department of Agriculture permit is required for
work with plant or animal pathogens (see Section V-G, *Footnotes and
References of Sections I-IV).*

**Section III-D-3.** Experiments Involving the Use of Infectious DNA or
RNA Viruses or Defective DNA or RNA Viruses in the Presence of Helper
Virus in Tissue Culture Systems

**Caution:** Special care should be used in the evaluation of
containment levels for experiments, which are likely to either, enhance
the pathogenicity (e.g., insertion of a host oncogene) or to extend the
host range (e.g., introduction of novel control elements) of viral
vectors under conditions that permit a productive infection. In such
cases, serious consideration should be given to increasing physical
containment by at least one level.

**Note:** Recombinant DNA or RNA molecules derived there from, which
contain less than two-thirds of the genome of any eukaryotic virus (all
viruses from a single Family (see Section V-I, *Footnotes and References
of Sections I-IV)* being considered identical (see Section V-K,
*Footnotes and References of Sections I-IV),* are considered defective
and may be used in the absence of helper under the conditions specified
in Section III-E-l, *Experiments Involving the Formation of Recombinant
DNA Molecules Containing No More than Two- Thirds of the Genome of any
Eukaryotic Virus.*

**Section III-D-3-a.** Experiments involving the use of infectious or
defective Risk Group 2 viruses (see Appendix **B-II,** *Risk Group* 2
*Agents)* in the presence of helper virus may be conducted at BL2.

**Section III-D-3-b.** Experiments involving the use of infectious or
defective Risk Group 3 viruses (see Appendix B-Ill-D, *Risk Group* 3
*(RG3) -Viruses and Prions)* in the presence of helper virus may be
conducted at BL3.

**Section III-D-3-c.** Experiments involving the use of infectious or
defective Risk Group 4 viruses (see Appendix B-IV-D, *Risk Group* 4
*(RG4) -Viral Agents)* in the presence of helper virus may be conducted
at BU.

**Section III-D-3-d.** Experiments involving the use of infectious or
defective restricted poxviruses (see Sections V-A and V-L, *Footnotes
and References of Sections I-IJI)* in the presence of helper virus shall
be determined on a case-by-case basis following NIH/ORDA review. A U.S.
Department of Agriculture permit is required for work with plant or
animal pathogens (see Section V-G, *Footnotes and References of Sections
I-IV).*

**Section III-D-3-e.** Experiments involving the use of infectious or
defective viruses in the presence of helper virus, which are not covered
in Sections III-D-3-a through III-D-3-d, may be conducted at BL1.

**Section III-D-4.** **Experiments Involving Whole Animals**

This section covers experiments involving whole animals in which the
animal\'s genome has been altered by stable introduction of recombinant
DNA, or DNA derived there from, into the germ-line (transgenic animals)
and experiments involving viable recombinant DNA-modified microorganisms
tested on whole animals. For the latter, other than viruses, which are
only vertically transmitted, the experiments may *not* be conducted at
BLI-N containment. A minimum containment ofBL2 or BL2-N is required.

**Caution** -Special care should be used in the evaluation of
containment conditions for some experiments with transgenic animals. For
example, such experiments might lead to the creation of novel mechanisms
or increased transmission of a recombinant pathogen or production of
undesirable traits in the host animal. In such cases, serious
consideration should be given to increasing the containment conditions.

**Section III-D-4-a.** Recombinant DNA, or DNA or RNA molecules derived
there from, from any source except for greater than two-thirds of
eukaryotic viral genome may be transferred to any non-human vertebrate
or any invertebrate organism and propagated under conditions of physical
containment comparable to BLI or BLI-N and appropriate to the organism
under study (see Section V-B, *Footnotes and References of Sections
I-IV).* Animals that contain sequences from viral vectors, which do not
lead to transmissible infection either directly or indirectly as a
result of complementation or recombination in animals, may be propagated
under conditions of physical containment comparable to BLI or BLI-N and
appropriate to the organism under study. Experiments involving the
introduction of other sequences from eukaryotic viral genomes into
animals are covered under Section Ill-D-4-b, *Experiments Involving
Whole Animals.* For experiments involving recombinant DNA-modified Risk
Groups 2, 3, 4, or restricted organisms, see Sections V-A, V-G, and V-L,
*Footnotes and References of Sections I-IV.* It is important that the
investigator demonstrate that the fraction of the viral genome being
utilized does not lead to productive infection. A U.S. Department of
Agriculture permit is required for work with plant or animal pathogens
(see Section V-G, *Footnotes and References of Sections I-IV).*

**Section III-D-4-b.** For experiments involving recombinant DNA, or DNA
or RNA derived there from, involving whole animals, including transgenic
animals, and not covered by Sections III-D-I, *Experiments Using Human
or Animal Pathogens (Risk Group* 2, *Risk Group* 3, *Risk Group* 4, *or
Restricted Agents as Host-Vector Systems,* or llI-D-4-a, *Experiments
Involving Whole Animals,* the appropriate containment shall be
determined by the Institutional Biosafety Committee.

**Section III-D-4-c.** Exceptions under Section III-D-4, *Experiments
Involving Whole Animals*

**Section III-D-4-c-(1).** Experiments involving the generation of
transgenic rodents that require BLI containment are described under
Section lll-E-3, *Experiments Involving Transgenic Rodents.*

**Section III-D-4-c-(2).** The purchase or transfer of transgenic
rodents is exempt from the *NIH Guidelines* under Section III-F, *Exempt
Experiments* (see Appendix C- VI, *The Purchase or Transfer of
Transgenic Rodents).*

**Section III-D-5. Experiments Involving Whole Plants**

Experiments to genetically engineer plants by recombinant DNA methods,
to use such plants for other experimental purposes (e.g., response to
stress), to propagate such plants, or to use plants together with
microorganisms or insects containing recombinant DNA, may be conducted
under the containment conditions described in Sections III-D-5-a through
III-D-5-e. If experiments involving whole plants are not described in
Section III-D-5 and do not fall under Sections III-A, III-B, III-D or
III F, they are included in Section III-E.

**NOTE** -For recombinant DNA experiments falling under Sections
III-D-5-a through III-D-5-d, physical containment requirements may be
reduced to the next lower level by appropriate biological containment
practices, such as conducting experiments on a virus with an obligate
insect vector in the absence of that vector or using a genetically
attenuated strain.

**Section III-D-5-a.** BL3-P (plants) or BL2-P + biological containment
is recommended for experiments involving most exotic (see Section V -W,
*Footnotes and References of Sections I-IV)* infectious agents with
recognized potential for serious detrimental impact on managed or
natural ecosystems when recombinant DNA techniques are associated with
whole plants.

**Section III-D-5-b.** BL3-P or BL2-P + biological containment is
recommended for experiments involving plants containing cloned genomes
of readily transmissible exotic (see Section V-W, *Footnotes and
References of Sections I-IV)* infectious agents with recognized
potential for serious detrimental effects on managed or natural
ecosystems in which there exists the possibility of reconstituting the
complete and functional genome of the infectious agent by genomic
complementation *in planta.*

**Section III-D-5-c.** BL4-P containment is recommended for experiments
with a small number of readily transmissible exotic (see Section V-W,
*Footnotes and References of Sections I-IV)* infectious agents, such as
the soybean rust fungus *(Phakospora pachyrhizi)* and maize streak or
other viruses in the presence of their specific arthropod vectors, that
have the potential of being serious pathogens of major U.S. crops.

**Section 1ll-D-5-d.** BL3-P containment is recommended for experiments
involving sequences encoding potent vertebrate toxins introduced into
plants or associated organisms. Recombinant DNA containing genes for the
biosynthesis of toxin molecules lethal for vertebrates at an LD~50~ of
\< 100 nanograms per kilogram body weight fall under Section III-B-I,
*Experiments Involving the Cloning of Toxin Molecules with LD~5o~ of
Less than 1*

*00 Nanograms Per Kilogram Body Weight,* and require NIH/ORDA and
Institutional Biosafety Committee approval before initiation.

**Section III-D-5-e.** BL3-P or BL2-P + biological containment is
recommended for experiments with microbial pathogens of insects or small
animals associated with plants if the recombinant DNA-modified organism
has a recognized potential for serious detrimental impact on managed or
natural ecosystems.

**Section III-D-6. Experiments Involving More than 10 Liters of
Culture**

The appropriate containment will be decided by the Institutional
Biosafety Committee. Where appropriate, Appendix K, *Physical
Containment for Large Scale Uses of Organisms Containing Recombinant DNA
Molecules,* shall be used. Appendix K describes containment conditions
Good Large Scale Practice through BL3-Large Scale.

Section III-E. Experiments that Require Institutional Biosafety
Committee Notice Simultaneous with Initiation

Experiments not included in Sections III-A, III-B, III-C, III-D, III-F,
and their subsections are considered in Section III-E. All such
experiments may be conducted at BLI containment. For experiments in this
category, a registration document (see Section III-D, *Experiments that
Require Institutional Biosafety Committee Approval Before Initiation)*
shall be dated and signed by the investigator and filed with the local
Institutional Biosafety Committee at the time the experiment is
initiated. The Institutional Biosafety Committee reviews and approves
all such proposals, but Institutional Biosafety Committee review and
approval prior to initiation of the experiment is not required (see
Section IV-A, *Policy).* For example, experiments in which all
components derived from non-pathogenic prokaryotes and non-pathogenic
lower eukaryotes fall under Section III-E and may be conducted at BL 1
containment.

**Section Ill-E-l.** **Experiments Involving the Formation of
Recombinant DNA Molecules Containing No More than Two- Thirds of the
Genome of any Eukaryotic Virus**

Recombinant DNA molecules containing no more than two-thirds of the
genome of any eukaryotic virus (all viruses from a single Family (see
Section V-Q, *Footnotes and References of Sections I-IV)* being
considered identical (see Section V-S, *Footnotes and References of
Sections I-IV))* may be propagated and maintained in cells in tissue
culture using BLI containment. For such experiments, it must be
demonstrated that the cells lack helper virus for the specific Families
of defective viruses being used. If helper virus is present, procedures
specified under Section III-D-3, *Experiments Involving the Use of
Infectious Animal or Plant DNA or RNA Viruses or Defective Animal or
Plant DNA or RNA Viruses in the Presence of Helper Virus in Tissue
Culture Systems,* should be used. The DNA may contain fragments of the
genome of viruses from more than one Family but each fragment shall be
less than two-thirds of a genome.

## Section III-E-2. Experiments Involving Whole Plants 

This section covers experiments involving recombinant DNA-modified whole
plants, and/or experiments involving recombinant DNA-modified organisms
associated with whole plants, except those that fall under Section
III-A, III-B, III-D, or III-F. It should be emphasized that knowledge of
the organisms and judgment based on accepted scientific practices should
be used in all cases in selecting the appropriate level of containment.
For example, if the genetic modification has the objective of increasing
pathogenicity or converting a non-pathogenic organism into a pathogen,
then a higher level of containment may be appropriate depending on the
organism, its mode of dissemination, and its target organisms. By
contrast, a lower level of containment may be appropriate for small
animals associated with many types of recombinant DNA-modified plants.

**Section III-E-2-a.** BL1-P is recommended for all experiments with
recombinant DNA-containing plants and plant-associated microorganisms
not covered in Section III-E-2-b or other sections of the *NIH
Guidelines.* Examples of such experiments are those involving
recombinant DNA-modified plants that are not noxious weeds or that
cannot interbreed with noxious weeds in the immediate geographic area,
and experiments involving whole plants and recombinant DNA-modified
non-exotic (see Section V-M. *Footnotes and References of Sections
I-IV)* microorganisms that have no recognized potential for rapid and
widespread dissemination or for serious detrimental impact on managed or
natural ecosystems (e.g., *Rhizobium* spp. and *Agrobacterium* spp.).

**Section III-E-2-b.** BL2-P or BL1-P + biological containment is
recommended for the following experiments:

**Section ID-E-2-b-(1).** Plants modified by recombinant DNA that are
noxious weeds or can interbreed with noxious weeds in the immediate
geographic area.

**Section Ill-E-2-b-(2).** Plants in which the introduced DNA represents
the complete genome of a non-exotic infectious agent (see Section V-W,
*Footnotes and References of Sections I-IV).*

**Section III-E-2-b-(3).** Plants associated with recombinant
DNA-modified non-exotic microorganisms that have a recognized potential
for serious detrimental impact on managed or natural ecosystems (see
Section V -W, *Footnotes and References of Sections I-IV).*

**Section III-E-2-b-(4).** Plants associated with recombinant
DNA-modified exotic microorganisms that have no recognized potential for
serious natural ecosystems (see Section V-W, *Footnotes and References
of Sections I-IV).*

**Section III-E-2-b-(5).** Experiments with recombinant DNA-modified
arthropods or small animals associated with plants, or with arthropods
or small animals with recombinant DNA-modified microorganisms associated
with them if the recombinant DNA-modified microorganisms have no
recognized potential for serious detrimental impact on managed or
natural ecosystems (see Section V-W, *Footnotes and References of
Sections I-IV).*

## Section III-E-3. Experiments Involving Transgenic Rodents 

This section covers experiments involving the generation of rodents in
which the animal\'s genome has been altered by stable introduction of
recombinant DNA, or DNA derived there from, into the germ-line
(transgenic rodents). Only experiments that require BLI containment are
covered under this section; experiments that require BL2, BL3, BL4
containment are covered under Section III-D-4, *Experiments Involving
Whole Animals.*

**Section III-F. Exempt Experiments**

The following recombinant DNA molecules are exempt from the *NIH
Guidelines* and registration with the Institutional Biosafety Committee
is not required:

**Section III-F-l.** Those that are not in organisms or viruses.

**Section III-F-2.** Those that consist entirely of DNA segments from a
single nonchromosomal or viral DNA source, though one or more of the
segments may be a synthetic equivalent.

**Section III-F-3.** Those that consist entirely of DNA from a
prokaryotic host including its indigenous plasmids or viruses when
propagated only in that host (or a closely related strain of the same
species), or when transferred to another host by well established
physiological means.

**Section III-F-4.** Those that consist entirely of DNA from an
eukaryotic host including its chloroplasts, mitochondria, or plasmids
(but excluding viruses) when propagated only in that host (or a closely
related strain of the same species).

**Section 1lI-F-5.** Those that consist entirely of DNA segments from
different species that exchange DNA by known physiological processes,
though one or more of the segments may be a synthetic equivalent. A list
of such exchangers will be prepared and periodically revised by the NIH
Director with advice of the RAC after appropriate notice and opportunity
for public comment (see Section IV-C-l-b-(l)-(c), *Major Actions).* See
Appendices A-I through A-VI, *Exemptions Under Section
III-F-5\--Sublists of Natural Exchangers,* for a list of natural
exchangers that are exempt from the *NIH Guidelines.*

**Section III-F-6.** Those that do not present a significant risk to
health or the environment (see Section IV-C-I-b-(I)-(c), *Major
Actions),* as determined by the NIH Director, with the advice of the
RAC, and following appropriate notice and opportunity for public
comment. See Appendix C, *Exemptions under Section III-F-6* for other
classes of experiments which are exempt from the *NIH Guidelines.*

**APPENDIX A. EXEMPTIONS UNDER SECTION III-E-5\--SUBLISTS OF NATURAL
EXCHANGERS**

Certain specified recombinant DNA molecules that consist entirely of DNA
segments from different species that exchange DNA by known physiological
processes, though one or more of the segments may be a synthetic
equivalent are exempt from these *NIH Guidelines* (see Section III-E-5,
*Exempt Experiments).* Institutional Biosafety Committee registration is
not required for these exempt experiments. A list of such exchangers
will be prepared and periodically revised by the NIH Director with
advice from the RAC after appropriate notice and opportunity for public
comment (see Section IV-C-I-b-(I)-(c), *NIH Director- -Specific
Responsibilities).* For a list of natural exchangers that are exempt
from the *NIH Guidelines,* see Appendices A-I through A-VI, *Exemptions
Under Section III-E-5 Sublists of Natural Exchangers.* Section Ill-E-5,
*Exempt Experiments,* describes recombinant DNA molecules that are: (I)
composed entirely of DNA segments from one or more of the organisms
within a sublist, and (2) to be propagated in any of the organisms
within a sublist (see *Classification of Bergey\'s Manual of
Determinative Bacteriology;* 8th edition, R. E. Buchanan and N. E.
Gibbons, editors, Williams and Wilkins Company; Baltimore, Maryland
1984). Although these experiments are exempt, it is recommended that
they be performed at the appropriate biosafety level for the host or
recombinant organism (see *Biosafety in Microbiological and Biomedical
Laboratories,* 3rd edition, May 1993, U.S. DHHS, Public Health Service,
Centers for Disease Control and Prevention, Atlanta, Georgia, and NIH
Office of Biosafety, Bethesda, Maryland).

## [Appendix A-I. Sublist A]{.underline} 

Genus *Escherichia*

*Genus Shigella*

Genus *Salmonella* -including *Arizona*

*Genus Enterobacter*

Genus *Citrobacter* -including *Levinea*

*Genus Klebsiella* -including *oxytoca*

*Genus Erwinia*

*Pseudomonas ael1lginosa,Pseudomonas putida, Pseudomonas fluorescens,&
Pseudomonas mendocina*

*Serratia marcescens*

*Yersinia enterocolitica*

## [Appendix A-II. Sublist B]{.underline} 

*Bacillus subtilis*

### Bacillus licheniformis 

*Bacillus pumilus*

*Bacillus globigii*

*Bacillus niger*

*Bacillus nato*

*Bacillus amyloliquefaciens*

*Bacillus aterrimus*

## [Appendix A-III. Sublist C]{.underline} 

*Streptomyces aureofaciens*

*Streptomyces rimosus*

*Streptomyces coe/icolor*

## [Appendix A-IV. Sublist D]{.underline} 

*Streptomyces griseus*

*Streptomyces cyaneus*

*Streptomyces venezuelae*

## [Appendix A-V. Sublist E]{.underline} 

One way transfer of Streptococcus mutans or Streptococcus lactis **DNA**
into Streptococcus sanguis

## [Appendix A-VI. Sublist F]{.underline} 

*Streptococcus sanguis*

*Streptococcus pneumoniae*

*Streptococcus faecalis*

*Streptococcus pyogenes*

*Streptococcus mutans*

#### APPENDIX C. EXEMPTIONS UNDER SECTION III-F-6

**Section III-F-6** states that exempt from these *NIH Guidelines* are
\"those that do not present a significant risk to health or the
environment (see Section *N* -C-I-b-( I )-( c), *NIB Director\--Specific
Responsibilities),* as determined by the NIH Director, with the advice
of the RAC, and following appropriate notice and opportunity for public
comment. See Appendix C, *Exemptions under Sections III-F-6,* for other
classes of experiments which are exempt from the *NIH Guidelines.\"* The
following classes of experiments are exempt under Section III-F-6:

## Appendix C-I. Recombinant DNA in Tissue Culture 

Recombinant DNA molecules containing less than one-half of any
eukaryotic viral genome (all viruses from a single family (see Appendix
C-VI-D, *Footnotes and References of Appendix* C) being considered
identical (see Appendix C-VI-E, *Footnotes and References of Appendix*
C), that are propagated and maintained in cells in tissue culture are
exempt from these *NIH Guidelines* with the exceptions listed in
Appendix C-I-A.

## Appendix C-I-A. Exceptions 

The following categories are not exempt from the *NIH Guidelines:* (i)
experiments described in Section III-A which require Institutional
Biosafety Committee approval, RAC review, and NIH Director approval
before initiation, (ii) experiments described in Section III-B which
require NIH/ORDA and Institutional Biosafety Committee approval before
initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or
restricted organisms (see Appendix B, *Classification of Human Etiologic
Agents on the Basis of Hazard,* and Sections V-G and V-L, *Footnotes and
References of Sections I through IV)* or cells known to be infected with
these agents, (iv) experiments involving the deliberate introduction of
genes coding for the biosynthesis of molecules that are toxic for
vertebrates (see Appendix F, *Containment Conditions for Cloning of
Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates),*
and (v) whole plants regenerated from plant cells and tissue cultures
are covered by the exemption provided they remain axenic cultures even
though they differentiate into embryonic tissue and regenerate into
plantlets.

**Appendix C-II. *Escherichia coli* K-12 Host-Vector Systems**

Experiments which use *Escherichia coli* K-12 host-vector systems, with
the exception of those experiments listed in Appendix C-II-A, are exempt
from the *NIH Guidelines* provided that: (i) the *Escherichia coli* host
does not contain conjugation proficient plasmids or generalized
transducing phages; or (ii) lambda or lambdoid or Ff bacteriophages or
non-conjugative plasmids (see Appendix C-VI-B, *Footnotes and References
of Appendix* C) shall be used as vectors. However, experiments involving
the insertion into *Escherichia coli* K-12 of DNA from prokaryotes that
exchange genetic information (see Appendix C- VI-C, *Footnotes and
References of Appendix* C) with *Escherichia coli* may be performed with
any *Escherichia coli* K-12 vector (e.g., conjugative plasmid). When a
non-conjugative vector is used, the *Escherichia coli* K-12 host may
contain conjugation- proficient plasmids either autonomous or
integrated, or generalized transducing phages. For these exempt
laboratory experiments, Biosafety Level (BL) I physical containment
conditions are recommended. For large scale fermentation experiments,
the appropriate physical containment conditions need be no greater than
those for the host organism unmodified by recombinant DNA techniques;
the Institutional Biosafety Committee can specify higher containment if
deemed necessary.

## Appendix C-II-A. Exceptions 

The following categories are not exempt from the *NIH Guidelines:* (i)
experiments described in Section III-A which require Institutional
Biosafety Committee approval, RAC review, and NIH Director approval
before initiation, (ii) experiments described in Section III-B which
require NIH/ORDA and Institutional Biosafety Committee approval before
initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or
restricted organisms (see Appendix B, *Classification of Human Etiologic
Agents* on *the Basis of Hazard,* and Sections v-a and V-L, *Footnotes
and References of Sections I through IV)* or cells known to be infected
with these agents may be conducted under containment conditions
specified in Section III-D-2 with prior Institutional Biosafety
Committee review and approval, (iv) large scale experiments (e.g., more
than 10 liters of culture), and (v) experiments involving the cloning of
toxin molecule genes coding for the biosynthesis of molecules toxic for
vertebrates (see Appendix F, *Containment Conditions for Cloning of
Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates).*

**Appendix C-III. *Saccharomyces* Host-Vector Systems**

Experiments involving *Saccharomyces cerevisiae* and *Saccharomyces
uvarum* host-vector systems, with the exception of experiments listed in
Appendix C-III-A, are exempt from the *NIH Guidelines.* For these exempt
experiments, BLI physical containment is recommended. For large scale
fermentation experiments, the appropriate physical containment
conditions need be no greater than those for the host organism
unmodified by recombinant DNA techniques; the Institutional Biosafety
Committee can specify higher containment if deemed necessary.

## Appendix C-ID-A. Exceptions 

The following categories are not exempt from the *NIH Guidelines:* (i)
experiments described in Section III-A which require Institutional
Biosafety Committee approval, RAC review, and NIH Director approval
before initiation, (ii) experiments described in Section III-B which
require NIH/ORDA and Institutional Biosafety Committee approval before
initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or
restricted organisms (see Appendix B, *Classification of Human Etiologic
Agents on the Basis of Hazard,* and Sections V -G and V -L, *Footnotes
and References of Sections I through IV)* or cells known to be infected
with these agents may be conducted under containment conditions
specified in Section m-D-2 with prior Institutional Biosafety Committee
review and approval, (iv) large scale experiments (e.g., more than 10
liters of culture), and (v) experiments involving the deliberate cloning
of genes coding for the biosynthesis of molecules toxic for vertebrates
(see Appendix F, *Containment Conditions for Cloning of Genes Coding for
the Biosynthesis of Molecules Toxic for Vertebrates).*

Appendix C-IV. *Bacillus subtilis or Bacillus licheniformis* Host-Vector
Systems

Any asporogenic *Bacillus subtilis* or asporogenic *Bacillus
licheniformis* strain which does not revert to a spore-former with a
frequency greater than 10-7 may be used for cloning DNA with the
exception of those experiments listed in Appendix C-IV-A, *Exceptions.*
For these exempt laboratory experiments, BL1 physical containment
conditions are recommended. For large scale fermentation experiments,
the appropriate physical containment conditions need be no greater than
those for the host organism unmodified by recombinant DNA techniques;
the Institutional Biosafety Committee can specify higher containment if
it deems necessary.

## Appendix C-IV-A. Exceptions 

The following categories are not exempt from the *NIH Guidelines:* (i)
experiments described in Section III-A which require Institutional
Biosafety Committee approval, RAC review, and NIH Director approval
before initiation, (ii) experiments described in Section III-B which
require NlH/ORDA and Institutional Biosafety Committee approval before
initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or
restricted organisms (see Appendix B, *Classification of Human Etiologic
Agents on the Basis of Hazard,* and Sections V-G and V-L, *Footnotes and
References of Sections I through* IV) or cells known to be infected with
these agents may be conducted under containment conditions specified in
Section III-D-2 with prior Institutional Biosafety Committee review and
approval, (iv) large scale experiments (e.g., more than 10 liters of
culture), and (v) experiments involving the deliberate cloning of genes
coding for the biosynthesis of molecules toxic for vertebrates (see
Appendix F, *Containment Conditions for Cloning of Genes Coding for the
Biosynthesis of Molecules Toxic for Vertebrates).*

**Appendix C-V. Extra chromosomal Elements of Gram Positive Organisms**

Recombinant DNA molecules derived entirely from extra chromosomal
elements of the organisms listed below (including shuttle vectors
constructed from vectors described in Appendix C), propagated and
maintained in organisms listed below are exempt from these *NIH
Guidelines.*

*Bacillus amyloliquefaciens*

*Bacillus amylosacchariticus*

*Bacillus anthracis*

*Bacillus aterrimus*

*Bacillus brevis*

*Bacillus cereus*

*Bacillus globigii*

*Bacillus licheniformis*

*Bacillus megaterium*

*Bacillus natto*

*Bacillus niger*

*Bacillus pumilus*

*Bacillus sphaericus*

*Bacillus stearothermophilis*

*Bacillus subtilis*

*Bacillus thuringiensis*

*Clostridium acetobutylicum*

*Lactobacillus casei*

*Listeria grayi*

*Listeria monocytogenes*

*Listeria murrayi*

*Pediococcus acidilactici*

*Pediococcus damnosus*

*Pediococcus pentosaceus*

*Staphylococcus aureus*

*Staphylcoccus carnosus*

*Staphylococcus epidermidis*

*Streptococcus agalactiae*

*Streptococcus anginosus*

*Streptococcus avium*

*Streptococcus cremoris*

*Streptococcus dorans*

*Streptococcus equisimilis*

*Streptococcus faecalis*

*Streptococcus ferus*

*Streptococcus lactis*

*Streptococcus mitior*

*Streptococcus mutans*

*Streptococcus pneumoniae*

*Streptococcus pyogenes*

*Streptococcus salivarious*

*Streptococcus sanguis*

*Streptococcus sobrinus*

*Streptococcus thermophylus*

**Appendix C-V-A. Exceptions**

The following categories are not exempt from the *NIH Guidelines:* (i)
experiments described in Section III-A which require Institutional
Biosafety Committee approval, RAC review, and NIH Director approval
before initiation, (ii) experiments described in Section III-B which
require NlH/ORDA and Institutional Biosafety Committee approval before
initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or
restricted organisms (see Appendix B, *Classification of Human Etiologic
Agents on the Basis of Hazard,* and Sections V-G and V-L, *Footnotes and
References of Sections I through IV)* or cells known to be infected with
these agents may be conducted under containment conditions specified in
Section III-D-2 with prior Institutional Biosafety Committee review and
approval, (iv) large scale experiments (e.g., more than 10 liters of
culture), and (v) experiments involving the deliberate cloning of genes
coding for the biosynthesis of molecules toxic for vertebrates (see
Appendix F, *Containment Conditions for Cloning of Genes Coding for the
Biosynthesis of Molecules Toxic for Vertebrates).*

**Appendix C-VI**. **The Purchase or Transfer of Transgenic** **Rodents
the purchase or transfer of transgenic rodents for experiments that
require BLI containment (See Appendix G-II-M, *Footnotes and References
of Appendix* G) are exempt from the *NIH Guidelines. ***

## Appendix C-VII. Footnotes and References of Appendix C 

**Appendix C-VII-A**. The NIH Director, with advice of the RAC, may
revise the classification for the purposes of these *NIH Guidelines*
(see Section IV -C-I-b-(2)-(b), *NIH Director\--Specific
Responsibilities).* The list of organisms in each Risk Group is located
in Appendix B.

**Appendix C-VII-A-l.** The NIH Director, with advice of the RAC, may
revise the classification for the purposes of these *NIH Guidelines*
(see Section IV -C-l-b-(2 )-(b), *NIH Director\--Specific
Responsibilities).* The revised list of organisms in each class is
reprinted in Appendix B.

**Appendix C- VII-B.** A subset of non-conjugative plasmid vectors are
poorly mobilizable (e.g., pBR322, pBR313). Where practical, these
vectors should be employed.

**Appendix C-VII-C.** Defined as observable under optimal laboratory
conditions by transformation, transduction, phage infection, and/or
conjugation with transfer of phage, plasmid, and/or chromosomal genetic
information. Note that this definition of exchange may be less stringent
than that applied to exempt organisms under Section III-F-5, *Exempt
Experiments.*

**Appendix C-VII-D.** As classified in the *Third Report of the
International Committee on Taxonomy of Viruses: Classification and
Nomenclature of Viruses,* R. E. F. Matthews (ed.), Intervirology 12
(129-296),1979.

**Appendix C-VII-E**. i.e., the total of all genomes within a Family
shall not exceed one-half of the genome.