NIH GUIDELINES FOR RESEARCHINVOLVING
RECOMBINANT OR SYNTHETIC
NUCLEIC ACID MOLECULES(NIH GUIDELINES)
April 2024

DEPARTMENT OF HEALTH AND HUMAN SERVICES
National Institutes of Health************************************************************************************************************************
Visit the NIH OSP Web site at:https://osp.od.nih.gov

NIH OFFICE OF SCIENCE POLICY CONTACT INFORMATION:
Office of Science Policy, National Institutes of Health, 6705 Rockledge Drive, Suite 630, MSC 7985,
Bethesda, MD 20892-7985 (20817 for non-USPS mail), (301) 496-9838; (301) 496-9839 (fax).
For inquiries, information requests, and report submissions:

NIHGuidelines@od.nih.gov

These NIH Guidelines shall supersede all earlier versions until further notice.
************************************************************************************************************************

Page 2 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

FEDERAL REGISTER NOTICES
Effective June 24, 1994, Published in Federal Register, July 5, 1994 (59 FR 34472)
Amendment Effective July 28, 1994, Federal Register, August 5, 1994 (59 FR 40170)
Amendment Effective April 17, 1995, Federal Register, April 27, 1995 (60 FR 20726)
Amendment Effective December 14, 1995, Federal Register, January 19, 1996 (61 FR 1482)
Amendment Effective March 1, 1996, Federal Register, March 12, 1996 (61 FR 10004)
Amendment Effective January 23, 1997, Federal Register, January 31, 1997 (62 FR 4782)
Amendment Effective September 30, 1997, Federal Register, October 14, 1997 (62 FR 53335)
Amendment Effective October 20, 1997, Federal Register, October 29, 1997 (62 FR 56196)
Amendment Effective October 22, 1997, Federal Register, October 31, 1997 (62 FR 59032)
Amendment Effective February 4, 1998, Federal Register, February 17, 1998 (63 FR 8052)
Amendment Effective April 30, 1998, Federal Register, May 11, 1998 (63 FR 26018)
Amendment Effective April 29, 1999, Federal Register, May 11, 1999 (64 FR 25361)
Amendment Effective October 2, 2000, Federal Register, October 10, 2000 (65 FR 60328)
Amendment Effective December 28, 2000, Federal Register, January 5, 2001 (66 FR 1146)
Amendment Effective December 11, 2001, Federal Register, December 11, 2001 (66 FR 64051)
Amendment Effective December 19, 2001, Federal Register, November 19, 2001 (66 FR 57970)
Amendment Effective January 10, 2002, Federal Register, December 11, 2001 (66 FR 64052)
Amendment Effective January 24, 2002, Federal Register, November 19, 2001 (66 FR 57970)
Amendment Effective September 22, 2009, Federal Register, September 22, 2009 (74 FR 48275)
Amendment Effective January 19, 2011, Federal Register, January 19, 2011 (76 FR 3150)
Amendment Effective May 12, 2011, Federal Register, May 12, 2011 (76 FR 27653)
Amendment Effective October 11, 2011, Federal Register, October 11, 2011 (76 FR 62816)
Amendment Effective February 21, 2013, Federal Register, February 21, 2013 (78 FR 12074)
Amendment Effective March 5, 2013, Federal Register, September 5, 2012 (77 FR 54584)
Amendment Effective November 6, 2013, Federal Register, November 6, 2013 (78 FR 66751)
Amendment Effective April 27, 2016, Federal Register, March 22, 2016 (81 FR 15315)
Amendment Effective April 15, 2016, Federal Register, April 15, 2016 (81 FR 22286)
Amendment Effective April 25, 2019, Federal Register, April 26, 2019 (84 FR 17858)
Amendment Effective September 30, 2024, Federal Register, April 5, 2024 (89 FR 24016)

Page 3 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

TABLE OF CONTENTSNIH Office of Science Policy Contact Information: ................................................................................. 1
SECTION I.
SCOPE OF THE NIH GUIDELINES ............................................................... 10
Section I-A.
Purpose........................................................................................................... 10
Section I-B.
Definition of Recombinant and Synthetic Nucleic Acid Molecules ................. 10
Section I-C.
General Applicability ....................................................................................... 10
Section I-D.
Compliance with the NIH Guidelines .............................................................. 11
Section I-E.
General Definitions ......................................................................................... 11
SECTION II.
Section II-A.
Section II-A-1.
Section II-A-2.
Section II-A-3.
Section II-B.

SAFETY CONSIDERATIONS ........................................................................ 12
Risk Assessment ............................................................................................ 12
Risk Groups .................................................................................................... 12
Criteria for Risk Groups .................................................................................. 12
Comprehensive Risk Assessment .................................................................. 12
Containment.................................................................................................... 14

SECTION III.
Section III-A.

EXPERIMENTS COVERED BY THE NIH GUIDELINES .............................. 16
Experiments that Require NIH Director Approval and Institutional Biosafety
Committee Approval Before Initiation (See Section IV-C-1-b-(1), Major
Actions). .......................................................................................................... 16
Major Actions under the NIH Guidelines ........................................................ 16
Experiments That Require NIH OSP and Institutional Biosafety Committee
Approval Before Initiation................................................................................ 17
Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than
100 Nanograms per Kilogram Body Weight ................................................... 17
Experiments that have been Approved (under Section III-A-1-a) as Major
Actions under the NIH Guidelines................................................................... 17
Experiments Involving Human Gene Transfer that Require Institutional
Biosafety Committee Approval Prior to Initiation ............................................ 17
Experiments Involving the Deliberate Transfer of Recombinant or Synthetic
Nucleic Acid Molecules, or DNA or RNA Derived from Recombinant orSynthetic Nucleic Acid Molecules, into One or More Human ResearchParticipants ..................................................................................................... 17
Experiments that Require Institutional Biosafety Committee Approval Before
Initiation........................................................................................................... 18
Experiments Using Risk Group 2, Risk Group 3, Risk Group 4, or Restricted
Agents as Host-Vector Systems (See Section II-A, Risk Assessment).......... 18
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 .................................................................... 18
Experiments Involving the Use of Infectious DNA or RNA Viruses or Defective
DNA or RNA Viruses in the Presence of a Helper System in Tissue Culture
Systems .......................................................................................................... 19
Experiments Involving Whole Animals ........................................................... 19
Experiments Involving Whole Plants .............................................................. 20
Experiments Involving More than 10 Liters of Culture .................................... 21
Experiments Involving Influenza Viruses ........................................................ 21
Experiments Involving Gene Drive Modified Organisms ................................ 21
Experiments that Require Institutional Biosafety Committee Notice
Simultaneous with Initiation ............................................................................ 22
Experiments Involving the Formation of Recombinant or Synthetic Nucleic
Acid Molecules Containing No More than Two-Thirds of the Genome of any
Eukaryotic Virus .............................................................................................. 23
Experiments Involving Whole Plants .............................................................. 23
Experiments Involving Transgenic Rodents ................................................... 24

Section III-A-1.
Section III-B.
Section III-B-1.
Section III-B-2.
Section III-C.
Section III-C-1.

Section III-D.
Section III-D-1.
Section III-D-2.
Section III-D-3.
Section III-D-4.
Section III-D-5.
Section III-D-6.
Section III-D-7.
Section III-D-8.
Section III-E.
Section III-E-1.
Section III-E-2.
Section III-E-3.

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Section III-F.
SECTION IV.
Section IV-A.
Section IV-B.
Section IV-B-1.
Section IV-B-2.
Section IV-B-2-a.
Section IV-B-2-b.
Section IV-B-3.
Section IV-B-4.
Section IV-B-5.
Section IV-B-6.
Section IV-B-7.
Section IV-B-7-a.
Section IV-B-7-b.
Section IV-B-7-c.
Section IV-B-7-d.
Section IV-B-7-e.
Section IV-C.
Section IV-C-1.
Section IV-C-1-a.
Section IV-C-1-b.
Section IV-C-1-b-(1).
Section IV-C-1-b-(2).
Section IV-C-2.
Section IV-C-3.
Section IV-D.
Section IV-D-1.
Section IV-D-2.
Section IV-D-3.
Section IV-D-4.
Section IV-D-5.
Section IV-D-5-a.
Section IV-D-5-b.

(April 2024)

Exempt Experiments ....................................................................................... 24
ROLES AND RESPONSIBILITIES ................................................................ 25
Policy .............................................................................................................. 25
Responsibilities of the Institution .................................................................... 25
General Information ........................................................................................ 25
Institutional Biosafety Committee (IBC) .......................................................... 27
Membership and Procedures.......................................................................... 27
Functions ........................................................................................................ 28
Biological Safety Officer (BSO)....................................................................... 29
Plant, Plant Pathogen, or Plant Pest Containment Expert ............................. 30
Animal Containment Expert ............................................................................ 30
Human Gene Therapy Expertise .................................................................... 30
Principal Investigator (PI)................................................................................ 30
General Responsibilities ................................................................................. 30
Information to Be Submitted by the Principal Investigator to NIH OSP .......... 31
Submissions by the Principal Investigator to the Institutional Biosafety
Committee....................................................................................................... 31
Responsibilities of the Principal Investigator Prior to Initiating Research....... 32
Responsibilities of the Principal Investigator During the Conduct of the
Research......................................................................................................... 32
Responsibilities of the National Institutes of Health (NIH) .............................. 32
NIH Director .................................................................................................... 32
General Responsibilities ................................................................................. 32
Specific Responsibilities ................................................................................. 33
Major Actions .................................................................................................. 33
Minor Actions .................................................................................................. 33
Office of Science Policy (OSP) ....................................................................... 34
Other NIH Components .................................................................................. 34
Voluntary Compliance..................................................................................... 34
Basic Policy - Voluntary Compliance .............................................................. 34
Institutional Biosafety Committee Approval - Voluntary Compliance ............. 35
Certification of Host-Vector Systems - Voluntary Compliance ....................... 35
Requests for Exemptions and Approvals - Voluntary Compliance ................. 35
Protection of Proprietary Data - Voluntary Compliance .................................. 35
General ........................................................................................................... 35
Pre-submission Review .................................................................................. 36

SECTION V.

FOOTNOTES AND REFERENCES OF SECTIONS I THROUGH IV............ 36

APPENDIX A.

EXEMPTIONS UNDER SECTION III-F-6--SUBLISTS OF NATURALEXCHANGERS .............................................................................................. 37
Sublist A .......................................................................................................... 38
Sublist B .......................................................................................................... 38
Sublist C.......................................................................................................... 38
Sublist D.......................................................................................................... 38
Sublist E .......................................................................................................... 39
Sublist F .......................................................................................................... 39

Appendix A-I.
Appendix A-II.
Appendix A-III.
Appendix A-IV.
Appendix A-V.
Appendix A-VI.
APPENDIX B.
Appendix B-I.
Appendix B-II.
Appendix B-II-A.
Appendix B-II-B.
Appendix B-II-C.
Appendix B-II-D.

CLASSIFICATION OF HUMAN ETIOLOGIC AGENTS ON THE BASIS OF
HAZARD ......................................................................................................... 39
Risk Group 1 (RG1) Agents ............................................................................ 39
Risk Group 2 (RG2) Agents ............................................................................ 40
Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia ......................... 40
Risk Group 2 (RG2) - Fungal Agents ............................................................. 41
Risk Group 2 (RG2) - Parasitic Agents ........................................................... 41
Risk Group 2 (RG2) - Viruses ......................................................................... 42

Page 5 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Appendix B-III.
Appendix B-III-A.
Appendix B-III-B.
Appendix B-III-C.
Appendix B-III-D.
Appendix B-IV.
Appendix B-IV-A.
Appendix B-IV-B.
Appendix B-IV-C.
Appendix B-IV-D.
Appendix B-V.
Appendix B-V-1.

Risk Group 3 (RG3) Agents ............................................................................ 43
Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia .......................... 43
Risk Group 3 (RG3) - Fungal Agents ............................................................. 44
Risk Group 3 (RG3) - Parasitic Agents ........................................................... 44
Risk Group 3 (RG3) - Viruses and Prions ...................................................... 44
Risk Group 4 (RG4) Agents ............................................................................ 45
Risk Group 4 (RG4) - Bacterial Agents .......................................................... 45
Risk Group 4 (RG4) - Fungal Agents ............................................................. 45
Risk Group 4 (RG4) - Parasitic Agents ........................................................... 45
Risk Group 4 (RG4) - Viral Agents ................................................................. 45
Animal Viral Etiologic Agents in Common Use ............................................... 46
Murine Retroviral Vectors ............................................................................... 46

APPENDIX C.
Appendix C-I.
Appendix C-I-A.
Appendix C-II.
Appendix C-II-A.
Appendix C-III.
Appendix C-III-A.
Appendix C-IV.
Appendix C-IV-A
Appendix C-V.
Appendix C-V-A.
Appendix C-VI.
Appendix C-VI-A.
Appendix C-VII.
Appendix C-VIII.
Appendix C-IX.

EXEMPTIONS UNDER SECTION III-F-8 ....................................................... 46
Recombinant or Synthetic Nucleic Acid Molecules in Tissue Culture ............ 47
Exceptions ...................................................................................................... 47
Escherichia coli K-12 Host-Vector Systems ................................................... 47
Exceptions ...................................................................................................... 47
Saccharomyces Host-Vector Systems ........................................................... 48
Exceptions ...................................................................................................... 48
Kluyveromyces Host-Vector Systems ............................................................ 48
Exceptions ...................................................................................................... 48
Bacillus subtilis or Bacillus licheniformis Host-Vector Systems...................... 48
Exceptions ...................................................................................................... 48
Extrachromosomal Elements of Gram Positive Organisms ........................... 49
Exceptions ...................................................................................................... 50
The Purchase or Transfer of Transgenic Rodents ......................................... 50
Generation of BL1 Transgenic Rodents via Breeding .................................... 50
Footnotes and References of Appendix C...................................................... 50

APPENDIX D.

MAJOR ACTIONS TAKEN UNDER THE NIH GUIDELINES ........................ 51

APPENDIX E.

CERTIFIED HOST-VECTOR SYSTEMS (See Appendix I, BiologicalContainment) ................................................................................................. 72
Bacillus subtilis................................................................................................ 72
Bacillus subtilis Host-Vector 1 Systems ......................................................... 72
Bacillus subtilis Host-Vector 2 Systems ......................................................... 72
Saccharomyces cerevisiae ............................................................................. 72
Saccharomyces cerevisiae Host-Vector 2 Systems ....................................... 72
Escherichia coli ............................................................................................... 72
Escherichia coli (EK2) Plasmid Systems ........................................................ 73
Escherichia coli (EK2) Bacteriophage Systems ............................................. 73
Neurospora crassa ......................................................................................... 73
Neurospora crassa Host-Vector 1 Systems ................................................... 73
Streptomyces .................................................................................................. 73
Streptomyces Host-Vector 1 Systems ............................................................ 73
Pseudomonas putida ...................................................................................... 73
Pseudomonas putida Host-Vector 1 Systems ................................................ 73

Appendix E-I.
Appendix E-I-A.
Appendix E-I-B.
Appendix E-II.
Appendix E-II-A.
Appendix E-III.
Appendix E-III-A.
Appendix E-III-B.
Appendix E-IV.
Appendix E-IV-A.
Appendix E-V.
Appendix E-V-A.
Appendix E-VI.
Appendix E-VI-A.
APPENDIX F.
Appendix F-I.
Appendix F-II.
Appendix F-III.
Appendix F-IV.

CONTAINMENT CONDITIONS FOR CLONING OF GENES CODING FOR
THE BIOSYNTHESIS OF MOLECULES TOXIC FOR VERTEBRATES ...... 74
General Information ........................................................................................ 74
Cloning of Toxin Molecule Genes in Escherichia coli K-12 ............................ 74
Cloning of Toxic Molecule Genes in Organisms Other Than Escherichia coli
K-12 ................................................................................................................ 74
Specific Approvals .......................................................................................... 74

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APPENDIX G.
Appendix G-I.
Appendix G-II.
Appendix G-II-A.
Appendix G-II-A-1.
Appendix G-II-A-2.
Appendix G-II-A-3.
Appendix G-II-A-4.
Appendix G-II-B.
Appendix G-II-B-1.
Appendix G-II-B-2.
Appendix G-II-B-3.
Appendix G-II-B-4.
Appendix G-II-C.
Appendix G-II-C-1.
Appendix G-II-C-2.
Appendix G-II-C-2-t.
Appendix G-II-C-3.
Appendix G-II-C-4.
Appendix G-II-C-5.

Appendix G-II-C-5-a.
Appendix G-II-C-5-b.
Appendix G-II-C-5-c.
Appendix G-II-D.
Appendix G-II-D-1.
Appendix G-II-D-2.
Appendix G-II-D-2-m.
Appendix G-II-D-3.
Appendix G-II-D-4.
Appendix G-III.

(April 2024)

PHYSICAL CONTAINMENT .......................................................................... 75
Standard Practices and Training .................................................................... 75
Physical Containment Levels.......................................................................... 75
Biosafety Level 1 (BL1) (See Appendix G-III-M, Footnotes and References ofAppendix G) .................................................................................................... 76Standard Microbiological Practices (BL1)....................................................... 76
Special Practices (BL1) .................................................................................. 76
Containment Equipment (BL1) ....................................................................... 76
Laboratory Facilities (BL1) .............................................................................. 77
Biosafety Level 2 (BL2) (See Appendix G-III-N, Footnotes and References ofAppendix G) .................................................................................................... 77
Standard Microbiological Practices (BL2)....................................................... 77
Special Practices (BL2) .................................................................................. 77
Containment Equipment (BL2) ....................................................................... 78
Laboratory Facilities (BL2) .............................................................................. 79
Biosafety Level 3 (BL3) (See Appendix G-III-P, Footnotes and References ofAppendix G) .................................................................................................... 79
Standard Microbiological Practices (BL3)....................................................... 79
Special Practices (BL3) .................................................................................. 79
Alternative Selection of Containment Equipment (BL3) ................................. 81
Containment Equipment (BL3) ....................................................................... 82
Laboratory Facilities (BL3) .............................................................................. 82
Biosafety Level 3 Enhanced for Research Involving Risk Group 3 Influenza
Viruses. (See Appendices G-II-C-2-n, G-II-C-2-r, and G-II-C-4-i for
additional guidance for facilities, waste handling, and serum collection forresearch involving mammaliantransmissible HPAI H5N1 virus................... 83
Containment, Practices, and Training for Research with Risk Group 3
Influenza Viruses (BL3 Enhanced). ................................................................ 83
Containment for Animal Research. ................................................................. 84
Occupational Health ....................................................................................... 84
Biosafety Level 4 (BL4)................................................................................... 86Standard Microbiological Practices (BL4)....................................................... 86
Special Practices (BL4) .................................................................................. 86
Alternative Selection of Containment Equipment (BL4) ................................. 88
Containment Equipment (BL4) ....................................................................... 88
Laboratory Facilities (BL4) .............................................................................. 88
Footnotes and References of Appendix G ..................................................... 90

APPENDIX H.
Appendix H-III.

SHIPMENT ..................................................................................................... 92
Footnotes and References of Appendix H...................................................... 93

APPENDIX I.

BIOLOGICAL CONTAINMENT (See Appendix E, Certified Host-Vector
Systems) ........................................................................................................ 93
Levels of Biological Containment.................................................................... 93
Host-Vector 1 Systems ................................................................................... 93
Escherichia coli K-12 Host-Vector 1 Systems (EK1) ...................................... 93
Other Host-Vector 1 Systems ......................................................................... 93
Host-Vector 2 Systems (EK2) ......................................................................... 94
Certification of Host-Vector Systems .............................................................. 94
Responsibility .................................................................................................. 94
Data to be Submitted for Certification ............................................................. 94
Host-Vector 1 Systems Other than Escherichia coli K-12 .............................. 94
Host-Vector 2 Systems ................................................................................... 95
Footnotes and References of Appendix I ....................................................... 95

Appendix I-I.
Appendix I-I-A.
Appendix I-I-A-1.
Appendix I-I-A-2.
Appendix I-I-B.
Appendix I-II.
Appendix I-II-A.
Appendix I-II-B.
Appendix I-II-B-1.
Appendix I-II-B-2.
Appendix I-III.
APPENDIX J.

BIOTECHNOLOGY RESEARCH SUBCOMMITTEE .................................... 96

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APPENDIX K.
Appendix K-I.
Appendix K-II.
Appendix K-III.
Appendix K-IV.
Appendix K-V.
Appendix K-VI.
Appendix K-VII.
APPENDIX L.
Appendix L-I.
Appendix L-II.
Appendix L-II-A.
Appendix L-II-A-1.
Appendix L-II-A-1-a.
Appendix L-II-A-1-b.
Appendix L-II-A-1-c.
Appendix L-II-A-1-d.
Appendix L-II-A-1-e.
Appendix L-II-A-2.
Appendix L-II-A-2-a.
Appendix L-II-A-2-b.
Appendix L-II-B.
Appendix L-II-B-1.
Appendix L-II-B-1-a.
Appendix L-II-B-1-b.
Appendix L-II-B-1-c.
Appendix L-II-B-1-d.
Appendix L-II-B-1-e.
Appendix L-II-B-1-f.
Appendix L-II-B-1-g.
Appendix L-II-B-1-h.
Appendix L-II-B-2.
Appendix L-II-B-2-a.
Appendix L-II-B-2-b.
Appendix L-II-B-2-c.
Appendix L-II-B-2-d.
Appendix L-II-B-2-e.
Appendix L-II-C.
Appendix L-II-C-1.
Appendix L-II-C-1-a.
Appendix L-II-C-1-b.
Appendix L-II-C-1-c.
Appendix L-II-C-1-d.
Appendix L-II-C-1-e.
Appendix L-II-C-1-f.
Appendix L-II-C-1-g.
Appendix L-II-C-1-h.
Appendix L-II-C-1-i.
Appendix L-II-C-1-j.
Appendix L-II-C-2.
Appendix L-II-C-2-a.

(April 2024)

PHYSICAL CONTAINMENT FOR LARGE SCALE USES OF ORGANISMSCONTAINING RECOMBINANT OR SYNTHETIC NUCLEIC ACID
MOLECULES ................................................................................................. 97
Selection of Physical Containment Levels...................................................... 97
Good Large Scale Practice (GLSP) ................................................................ 98
Biosafety Level 1 (BL1) - Large Scale ............................................................ 98
Biosafety Level 2 (BL2) - Large Scale ............................................................ 99
Biosafety Level 3 (BL3) - Large Scale .......................................................... 101
Footnotes of Appendix K .............................................................................. 105
Definitions to Accompany Containment Grid and Appendix K ..................... 105
PHYSICAL AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT ORSYNTHETIC NUCLEIC ACID MOLECULE RESEARCH INVOLVING
PLANTS........................................................................................................ 106
General Plant Biosafety Levels..................................................................... 107
Physical Containment Levels........................................................................ 107
Biosafety Level 1 - Plants (BL1-P) ................................................................ 107
Standard Practices (BL1-P) .......................................................................... 107
Greenhouse Access (BL1-P) ........................................................................ 107
Records (BL1-P) ........................................................................................... 107
Decontamination and Inactivation (BL1-P) ................................................... 108
Control of Undesired Species and Motile Macroorganisms (BL1-P) ............ 108
Concurrent Experiments Conducted in the Greenhouse (BL1-P) ................ 108
Facilities (BL1-P) .......................................................................................... 108
Definitions (BL1-P) ........................................................................................ 108
Greenhouse Design (BL1-P) ........................................................................ 108
Biosafety Level 2 - Plants (BL2-P) ................................................................ 108
Standard Practices (BL2-P) .......................................................................... 108
Greenhouse Access (BL2-P) ........................................................................ 108
Records (BL2-P) ........................................................................................... 109
Decontamination and Inactivation (BL2-P) ................................................... 109
Control of Undesired Species and Motile Macroorganisms (BL2-P) ............ 109
Concurrent Experiments Conducted in the Greenhouse (BL2-P) ................ 109
Signs (BL2-P)................................................................................................ 109
Transfer of Materials (BL2-P) ....................................................................... 110
Greenhouse Practices Manual (BL2-P) ........................................................ 110
Facilities (BL2-P) .......................................................................................... 110
Definitions (BL2-P) ........................................................................................ 110
Greenhouse Design (BL2-P) ........................................................................ 110
Autoclaves (BL2-P) ....................................................................................... 110
Supply and Exhaust Air Ventilation Systems (BL2-P) .................................. 110
Other (BL2-P)................................................................................................ 110
Biosafety Level 3 - Plants (BL3-P) ................................................................ 111
Standard Practices (BL3-P) .......................................................................... 111
Greenhouse Access (BL3-P) ........................................................................ 111
Records (BL3-P) ........................................................................................... 111
Decontamination and Inactivation (BL3-P) ................................................... 111
Control of Undesired Species and Motile Macroorganisms (BL3-P) ............ 111
Concurrent Experiments Conducted in the Greenhouse (BL3-P) ................ 111
Signs (BL3-P)................................................................................................ 111
Transfer of Materials (BL3-P) ....................................................................... 112
Greenhouse Practices Manual (BL3-P) ........................................................ 112
Protective Clothing (BL3-P) .......................................................................... 112
Other (BL3-P)................................................................................................ 112
Facilities (BL3-P) .......................................................................................... 112
Definitions (BL3-P) ........................................................................................ 112

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Appendix L-II-C-2-b.
Appendix L-II-C-2-c.
Appendix L-II-C-2-d.
Appendix L-II-C-2-e.
Appendix L-II-D.
Appendix L-II-D-1.
Appendix L-II-D-1-a.
Appendix L-II-D-1-b.
Appendix L-II-D-1-c.
Appendix L-II-D-1-d.
Appendix L-II-D-1-e.
Appendix L-II-D-1-f.
Appendix L-II-D-1-g.
Appendix L-II-D-1-h.
Appendix L-II-D-1-i.
Appendix L-II-D-2.
Appendix L-II-D-2-a.
Appendix L-II-D-2-b.
Appendix L-II-D-2-c.
Appendix L-II-D-2-d.
Appendix L-III.
Appendix L-III-A.
Appendix L-III-B.
Appendix L-III-C.
APPENDIX M.
Appendix M-I.
Appendix M-I-A.
Appendix M-I-B.
Appendix M-II.
Appendix M-II-A.
Appendix M-II-A-1.
Appendix M-II-A-1-a.
Appendix M-II-A-1-b.
Appendix M-II-A-2.
Appendix M-II-B.
Appendix M-II-B-1.
Appendix M-II-B-1-a.
Appendix M-II-B-1-b.
Appendix M-II-B-1-c.
Appendix M-II-B-1-d.
Appendix M-II-B-1-e.
Appendix M-II-B-1-f.
Appendix M-II-B-1-g.
Appendix M-II-B-2.
Appendix M-II-C.
Appendix M-II-C-1.
Appendix M-II-C-1-a.
Appendix M-II-C-1-b.
Appendix M-II-C-1-c.
Appendix M-II-C-1-d.
Appendix M-II-C-1-e.

(April 2024)

Greenhouse Design (BL3-P) ........................................................................ 113
Autoclaves (BL3-P) ....................................................................................... 113
Supply and Exhaust Air Ventilation Systems (BL3-P) .................................. 113
Other (BL3-P)................................................................................................ 113
Biosafety Level 4 - Plants (BL4-P) ................................................................ 114
Standard Practices (BL4-P) .......................................................................... 114
Greenhouse Access (BL4-P) ........................................................................ 114
Records (BL4-P) ........................................................................................... 114
Decontamination and Inactivation (BL4-P) ................................................... 114
Control of Undesired Species and Motile Macroorganisms (BL4-P) ............ 115
Concurrent Experiments Conducted in the Greenhouse (BL4-P) ................ 115
Signs (BL4-P)................................................................................................ 115
Transfer of Materials (BL4-P) ....................................................................... 115
Greenhouse Practices Manual (BL4-P) ........................................................ 116
Protective Clothing (BL4-P) .......................................................................... 116
Facilities (BL4-P) .......................................................................................... 116
Greenhouse Design (BL4-P) ........................................................................ 116
Autoclaves (BL4-P) ....................................................................................... 116
Supply and Exhaust Air Ventilation Systems (BL4-P) .................................. 117
Other (BL4-P)................................................................................................ 117
Biological Containment Practices ................................................................. 117
Biological Containment Practices (Plants).................................................... 117
Biological Containment Practices (Microorganisms) .................................... 118
Biological Containment Practices (Macroorganisms) ................................... 118
PHYSICAL AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT ORSYNTHETIC NUCLEIC ACID MOLECULE RESEARCH INVOLVINGANIMALS...................................................................................................... 118
General Considerations ................................................................................ 119
Containment Levels ...................................................................................... 119
Disposal of Animals (BL1-N through BL4-N) ................................................ 119
Physical and Biological Containment Levels ................................................ 119
Biosafety Level 1 - Animals (BL1-N) ............................................................. 119
Standard Practices (BL1-N) .......................................................................... 119
Animal Facility Access (BL1-N) .................................................................... 119
Other (BL1-N) ............................................................................................... 119
Animal Facilities (BL1-N) .............................................................................. 119
Biosafety Level 2 - Animals (BL2-N) (See Appendix M-III-A, Footnotes andReferences for Appendix M) ......................................................................... 120
Standard Practices (BL2-N) .......................................................................... 120
Animal Facility Access (BL2-N) .................................................................... 120
Decontamination and Inactivation (BL2-N) ................................................... 120
Signs (BL2-N) ............................................................................................... 120
Protective Clothing (BL2-N) .......................................................................... 120
Records (BL2-N) ........................................................................................... 120
Transfer of Materials (BL2-N) ....................................................................... 121
Other (BL2-N) ............................................................................................... 121
Animal Facilities (BL2-N) .............................................................................. 122
Biosafety Level 3 - Animals (BL3-N) (See Appendix M-III-B, Footnotes andReferences for Appendix M) ......................................................................... 122
Standard Practices (BL3-N) .......................................................................... 122
Animal Facility Access (BL3-N) .................................................................... 122
Decontamination and Inactivation (BL3-N) ................................................... 122
Signs (BL3-N) ............................................................................................... 123
Protective Clothing (BL3-N) .......................................................................... 123
Records (BL3-N) ........................................................................................... 123

Page 9 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules

Appendix M-II-C-1-f.
Appendix M-II-C-1-g.
Appendix M-II-C-2.
Appendix M-II-D.
Appendix M-II-D-1.
Appendix M-II-D-1-a.
Appendix M-II-D-1-b.
Appendix M-II-D-1-c.
Appendix M-II-D-1-d.
Appendix M-II-D-1-e.
Appendix M-II-D-1-f.
Appendix M-II-D-1-g.
Appendix M-II-D-2.
Appendix M-III.

(April 2024)

Transfer of Materials (BL3-N) ....................................................................... 123
Other (BL3-N) ............................................................................................... 124
Animal Facilities (BL3-N) .............................................................................. 125
Biosafety Level 4 - Animals (BL4-N) (See Appendix M-III-C, Footnotes andReferences for Appendix M) ......................................................................... 126
Standard Practices (BL4-N) .......................................................................... 126
Animal Facility Access (BL4-N) .................................................................... 126
Decontamination and Inactivation (BL4-N) ................................................... 126
Signs (BL4-N) ............................................................................................... 127
Protective Clothing (BL4-N) .......................................................................... 127
Records (BL4-N) ........................................................................................... 128
Transfer of Materials (BL4-N) ....................................................................... 128
Other (BL4-N) ............................................................................................... 129
Animal Facilities (BL4-N) .............................................................................. 130
Footnotes and References for Appendix M .................................................. 131************************************************************************************************************************
LIST OF TABLES
Appendix B - Table 1.

Basis for the Classification of Biohazardous Agents by Risk Group .............. 39

Appendix G - Table 1.

Possible Alternate Combinations Of Physical And Biological Containment
Safeguards ...................................................................................................... 98

Appendix K - Table 1.

Comparison of Good Large Scale Practice (GLSP) and Biosafety Level (BL) Large Scale (LS) Practice ............................................................................. 104************************************************************************************************************************

Page 10 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules

SECTION I.

SCOPE OF THE NIH GUIDELINES

Section I-A.

Purpose(April 2024)

The purpose of the NIH Guidelines is to specify the biosafety practices and containment principles forconstructing and handling: (i) recombinant nucleic acid molecules, (ii) synthetic nucleic acid molecules,
including those that are chemically or otherwise modified but can base pair with naturally occurring
nucleic acid molecules, and (iii) cells, organisms, and viruses containing such molecules.
Section I-A-1. Any nucleic acid molecule experiment, which according to the NIH Guidelines requires
approval by NIH, must be submitted to NIH or to another Federal agency that has jurisdiction for review
and approval. Once approvals, or other applicable clearances, have been obtained from a Federal
agency other than NIH (whether the experiment is referred to that agency by NIH or sent directly there by
the submitter), the experiment may proceed without the necessity for NIH review or approval.
Section I-A-1-a. For experiments involving the deliberate transfer of recombinant or synthetic nucleic
acid molecules, or DNA or RNA derived from recombinant or synthetic nucleic acid molecules, into humanresearch participants (human gene transfer), no human gene transfer experiment shall be initiated (seedefinition of initiation in Section I-E-4) until Institutional Biosafety Committee (IBC) approval (from theclinical trial site) has been obtained and all applicable regulatory authorization(s) and approvals havebeen obtained.
Section I-B.

Definition of Recombinant and Synthetic Nucleic Acid Molecules

In the context of the NIH Guidelines, recombinant and synthetic nucleic acids are defined as:
(i)
(ii)
(iii)

molecules that a) are constructed by joining nucleic acid molecules and b) that can replicate
in a living cell, i.e., recombinant nucleic acids;
nucleic acid molecules that are chemically or by other means synthesized or amplified,including those that are chemically or otherwise modified but can base pair with naturally
occurring nucleic acid molecules, i.e., synthetic nucleic acids, ormolecules that result from the replication of those described in (i) or (ii) above.

Section I-C.

General Applicability

Section I-C-1. The NIH Guidelines are applicable to:
Section I-C-1-a. All recombinant or synthetic nucleic acid research within the United States (U.S.) or its
territories that is within the category of research described in either Section I-C-1-a-(1) or Section I-C-1-a(2).
Section I-C-1-a-(1). Research that is conducted at or sponsored by an institution that receives any
support for recombinant or synthetic nucleic acid research from NIH, including research performed
directly by NIH. An individual who receives support for research involving recombinant or synthetic
nucleic acids must be associated with or sponsored by an institution that assumes the responsibilities
assigned in the NIH Guidelines.
Section I-C-1-a-(2). Research that involves testing in humans of materials containing recombinant orsynthetic nucleic acids developed with NIH funds, if the institution that developed those materials
sponsors or participates in those projects. Participation includes research collaboration or contractual
agreements, not mere provision of research materials.
Section I-C-1-b. All recombinant or synthetic nucleic acid research performed abroad that is within the
category of research described in either Section I-C-1-b-(1) or Section I-C-1-b-(2).
Section I-C-1-b-(1). Research supported by NIH funds.

Page 11 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Section I-C-1-b-(2). Research that involves testing in humans of materials containing recombinant orsynthetic nucleic acids developed with NIH funds, if the institution that developed those materials
sponsors or participates in those projects. Participation includes research collaboration or contractual
agreements, not mere provision of research materials.
Section I-C-1-b-(3). If the host country has established rules for the conduct of recombinant or synthetic
nucleic acid molecule research, then the research must be in compliance with those rules. If the hostcountry does not have such rules, the proposed research must be reviewed and approved by an NIHapproved Institutional Biosafety Committee or equivalent review body and accepted in writing by anappropriate national governmental authority of the host country. The safety practices that are employedabroad must be reasonably consistent with the NIH Guidelines.
Section I-D.

Compliance with the NIH Guidelines

As a condition for NIH funding of recombinant or synthetic nucleic acid molecule research, institutions
shall ensure that such research conducted at or sponsored by the institution, irrespective of the source offunding, shall comply with the NIH Guidelines.
Information concerning noncompliance with the NIH Guidelines may be brought forward by any person. Itshould be delivered to both NIH OSP and the relevant institution. The institution, generally through theInstitutional Biosafety Committee, shall take appropriate action. The institution shall forward a complete
report of the incident recommending any further action to the Office of Science Policy, National Institutesof Health, preferably by e-mail to: NIHGuidelines@od.nih.gov; additional contact information is alsoavailable here and on the OSP website (www.osp.od.nih.gov).
In cases where NIH proposes to suspend, limit, or terminate financial assistance because ofnoncompliance with the NIH Guidelines, applicable DHHS and Public Health Service procedures shallgovern.
The policies on compliance are as follows:
Section I-D-1. All NIH-funded projects involving recombinant or synthetic nucleic acid molecules mustcomply with the NIH Guidelines. Non-compliance may result in: (i) suspension, limitation, or terminationof financial assistance for the noncompliant NIH-funded research project and of NIH funds for otherrecombinant or synthetic nucleic acid molecule research at the institution, or (ii) a requirement for prior
NIH approval of any or all recombinant or synthetic nucleic acid molecule projects at the institution.
Section I-D-2. All non-NIH funded projects involving recombinant or synthetic nucleic acid molecule
conducted at or sponsored by an institution that receives NIH funds for projects involving such techniquesmust comply with the NIH Guidelines. Noncompliance may result in: (i) suspension, limitation, or
termination of NIH funds for recombinant or synthetic nucleic acid molecule research at the institution, or(ii) a requirement for prior NIH approval of any or all recombinant or synthetic nucleic acid molecule
projects at the institution.
Section I-E.
General Definitions
The following terms, which are used throughout the NIH Guidelines, are defined as follows:
Section I-E-1. An "institution" is any public or private entity (including Federal, state, and local
government agencies).
Section I-E-2. An "Institutional Biosafety Committee" is a committee that: (i) meets the requirements formembership specified in Section IV-B-2, Institutional Biosafety Committee (IBC), and (ii) reviews,
approves, and oversees projects in accordance with the responsibilities defined in Section IV-B-2,
Institutional Biosafety Committee (IBC).

Page 12 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Section I-E-3. The "Office of Science Policy (OSP)" is the office within the NIH that is responsible for: (i)reviewing and coordinating all activities relating to the NIH Guidelines, and (ii) performing other duties as
defined in Section IV-C-2, Office of Science Policy (OSP).
Section I-E-4. “Initiation” of research is the introduction of recombinant or synthetic nucleic acidmolecules into organisms, cells, or viruses.
Section I-E-5. The "NIH Director" is the Director of the National Institutes of Health, or any other officer or
employee of NIH to whom authority has been delegated.
Section I-E-6. "Deliberate release" is defined as a planned introduction of recombinant or synthetic
nucleic acid molecule-containing microorganisms, plants, or animals into the environment.
Section I-E-7. “Gene drive” is defined as a technology whereby a particular heritable element biases
inheritance in its favor, resulting in the heritable element becoming more prevalent than predicted by
Mendelian laws of inheritance in a population over successive generations.
***********************************************************************************************************************SECTION II.

SAFETY CONSIDERATIONS

Section II-A.

Risk Assessment

Section II-A-1. Risk Groups
Risk assessment is ultimately a subjective process. The investigator must make an initial risk
assessment based on the Risk Group (RG) of an agent (see Appendix B, Classification of Human
Etiologic Agents on the Basis of Hazard). Agents are classified into four Risk Groups (RGs) according totheir relative pathogenicity for healthy adult humans by the following criteria: (1) Risk Group 1 (RG1)
agents are not associated with disease in healthy adult humans. (2) Risk Group 2 (RG2) agents areassociated with human disease which is rarely serious and for which preventive or therapeuticinterventions are often available. (3) Risk Group 3 (RG3) agents are associated with serious or lethal
human disease for which preventive or therapeutic interventions may be available. (4) Risk Group 4(RG4) agents are likely to cause serious or lethal human disease for which preventive or therapeuticinterventions are not usually available.
Section II-A-2. Criteria for Risk Groups
Classification of agents in Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard,
is based on the potential effect of a biological agent on a healthy human adult and does not account for
instances in which an individual may have increased susceptibility to such agents, e.g., preexisting
diseases, medications, compromised immunity, pregnancy or breast feeding (which may increase
exposure of infants to some agents).
Personnel may need periodic medical surveillance to ascertain fitness to perform certain activities; they
may also need to be offered prophylactic vaccines and boosters (see Section IV-B-1-f, Responsibilities of
the Institution, General Information).
Section II-A-3. Comprehensive Risk Assessment
In deciding on the appropriate containment for an experiment, the first step is to assess the risk of theagent itself. Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, classifies
agents into Risk Groups based on an assessment of their ability to cause disease in humans and theavailable treatments for such disease. Once the Risk Group of the agent is identified, this should be
followed by a thorough consideration of how the agent is to be manipulated. Factors to be considered indetermining the level of containment include agent factors such as: virulence, pathogenicity, infectiousdose, environmental stability, route of spread, communicability, operations, quantity, availability ofPage 13 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

vaccine or treatment, and gene product effects such as toxicity, physiological activity, and allergenicity.
Any strain that is known to be more hazardous than the parent (wild-type) strain should be considered forhandling at a higher containment level. Certain attenuated strains or strains that have been demonstrated
to have irreversibly lost known virulence factors may qualify for a reduction of the containment levelcompared to the Risk Group assigned to the parent strain (see Section V-B, Footnotes and References ofSections I-IV).
While the starting point for the risk assessment is based on the identification of the Risk Group of theparent agent, as technology moves forward, it may be possible to develop an organism containing genetic
sequences from multiple sources such that the parent agent may not be obvious. In such cases, the risk
assessment should include at least two levels of analysis. The first involves a consideration of the Risk
Groups of the source(s) of the sequences and the second involves an assessment of the functions that
may be encoded by these sequences (e.g., virulence or transmissibility). It may be prudent to firstconsider the highest Risk Group classification of all agents that are the source of sequences included in
the construct. Other factors to be considered include the percentage of the genome contributed by eachparent agent and the predicted function or intended purpose of each contributing sequence. The initialassumption should be that all sequences will function as they did in the original host context.
The Principal Investigator and Institutional Biosafety Committee must also be cognizant that the
combination of certain sequences in a new biological context may result in an organism whose risk profile
could be higher than that of the contributing organisms or sequences. The synergistic function of thesesequences may be one of the key attributes to consider in deciding whether a higher containment level iswarranted, at least until further assessments can be carried out. A new biosafety risk may occur with anorganism formed through combination of sequences from a number of organisms or due to the synergistic
effect of combining transgenes that results in a new phenotype.
A final assessment of risk based on these considerations is then used to set the appropriate containment
conditions for the experiment (see Section II-B, Containment). The appropriate containment level may beequivalent to the Risk Group classification of the agent or it may be raised or lowered as a result of theabove considerations. The Institutional Biosafety Committee must approve the risk assessment and thebiosafety containment level for recombinant or synthetic nucleic acid experiments described in Sections
III-A, Experiments that Require NIH Director Approval and Institutional Biosafety Committee Approval,
Before Initiation; III-B, Experiments that Require NIH OSP and Institutional Biosafety Committee Approval
Before Initiation; III-C, Experiments Involving Human Gene Transfer that Require Institutional Biosafety
Committee Approval Prior to Initiation; III-D, Experiments that Require Institutional Biosafety Committee
Approval Before Initiation.
Research involving gene drive modified organisms may require risk assessments that incorporate abroader scope of considerations because of greater uncertainty of the technology and potential
uncertainty of the impact of the newly modified organism. Specific attention must be paid to risks of anunintended release from the laboratory and the potential impact on humans, other populations oforganisms, and the environment.
Considerations for conducting risk assessments for research involving gene drive modified organisms
might include:
1) The specific types of manipulations based on:a) Function or intended function of the genetic/gene drive construct (i.e., a designed orengineered assembly of sequences);b) Source of the genetic material (e.g., sequences of transgenes) in the construct;c) The modifications to the construct;d) Whether it is possible to predict the consequences of a construct, including therecognition of an unintended gene drive (i.e., construct not specifically designed as agene drive but nonetheless having properties of a gene drive) and the possible
consequences of escape into the environment;e) The potential ability of the gene drive to spread or persist in local populations;Page 14 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

2) Options for approaches to risk mitigation for specific types of risks in experiments or when dealing
with a high degree of uncertainty about risks;
3) Considerations for implementing more stringent containment measures until biosafety data are
accrued to support lowering containment.
Careful consideration should be given to the types of manipulation planned for some higher Risk Groupagents. For example, the RG2 dengue viruses may be cultured under the Biosafety Level (BL) 2
containment (see Section II-B); however, when such agents are used for animal inoculation ortransmission studies, a higher containment level is recommended. Similarly, RG3 agents such as
Venezuelan equine encephalomyelitis and yellow fever viruses should be handled at a higher
containment level for animal inoculation and transmission experiments.
Individuals working with human immunodeficiency virus (HIV), hepatitis B virus (HBV) or other bloodborne
pathogens should consult the applicable Occupational Safety and Health Administration (OSHA)
regulation, 29 CFR 1910.1030, and OSHA publication 3127 (1996 revised). BL2 containment isrecommended for activities involving all blood-contaminated clinical specimens, body fluids, and tissues
from all humans, or from HIV- or HBV-infected or inoculated laboratory animals. Activities such as the
production of research-laboratory scale quantities of HIV or other bloodborne pathogens, manipulating
concentrated virus preparations, or conducting procedures that may produce droplets or aerosols, areperformed in a BL2 facility using the additional practices and containment equipment recommended for
BL3. Activities involving industrial scale volumes or preparations of concentrated HIV are conducted in a
BL3 facility, or BL3 Large Scale if appropriate, using BL3 practices and containment equipment.
Exotic plant pathogens and animal pathogens of domestic livestock and poultry are restricted and may
require special laboratory design, operation and containment features not addressed in Biosafety in
Microbiological and Biomedical Laboratories (see Section V-C, Footnotes and References of Sections I
through IV). For information regarding the importation, possession, or use of these agents see Sections
V-G and V-H, Footnotes and References of Sections I through IV.
Section II-B.

Containment

Effective biological safety programs have been operative in a variety of laboratories for many years.
Considerable information already exists about the design of physical containment facilities and selectionof laboratory procedures applicable to organisms carrying additional recombinant or synthetic nucleic acidmolecules (see Section V-B, Footnotes and References of Sections I-IV). The existing programs rely
upon mechanisms that can be divided into two categories: (i) a set of standard practices that are
generally used in microbiological laboratories; and (ii) special procedures, equipment, and laboratory
installations that provide physical barriers that are applied in varying degrees according to the estimatedbiohazard. Four biosafety levels are described in Appendix G, Physical Containment. These biosafetylevels consist of combinations of laboratory practices and techniques, safety equipment, and laboratory
facilities appropriate for the operations performed and are based on the potential hazards imposed by theagents used and for the laboratory function and activity. Biosafety Level 4 provides the most stringent
containment conditions, Biosafety Level 1 the least stringent.
Experiments involving recombinant or synthetic nucleic acid molecules lend themselves to a thirdcontainment mechanism, namely, the application of highly specific biological barriers. Natural barriers
exist that limit either: (i) the infectivity of a vector or vehicle (plasmid or virus) for specific hosts, or (ii) its
dissemination and survival in the environment. Vectors, which provide the means for recombinant orsynthetic nucleic acid molecule and/or host cell replication, can be genetically designed to decrease, by
many orders of magnitude, the probability of dissemination of recombinant or synthetic nucleic acidmolecule outside the laboratory (see Appendix I, Biological Containment).
Since these three means of containment are complementary, different levels of containment can beestablished that apply various combinations of the physical and biological barriers along with a constantuse of standard practices. Categories of containment are considered separately in order that suchcombinations can be conveniently expressed in the NIH Guidelines.

Page 15 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Physical containment conditions within laboratories, described in Appendix G, Physical Containment, may
not always be appropriate for all organisms because of their physical size, the number of organisms
needed for an experiment, or the particular growth requirements of the organism. Likewise, biologicalcontainment for microorganisms described in Appendix I, Biological Containment, may not be appropriate
for all organisms, particularly higher eukaryotic organisms. However, significant information exists about
the design of research facilities and experimental procedures that are applicable to organisms containing
additional recombinant or synthetic nucleic acid molecules that are either integrated into the genome orinto microorganisms associated with the higher organism as a symbiont, pathogen, or other relationship.
This information describes facilities for physical containment of organisms used in non-traditional
laboratory settings and special practices for limiting or excluding the unwanted establishment, transfer ofgenetic information, and dissemination of organisms beyond the intended location, based on both
physical and biological containment principles. Research conducted in accordance with these conditions
effectively confines the organism.
For research involving plants, four biosafety levels (BL1-P through BL4-P) are described in Appendix L,
Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research
Involving Plants. BL1-P is designed to provide a moderate level of containment for experiments for which
there is convincing biological evidence that precludes the possibility of survival, transfer, or disseminationof recombinant or synthetic nucleic acid molecules into the environment, or in which there is norecognizable and predictable risk to the environment in the event of accidental release. BL2-P isdesigned to provide a greater level of containment for experiments involving plants and certain associatedorganisms in which there is a recognized possibility of survival, transmission, or dissemination ofrecombinant or synthetic nucleic acid molecule containing organisms, but the consequence of such an
inadvertent release has a predictably minimal biological impact. BL3-P and BL4-P describe additional
containment conditions for research with plants and certain pathogens and other organisms that require
special containment because of their recognized potential for significant detrimental impact on managed
or natural ecosystems. BL1-P relies upon accepted scientific practices for conducting research in mostordinary greenhouse or growth chamber facilities and incorporates accepted procedures for good pest
control and cultural practices. BL1-P facilities and procedures provide a modified and protectedenvironment for the propagation of plants and microorganisms associated with the plants and a degree ofcontainment that adequately controls the potential for release of biologically viable plants, plant parts, andmicroorganisms associated with them. BL2-P and BL3-P rely upon accepted scientific practices for
conducting research in greenhouses with organisms infecting or infesting plants in a manner thatminimizes or prevents inadvertent contamination of plants within or surrounding the greenhouse. BL4-P
describes facilities and practices known to provide containment of certain exotic plant pathogens.
For research involving animals, which are of a size or have growth requirements that preclude the use of
conventional primary containment systems used for small laboratory animals, four biosafety levels (BL1-N
through BL4-N) are described in Appendix M, Physical and Biological Containment for Recombinant orSynthetic Nucleic Acid Molecule Research Involving Animals. BL1-N describes containment for animals
that have been modified by stable introduction of recombinant or synthetic nucleic acid molecules, orDNA derived therefrom, into the germ-line (transgenic animals) and experiments involving viable
recombinant or synthetic nucleic acid molecule-modified microorganisms and is designed to eliminate thepossibility of sexual transmission of the modified genome or transmission of recombinant or synthetic
nucleic acid molecule-derived viruses known to be transmitted from animal parent to offspring only bysexual reproduction. Procedures, practices, and facilities follow classical methods of avoiding genetic
exchange between animals. BL2-N describes containment which is used for transgenic animals
associated with recombinant or synthetic nucleic acid molecule-derived organisms and is designed toeliminate the possibility of vertical or horizontal transmission. Procedures, practices, and facilities follow
classical methods of avoiding genetic exchange between animals or controlling arthropod transmission.
BL3-N and BL4-N describe higher levels of containment for research with certain transgenic animals
involving agents which pose recognized hazard.
In constructing the NIH Guidelines, it was necessary to define boundary conditions for the different levelsof physical and biological containment and for the classes of experiments to which they apply. ThesePage 16 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

definitions do not take into account all existing and anticipated information on special procedures that willallow particular experiments to be conducted under different conditions than indicated here without
affecting risk. Individual investigators and Institutional Biosafety Committees are urged to devise simple
and more effective containment procedures and to submit recommended changes in the NIH Guidelines
to permit the use of these procedures.
***********************************************************************************************************************SECTION III.

EXPERIMENTS COVERED BY THE NIH GUIDELINESThis section describes six categories of experiments involving recombinant or synthetic nucleic acidmolecules: (i) those that require NIH Director approval and Institutional Biosafety Committee (IBC)
approval before initiation (see Section III-A), (ii) those that require NIH OSP and Institutional Biosafety
Committee approval before initiation (see Section III-B), (iii) those that require Institutional Biosafety
Committee approval before initiation of human gene transfer (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) thosethat 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 andinto 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 OSP (see Section IV-C-1-b-(2) and its subsections, Minor
Actions).
Section III-A.
Experiments that Require NIH Director Approval and Institutional Biosafety
Committee Approval Before Initiation (See Section IV-C-1-b-(1), Major Actions).
Section III-A-1. Major Actions under the NIH Guidelines
Experiments considered as Major Actions as defined in Section III-A-1-a under the NIH Guidelines cannot
be initiated without submission of relevant information on the proposed experiment to the Office of
Science Policy, National Institutes of Health, preferably by e-mail to: NIHGuidelines@od.nih.gov, thepublication of the proposal in the Federal Register for a minimum of 15 days of comment, and specificapproval by NIH. The containment conditions or stipulation requirements for such experiments will be 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.
Section III-A-1-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 suchacquisition could compromise the ability to control disease agents in humans, veterinary medicine, or
agriculture, will require NIH Director approval.
Consideration should be given as to whether the drug resistance trait to be used in the experiment would
render that microorganism resistant to the primary drug available to and/or indicated for certain
populations, for example children or pregnant women.
At the request of an Institutional Biosafety Committee, NIH OSP will make a determination regardingwhether a specific experiment involving the deliberate transfer of a drug resistance trait falls under
Section III-A-1-a and therefore requires NIH Director approval. An Institutional Biosafety Committee may
also consult with NIH OSP regarding experiments that do not meet the requirements of Section III-A-1-abut nonetheless raise important public health issues.

Page 17 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Section III-B.
Experiments That Require NIH OSP 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 OSP. The containment conditions for such experiments will be determined
by NIH OSP in consultation with ad hoc experts. Such experiments require Institutional Biosafety
Committee approval before initiation (see Section IV-B-2-b-(1), Institutional Biosafety Committee).
Section III-B-1. Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than 100
Nanograms per Kilogram Body Weight
Deliberate formation of recombinant or synthetic nucleic acid molecules containing genes for thebiosynthesis of toxin molecules lethal for vertebrates at an LD50 of less than 100 nanograms per kilogram
body weight (e.g., microbial toxins such as the botulinum toxins, tetanus toxin, diphtheria toxin, andShigella dysenteriae neurotoxin). Specific approval has been given for the cloning in Escherichia coli K12 of DNA containing genes coding for the biosynthesis of toxic molecules which are lethal to vertebrates
at 100 nanograms to 100 micrograms per kilogram body weight. Specific experiments already approved
under this section may be obtained from the Office of Science Policy, National Institutes of Health,preferably by submitting a request for this information to: NIHGuidelines@od.nih.gov; additional contact
information is also available here and on the OSP website (www.osp.od.nih.gov).
Section III-B-2. Experiments that have been Approved (under Section III-A-1-a) as Major Actions
under the NIH Guidelines
Upon receipt and review of an application from the investigator, NIH OSP may determine that a proposed
experiment is equivalent to an experiment that has previously been approved by the NIH Director as a
Major Action, including experiments approved prior to implementation of these changes. An experimentwill only be considered equivalent if, as determined by NIH OSP, there are no substantive differences andpertinent information has not emerged since submission of the initial III-A-1-a experiment that wouldchange the biosafety and public health considerations for the proposed experiments. If such a
determination is made by NIH OSP, these experiments will not require review and approval under Section
III-A.
Section III-C.
Experiments Involving Human Gene Transfer that Require Institutional Biosafety
Committee Approval Prior to Initiation
Section III-C-1. Experiments Involving the Deliberate Transfer of Recombinant or Synthetic
Nucleic Acid Molecules, or DNA or RNA Derived from Recombinant or Synthetic Nucleic Acid
Molecules, into One or More Human Research Participants
Human gene transfer is the deliberate transfer into human research participants of either:
1) Recombinant nucleic acid molecules, or DNA or RNA derived from recombinant nucleic acidmolecules, or2) Synthetic nucleic acid molecules, or DNA or RNA derived from synthetic nucleic acid molecules,
that meet any one of the following criteria:a) Contain more than 100 nucleotides; orb) Possess biological properties that enable introduction of stable genetic modifications into
the genome (e.g., cis elements involved in integration, gene editing); orc) Have the potential to replicate in a cell; ord) Can be translated or transcribed.

Research cannot be initiated until Institutional Biosafety Committee and all other applicable institutionaland regulatory authorization(s) and approvals have been obtained.

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The deliberate transfer of recombinant or synthetic nucleic acids into one human research participant,conducted under a Food and Drug Administration (FDA) regulated individual patient expanded access
Investigational New Drug (IND) or protocol, including for emergency use, is not research subject to the
NIH Guidelines and thus does not need to be submitted to an IBC for review and approval.
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 aregistration 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 proteinthat 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 todecrease the level of containment specified for experiments in this category will be considered by NIH(see Section IV-C-1-b-(2)-(c), Minor Actions).
Section III-D-1. 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-1-a. Experiments involving the introduction of recombinant or synthetic nucleic acidmolecules 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-1-b. Experiments involving the introduction of recombinant or synthetic nucleic acidmolecules into Risk Group 3 agents will usually be conducted at BL3 containment. Experiments withsuch agents will usually be conducted with whole animals at BL3 or BL3-N containment.
Section III-D-1-c. Experiments involving the introduction of recombinant or synthetic nucleic acidmolecules into Risk Group 4 agents shall be conducted at BL4 containment. Experiments with suchagents shall be conducted with whole animals at BL4 or BL4-N containment.
Section III-D-1-d. Containment conditions for experiments involving the introduction of recombinant orsynthetic nucleic acid molecules into restricted agents shall be set on a case-by-case basis following NIH
OSP review. A U.S. Department of Agriculture - Animal and Plant Health Inspection Service(USDA/APHIS) permit is required for work with plant or animal pathogens (see Section V-G and V-M,
Footnotes and References of Sections I-IV). Experiments with such agents shall be conducted withwhole animals at BL4 or BL4-N containment.
Section III-D-2. Experiments in Which DNA From Risk Group 2, Risk Group 3, Risk Group 4, orRestricted 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 IIA, 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 agiven recombinant. In the absence of such a demonstration, BL4 containment shall be used. The
Institutional Biosafety Committee may approve the specific lowering of containment for particularexperiments to BL1. Many experiments in this category are exempt from the NIH Guidelines (see Section
III-F, Exempt Experiments). Experiments involving the formation of recombinant or synthetic nucleic acid

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molecules for certain genes coding for molecules toxic for vertebrates require NIH OSP approval (seeSection III-B-1, Experiments Involving the Cloning of Toxin Molecules with LD50 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 istransferred into nonpathogenic prokaryotes or lower eukaryotes shall be determined by NIH OSP
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 a Helper System in Tissue Culture Systems
Caution: The potential for reversion or generation of replication competent virus should be consideredwhen generating or using defective viruses or vectors in the presence of helper systems (e.g., helper
viruses, packaging cell lines, transient transfection systems, replicon systems). Special care should be
used in the evaluation of containment levels for experiments which are likely to either enhance thepathogenicity (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 or synthetic nucleic acid molecules or nucleic acid molecules derived therefrom,which contain less than two-thirds of the genome of any eukaryotic virus (all viruses from a single Family(see Section V-J, Footnotes and References of Sections I-IV) being considered identical (see Section VK, Footnotes and References of Sections I-IV)), are considered defective and may be used in theabsence of helper under the conditions specified in Section III-E-1, Experiments Involving the Formationof Recombinant or Synthetic 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 (seeAppendix B-II, Risk Group 2 Agents) in the presence of a helper system may be conducted at BL2.
Section III-D-3-b. Experiments involving the use of infectious or defective Risk Group 3 viruses (seeAppendix B-III-D, Risk Group 3 (RG3) - Viruses and Prions) in the presence of a helper system may be
conducted at BL3.
Section III-D-3-c. Experiments involving the use of infectious or defective Risk Group 4 viruses (seeAppendix B-IV-D, Risk Group 4 (RG4) - Viral Agents) in the presence of a helper system may be
conducted at BL4.
Section III-D-3-d. Experiments involving the use of infectious or defective restricted poxviruses (seeSections V-A and V-L, Footnotes and References of Sections I-IV) in the presence of a helper systemshall be determined on a case-by-case basis following NIH OSP review. A U.S. Department ofAgriculture permit is required for work with plant or animal pathogens (see Section V-G, Footnotes andReferences of Sections I-IV).
Section III-D-3-e. Experiments involving the use of infectious or defective viruses in the presence of ahelper system 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 deliberate transfer of recombinant or synthetic nucleic acidmolecules, DNA or RNA derived from recombinant or synthetic nucleic acid molecules, or recombinant orsynthetic nucleic acid molecule-modified microorganisms into whole animals and experiments involving

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whole animals in which the animal's genome has been altered by recombinant or synthetic nucleic acidmolecules, or nucleic acids derived therefrom, into the germ-line (transgenic animals). Experiments
involving gene drive modified animals or experiments involving viable recombinant or synthetic nucleic
acid molecule-modified microorganisms, except for viruses that are only vertically transmitted, may not be
conducted at BL1-N containment. A minimum containment of BL2 or BL2-N is required (see Section III-D8).
Caution - Special care should be used in the evaluation of containment conditions for some experimentswith transgenic animals. For example, such experiments might lead to the creation of novel mechanisms(e.g., a gene drive; refer to Section III-D-8) 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 or synthetic nucleic acid molecules, or DNA or RNA molecules derived
therefrom, from any source except for greater than two-thirds of eukaryotic viral genome may betransferred to any non-human vertebrate or any invertebrate organism and propagated under conditionsof physical containment comparable to BL1 or BL1-N and appropriate to the organism under study (seeSection 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 ofcomplementation or recombination in animals, may be propagated under conditions of physicalcontainment comparable to BL1 or BL1-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 III-D-4-b, Experiments Involving Whole Animals. For experiments involving recombinant orsynthetic nucleic acid molecule-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 demonstratethat 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 or synthetic nucleic acid molecules, or DNA or
RNA derived therefrom, involving whole animals, including transgenic animals, and not covered by
Section III-D-1, Experiments Using Human or Animal Pathogens (Risk Group 2, Risk Group 3, Risk Group4, or Restricted Agents as Host-Vector Systems), or Section III-D-4-a, the appropriate containment shallbe determined by the Institutional Biosafety Committee. Experiments involving gene drive modified
animals generated by recombinant or synthetic nucleic acid molecules shall be conducted at a minimumof BL2 or BL2-N (see Section III-D-8).
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 BL1
containment are described under Section III-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-VII, The Purchase or Transfer of Transgenic
Rodents).
Section III-D-4-c-(3). Experiments involving the generation or use of gene drive modified animals requirea minimum of BL2 containment and are covered under III-D-8, Experiments Involving Gene Drive
Modified Organisms.
Section III-D-5. Experiments Involving Whole Plants
Experiments to genetically engineer plants by recombinant or synthetic nucleic acid molecule methods, touse such plants for other experimental purposes (e.g., response to stress), to propagate such plants, or touse plants together with microorganisms or insects containing recombinant or synthetic nucleic acidmolecules, may be conducted under the containment conditions described in Sections III-D-5-a throughPage 21 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

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. Experiments involving the generationor use of gene drive modified organisms require a minimum of BL2 containment and are described under
Section III-D-8, Experiments Involving Gene Drive Modified Organisms.
NOTE - For recombinant or synthetic nucleic acid molecule experiments falling under Sections III-D-5-athrough III-D-5-d, physical containment requirements may be reduced to the next lower level byappropriate 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-M, Footnotes and References of Sections I-IV) infectious agents withrecognized potential for serious detrimental impact on managed or natural ecosystems when recombinantor synthetic nucleic acid molecule 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-M, Footnotes andReferences 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 andfunctional 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 readilytransmissible exotic (see Section V-M, Footnotes and References of Sections I-IV) infectious agents,
such as the soybean rust fungus (Phakospora pachyrhizi) and maize streak or other viruses in thepresence of their specific arthropod vectors, that have the potential of being serious pathogens of major
U.S. crops.
Section III-D-5-d. BL3-P containment is recommended for experiments involving sequences encoding
potent vertebrate toxins introduced into plants or associated organisms. Recombinant or synthetic
nucleic acid molecules containing genes for the biosynthesis of toxin molecules lethal for vertebrates at
an LD50 of <100 nanograms per kilogram body weight fall under Section III-B-1, Experiments Involving theCloning of Toxin Molecules with LD50 of Less than 100 Nanograms Per Kilogram Body Weight, andrequire NIH OSP and Institutional Biosafety Committee approval before initiation.
Section III-D-5-e. BL3-P or BL2-P + biological containment is recommended for experiments withmicrobial pathogens of insects or small animals associated with plants if the recombinant or synthetic
nucleic acid molecule-modified organism has a recognized potential for serious detrimental impact onmanaged 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 or Synthetic Recombinant or synthetic nucleic acid Molecules, shall be used. Appendix K
describes containment conditions Good Large Scale Practice through BL3-Large Scale.
Section III-D-7. Experiments Involving Influenza Viruses
Experiments with influenza viruses generated by recombinant or synthetic methods (e.g., generation by
reverse genetics of chimeric viruses with reassorted segments, introduction of specific mutations) shall be
conducted at the biosafety level containment corresponding to the Risk Group of the virus that was thesource of the majority of segments in the recombinant or synthetic virus (e.g., experiments with viruses
containing a majority of segments from a RG3 virus shall be conducted at BL3). Experiments with
influenza viruses containing genes or segments from 1918-1919 H1N1 (1918 H1N1), human H2N2
(1957-1968) and highly pathogenic avian influenza H5N1 strains within the Goose/Guangdong/96-like H5lineage (HPAI H5N1), including, but not limited to, strains of HPAI H5N1 virus that are transmissible

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among mammals by respiratory droplets, as demonstrated in an appropriate animal model or clinicallyin humans (hereinafter referred to as mammalian-transmissible HPAI H5N1 virus), shall be conducted at
BL3 enhanced containment (see Appendix G-II-C-5, Biosafety Level 3 Enhanced for Research Involving
Risk Group 3 Influenza Viruses) unless indicated below.
Section III-D-7-a. Human H2N2 (1957-1968). Experiments with influenza viruses containing the H2
hemagglutinin (HA) segment shall be conducted at BL3 enhanced (see Appendix G-II-C-5, Biosafety
Level 3 Enhanced for Research Involving Risk Group 3 Influenza Viruses). Experiments with the H2 HAgene in cold-adapted, live attenuated vaccine strains (e.g., A/Ann Arbor/6/60 H2N2) may be conducted at
BL2 containment provided segments with mutations conferring temperature sensitivity and attenuation
are not altered in the recombinant or synthetic virus. Experiments with Risk Group 2 influenza viruses
containing genes from human H2N2 other than the HA gene can be worked on at BL2.
Section III-D-7-b. Highly Pathogenic Avian Influenza H5N1 strains within the
Goose/Guangdong/96-like H5 lineage (HPAI H5N1). Experiments involving influenza viruses
containing a majority of genes and/or segments from a HPAI H5N1 influenza virus shall be conducted at
BL3 enhanced containment, (see Appendix G-II-C-5, Biosafety Level 3 Enhanced for Research Involving
Risk Group 3 Influenza Viruses). Experiments involving influenza viruses containing a minority of genes
and/or segments from a HPAI H5N1 influenza virus shall be conducted at BL3 enhanced unless a risk
assessment performed by the IBC determines that they can be conducted safely at biosafety level 2 andafter they have been excluded pursuant to 9 CFR 121.3(e). NIH OSP is available to IBCs to provide
consultation with influenza virus experts when risk assessments are being made to determine theappropriate biocontainment for experiments with influenza viruses containing a minority of gene/segmentsfrom HPAI H5N1. Such experiments may be performed at BL3 enhanced containment or containment
may be lowered to biosafety level 2, the level of containment for most research with other influenza
viruses. (USDA/APHIS regulations and decisions on lowering containment also apply.) In deciding tolower containment, the IBC should consider whether, in at least two animal models (e.g., ferret, mouse,
Syrian golden hamster, cotton rat, non-human primates), there is evidence that the resulting influenza
virus shows reduced replication and virulence compared to the parental RG3 virus at relevant doses.
This should be determined by measuring biological indices appropriate for the specific animal model (e.g.,
severe weight loss, elevated temperature, mortality or neurological symptoms).
Section III-D-7-c. 1918 H1N1. Experiments involving influenza viruses containing any gene or segmentfrom 1918 H1N1 shall be conducted at BL3 enhanced containment (see Appendix G-II-C-5, Biosafety
Level 3 Enhanced for Research Involving Risk Group 3 Influenza Viruses).
Section III-D-7-d. Antiviral Susceptibility and Containment. The availability of antiviral drugs aspreventive and therapeutic measures is an important safeguard for experiments with 1918 H1N1, HPAI
H5N1, and human H2N2 (1957-1968). If an influenza virus containing genes from one of these viruses is
resistant to both classes of current antiviral agents, adamantanes and neuraminidase inhibitors, higher
containment may be required based on the risk assessment considering transmissibility to humans,
virulence, pandemic potential, alternative antiviral agents if available, etc.
Experiments with 1918 H1N1, human H2N2 (1957-1968) or HPAI H5N1 that are designed to create
resistance to neuraminidase inhibitors or other effective antiviral agents (including investigational antiviral
agents being developed for influenza) would be subject to Section III-A-1 (Major Actions). As per Section
I-A-1 of the NIH Guidelines, if the agent is a Select Agent, the NIH will defer to the appropriate Federal
agency (HHS or USDA Select Agent Divisions) on such experiments.
Section III-D-8. Experiments Involving Gene Drive Modified Organisms
Experiments involving gene drive modified organisms generated by recombinant or synthetic nucleic acidmolecules shall be conducted at a minimum of Biosafety Level (BL) 2, BL2-N (Animals) or BL2-P (plant)
containment.
Section III-E.

Experiments that Require Institutional Biosafety Committee Notice SimultaneousPage 23 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)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 BL1 containment. For experiments in thiscategory, 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 thelocal 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 andapproval 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 BL1 containment.
Section III-E-1. Experiments Involving the Formation of Recombinant or Synthetic Nucleic Acid
Molecules Containing No More than Two-Thirds of the Genome of any Eukaryotic Virus
Recombinant or synthetic nucleic acid molecules containing no more than two-thirds of the genome ofany eukaryotic virus (all viruses from a single Family being considered identical [see Section V-J,
Footnotes and References of Sections I-IV]) may be propagated and maintained in cells in tissue cultureusing BL1 containment. For such experiments, it must be demonstrated that the cells lack a helper
system for the specific Families of defective viruses being used. If a helper system 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 a Helper System in Tissue
Culture Systems, should be used. The DNA may contain fragments of the genome of viruses from morethan 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 nucleic acid molecule-modified whole plants, and/or
experiments involving recombinant or synthetic nucleic acid molecule-modified organisms associated withwhole plants, except those that fall under Section III-A, III-B, III-D, or III-F. It should be emphasized thatknowledge of the organisms and judgment based on accepted scientific practices should be used in allcases in selecting the appropriate level of containment. For example, if the genetic modification has theobjective of increasing pathogenicity or converting a non-pathogenic organism into a pathogen, then ahigher 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 or synthetic nucleic acid molecule-modified plants.
Section III-E-2-a. BL1-P is recommended for all experiments with recombinant or synthetic recombinantor synthetic nucleic acid molecule-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 or synthetic nucleic acid molecule-modified plants that are not noxious weeds orthat cannot interbreed with noxious weeds in the immediate geographic area, and experiments involving
whole plants and recombinant or synthetic nucleic acid molecule-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 III-E-2-b-(1). Plants modified by recombinant or synthetic nucleic acid molecules that arenoxious weeds or can interbreed with noxious weeds in the immediate geographic area.
Section III-E-2-b-(2). Plants in which the introduced DNA represents the complete genome of a nonexotic infectious agent (see Section V-M, Footnotes and References of Sections I-IV).

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Section III-E-2-b-(3). Plants associated with recombinant or synthetic nucleic acid molecule-modified
non-exotic microorganisms that have a recognized potential for serious detrimental impact on managed
or natural ecosystems (see Section V-M, Footnotes and References of Sections I-IV).
Section III-E-2-b-(4). Plants associated with recombinant or synthetic nucleic acid molecule-modified
exotic microorganisms that have no recognized potential for serious detrimental impact on managed or
natural ecosystems (see Section V-M, Footnotes and References of Sections I-IV).
Section III-E-2-b-(5). Experiments with recombinant or synthetic nucleic acid molecule-modified
arthropods or small animals associated with plants, or with arthropods or small animals with recombinantor synthetic nucleic acid molecule-modified microorganisms associated with them if the recombinant orsynthetic nucleic acid molecule-modified microorganisms have no recognized potential for serious
detrimental impact on managed or natural ecosystems (see Section V-M, Footnotes and References ofSections I-IV).
Section III-E-3. Experiments Involving Transgenic Rodents
This section covers experiments involving the generation or use of rodents in which the animal's genome
has been altered by stable introduction of recombinant or synthetic nucleic acid molecules, or nucleic
acids derived therefrom, into the germ-line (transgenic rodents). Only experiments that require BL1
containment are covered under this section; experiments that require BL2, BL3, or BL4 containment arecovered under Section III-D-4, Experiments Involving Whole Animals or Section III-D-8, Experiments
Involving Gene Drive Modified Organisms.
Section III-E-3-a. Experiments involving the breeding of certain BL1 transgenic rodents are exempt
under Section III-F, Exempt Experiments (See Appendix C-VIII, Generation of BL1 Transgenic Rodents
via Breeding).
Section III-F.

Exempt Experiments

The following recombinant or synthetic nucleic acid molecules are exempt from the NIH Guidelines andregistration with the Institutional Biosafety Committee is not required; however, other federal and state
standards of biosafety may still apply to such research (for example, the Centers for Disease Control andPrevention (CDC)/NIH publication Biosafety in Microbiological and Biomedical Laboratories).
Section III-F-1. Those synthetic nucleic acids that: (1) can neither replicate nor generate nucleic acids
that can replicate in any living cell (e.g., oligonucleotides or other synthetic nucleic acids that do notcontain an origin of replication or contain elements known to interact with either DNA or RNA
polymerase), and (2) are not designed to introduce a stable genetic modification, and (3) do not produce
a toxin that is lethal for vertebrates at an LD50 of less than 100 nanograms per kilogram body weight. If asynthetic nucleic acid is deliberately transferred into one or more human research participants and meetsthe criteria of Section III-C, it is not exempt under this Section.
Section III-F-2. Those that are not in organisms, cells, or viruses and that have not been modified ormanipulated (e.g., encapsulated into synthetic or natural vehicles) to render them capable of penetrating
cellular membranes.
Section III-F-3. Those that consist solely of the exact recombinant or synthetic nucleic acid sequence
from a single source that exists contemporaneously in nature.
Section III-F-4. Those that consist entirely of nucleic acids 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-5. Those that consist entirely of nucleic acids from a eukaryotic host including its
chloroplasts, mitochondria, or plasmids (but excluding viruses) when propagated only in that host (or aPage 25 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

closely related strain of the same species).
Section III-F-6. 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 after appropriate
notice and opportunity for public comment (see Section IV-C-1-b-(1)-(c), Major Actions). See AppendicesA-I through A-VI, Exemptions under Section III-F-6--Sublists of Natural Exchangers, for a list of natural
exchangers that are exempt from the NIH Guidelines.
Section III-F-7. Those genomic DNA molecules that have acquired a transposable element, provided thetransposable element does not contain any recombinant and/or synthetic DNA.
Section III-F-8. Those that do not present a significant risk to health or the environment (see Section IVC-1-b-(1)-(c), Major Actions), as determined by the NIH Director following appropriate notice andopportunity for public comment. See Appendix C, Exemptions under Section III-F-8 for other classes ofexperiments which are exempt from the NIH Guidelines.
***********************************************************************************************************************SECTION IV.

ROLES AND RESPONSIBILITIESSection IV-A.

PolicyThe safe conduct of experiments involving recombinant or synthetic nucleic acid molecules depends on
the individual conducting such activities. The NIH Guidelines cannot anticipate every possible situation.
Motivation and good judgment are the key essentials to protection of health and the environment. The
NIH Guidelines are intended to assist the institution, Institutional Biosafety Committee, Biological Safety
Officer, and the Principal Investigator in determining safeguards that should be implemented. The NIH
Guidelines will never be complete or final since all conceivable experiments involving recombinant orsynthetic nucleic acid molecules cannot be foreseen. The utilization of new genetic manipulation
techniques may enable work previously conducted using recombinant means to be accomplished faster,
more efficiently, or at larger scale. These techniques have not yet yielded organisms that present safetyconcerns that fall outside the current risk assessment framework used for recombinant nucleic acidresearch. Nonetheless, an appropriate risk assessment of experiments involving these techniques must
be conducted taking into account the way these approaches may alter the risk assessment. As newtechniques develop, the NIH Guidelines should be periodically reviewed to determine whether and how
such research should be explicitly addressed.
It is the responsibility of the institution and those associated with it to adhere to the intent of the NIH
Guidelines as well as to their specifics. Therefore, each institution (and the Institutional Biosafety
Committee acting on its behalf) is responsible for ensuring that all research with recombinant or synthetic
nucleic acid molecules conducted at or sponsored by that institution is conducted in compliance with the
NIH Guidelines. The following roles and responsibilities constitute an administrative framework in whichsafety is an essential and integral part of research involving recombinant or synthetic nucleic acidmolecules. Further clarifications and interpretations of roles and responsibilities will be issued by NIH as
necessary.
Section IV-B.

Responsibilities of the Institution

Section IV-B-1. General Information
Each institution conducting or sponsoring recombinant or synthetic nucleic acid molecule research whichis covered by the NIH Guidelines is responsible for ensuring that the research is conducted in full
conformity with the provisions of the NIH Guidelines. In order to fulfill this responsibility, the institution
shall:

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Section IV-B-1-a. Establish and implement policies that provide for the safe conduct of recombinant orsynthetic nucleic acid molecule research and that ensure compliance with the NIH Guidelines. As part ofits general responsibilities for implementing the NIH Guidelines, the institution may establish additional
procedures, as deemed necessary, to govern the institution and its components in the discharge of its
responsibilities under the NIH Guidelines. Such procedures may include: (i) statements formulated by
the institution for the general implementation of the NIH Guidelines, and (ii) any additional precautionary
steps the institution deems appropriate.
Section IV-B-1-b. Establish an Institutional Biosafety Committee that meets the requirements set forth in
Section IV-B-2-a and carries out the functions detailed in Section IV-B-2-b.
Section IV-B-1-c. Appoint a Biological Safety Officer (who is also a member of the Institutional Biosafety
Committee) if the institution: (i) conducts recombinant or synthetic nucleic acid molecule research at
Biosafety Level (BL) 3 or BL4, (ii) engages in large-scale (greater than 10 liters) research or (iii) conducts
any research involving gene drive modified organisms, which all must be conducted at BL2 or higher
containment. The Biological Safety Officer carries out the duties specified in Section IV-B-3.
Section IV-B-1-d. Appoint at least one individual with expertise in plant, plant pathogen, or plant pest
containment principles (who is a member of the Institutional Biosafety Committee) if the institution
conducts recombinant or synthetic nucleic acid molecule research that requires Institutional Biosafety
Committee approval in accordance with Appendix L, Physical and Biological Containment for
Recombinant or Synthetic Nucleic Acid Molecule Research Involving Plants.
Section IV-B-1-e. Appoint at least one individual with expertise in animal containment principles (who isa member of the Institutional Biosafety Committee) if the institution conducts recombinant or synthetic
nucleic acid molecule research that requires Institutional Biosafety Committee approval in accordancewith Appendix M, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid
Molecule Research Involving Animals.
Section IV-B-1-f. Ensure that when the institution participates in or sponsors recombinant or synthetic
nucleic acid molecule research involving human participants: (i) the Institutional Biosafety Committee has
adequate expertise and training (using ad hoc consultants as deemed necessary) and (ii) no human genetransfer experiment shall be initiated until Institutional Biosafety Committee approval has been obtained
and all other applicable institutional and regulatory authorization(s) and approvals have been obtained.
Institutional Biosafety Committee approval must be obtained from the clinical trial site.
Section IV-B-1-g. Assist and ensure compliance with the NIH Guidelines by Principal Investigators
conducting research at the institution as specified in Section IV-B-7.
Section IV-B-1-h. Ensure appropriate training for the Institutional Biosafety Committee Chair andmembers, Biological Safety Officer and other containment experts (when applicable), Principal
Investigators, and laboratory staff regarding laboratory safety and implementation of the NIH Guidelines.
The Institutional Biosafety Committee Chair is responsible for ensuring that Institutional Biosafety
Committee members are appropriately trained. The Principal Investigator is responsible for ensuring thatlaboratory staff are appropriately trained. The institution is responsible for ensuring that the Principal
Investigator has sufficient training; however, this responsibility may be delegated to the Institutional
Biosafety Committee.
Section IV-B-1-i. Determine the necessity for health surveillance of personnel involved in connection
with individual recombinant or synthetic nucleic acid molecule projects; and if appropriate, conduct a
health surveillance program for such projects. The institution shall establish and maintain a healthsurveillance program for personnel engaged in large-scale research or production activities involving
viable organisms containing recombinant or synthetic nucleic acid molecules which require BL3
containment at the laboratory scale. The institution shall establish and maintain a health surveillance
program for personnel engaged in animal research involving viable recombinant or synthetic nucleic acid

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molecule-containing microorganisms that require BL3 or greater containment in the laboratory. The
Laboratory Safety Monograph discusses various components of such a program (e.g., records of agentshandled, active investigation of relevant illnesses, and the maintenance of serial serum samples formonitoring serologic changes that may result from the employees' work experience). Certain medical
conditions may place a laboratory worker at increased risk in any endeavor where infectious agents arehandled. Examples cited in the Laboratory Safety Monograph include gastrointestinal disorders andtreatment with steroids, immunosuppressive drugs, or antibiotics. Workers with such disorders ortreatment should be evaluated to determine whether they should be engaged in research with potentiallyhazardous organisms during their treatment or illness. Copies of the Laboratory Safety Monograph areavailable from the Office of Science Policy, National Institutes of Health, preferably by submitting arequest for this information to: NIHGuidelines@od.nih.gov; additional contact information is also available
here and on the OSP website (www.osp.od.nih.gov).
Section IV-B-1-j. Report any significant problems, violations of the NIH Guidelines, or any significant
research-related accidents and illnesses to NIH OSP within thirty days, unless the institution determinesthat a report has already been filed by the Principal Investigator or Institutional Biosafety Committee.
Reports shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:
NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website(www.osp.od.nih.gov).
Section IV-B-2. Institutional Biosafety Committee (IBC)
The institution shall establish an Institutional Biosafety Committee whose responsibilities need not be
restricted to recombinant or synthetic nucleic acid molecule research. The Institutional Biosafety
Committee shall meet the following requirements:
Section IV-B-2-a.

Membership and Procedures

Section IV-B-2-a-(1). The Institutional Biosafety Committee must comprise no fewer than five members
so selected that they collectively have experience and expertise in recombinant or synthetic nucleic acidmolecule technology and the capability to assess the safety of recombinant or synthetic nucleic acidmolecule research and to identify any potential risk to public health or the environment. At least two
members shall not be affiliated with the institution (apart from their membership on the Institutional
Biosafety Committee) and who represent the interest of the surrounding community with respect to healthand protection of the environment (e.g., officials of state or local public health or environmental protection
agencies, members of other local governmental bodies, or persons active in medical, occupational health,or environmental concerns in the community). The Institutional Biosafety Committee shall include at least
one individual with expertise in plant, plant pathogen, or plant pest containment principles whenexperiments utilizing Appendix L, Physical and Biological Containment for Recombinant or Synthetic
Nucleic Acid Molecule Research Involving Plants, require prior approval by the Institutional Biosafety
Committee. The Institutional Biosafety Committee shall include at least one scientist with expertise inanimal containment principles when experiments utilizing Appendix M, Physical and BiologicalContainment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Animals, require
Institutional Biosafety Committee prior approval. When the institution conducts research involving gene
drive modified organisms the institution must ensure that the Institutional Biosafety Committee has
adequate expertise (e.g., specific species containment, ecological or environmental risk assessment)using ad hoc consultants if necessary. When the institution conducts recombinant or synthetic nucleic
acid molecule research at BL3, BL4, or Large Scale (greater than 10 liters) or research involving gene
drive modified organisms, a Biological Safety Officer is mandatory and shall be a member of theInstitutional Biosafety Committee (see Section IV-B-3, Biological Safety Officer). When the institution
conducts research with gene drive modified organisms, the impact on ecosystems should be assessed by
the Institutional Biosafety Committee (see Section V-N, Footnotes and References of Sections I-IV).
When the institution participates in or sponsors recombinant or synthetic nucleic acid molecule research
involving human research participants, the institution must ensure that the Institutional Biosafety
Committee has adequate expertise and training (using ad hoc consultants if necessary). Institutional
Biosafety Committee approval must be obtained from the clinical trial site.

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Note: Individuals, corporations, and institutions not otherwise covered by the NIH Guidelines, areencouraged to adhere to the standards and procedures set forth in Sections I through IV (see Section IVD, Voluntary Compliance. The policy and procedures for establishing an Institutional Biosafety
Committee under Voluntary Compliance, are specified in Section IV-D-2, Institutional Biosafety
Committee Approval).
Section IV-B-2-a-(2). In order to ensure the competence necessary to review and approve recombinantor synthetic nucleic acid molecule activities, it is recommended that the Institutional Biosafety Committee:(i) include persons with expertise in recombinant or synthetic nucleic acid molecule technology, biologicalsafety, and physical containment; (ii) include or have available as consultants persons knowledgeable ininstitutional commitments and policies, applicable law, standards of professional conduct and practice,community attitudes, and the environment, and (iii) include at least one member representing thelaboratory technical staff.
Section IV-B-2-a-(3). The institution shall file an annual report with NIH OSP which includes: (i) a rosterof all Institutional Biosafety Committee members clearly indicating the Chair, contact person, Biological
Safety Officer (if applicable), plant expert (if applicable), animal expert (if applicable), human genetherapy expertise or ad hoc consultant (if applicable); and (ii) biographical sketches of all Institutional
Biosafety Committee members (including community members).
Section IV-B-2-a-(4). No member of an Institutional Biosafety Committee may be involved (except toprovide information requested by the Institutional Biosafety Committee) in the review or approval of a
project in which he/she has been or expects to be engaged or has a direct financial interest.
Section IV-B-2-a-(5). The institution, that is ultimately responsible for the effectiveness of the Institutional
Biosafety Committee, may establish procedures that the Institutional Biosafety Committee shall follow in
its initial and continuing review and approval of applications, proposals, and activities.
Section IV-B-2-a-(6). When possible and consistent with protection of privacy and proprietary interests,
the institution is encouraged to open its Institutional Biosafety Committee meetings to the public.
Section IV-B-2-a-(7). Upon request, the institution shall make available to the public all Institutional
Biosafety Committee meeting minutes and any documents submitted to or received from funding
agencies which the latter are required to make available to the public. If public comments are made on
Institutional Biosafety Committee actions, the institution shall forward both the public comments and theInstitutional Biosafety Committee's response to the Office of Science Policy, National Institutes of Health,preferably by e-mail to: NIHGuidelines@od.nih.gov; additional contact information is also available here
and on the OSP website (www.osp.od.nih.gov).
Section IV-B-2-b.

Functions

On behalf of the institution, the Institutional Biosafety Committee is responsible for:
Section IV-B-2-b-(1). Reviewing recombinant or synthetic nucleic acid molecule research conducted ator sponsored by the institution for compliance with the NIH Guidelines as specified in Section III,
Experiments Covered by the NIH Guidelines, and approving those research projects that are found to
conform with the NIH Guidelines. This review shall include: (i) independent assessment of thecontainment levels required by the NIH Guidelines for the proposed research; (ii) assessment of the
facilities, procedures, practices, and training and expertise of personnel involved in recombinant orsynthetic nucleic acid molecule research; (iii) for recombinant or synthetic nucleic acid molecule research
involving human research participants assessment focused on biosafety issues (e.g., administration,
shedding). IBC oversight may conclude after the last participant is administered the final dose of
product. However, IBCs may choose to establish other end points for oversight, based on their biosafetyassessment of the proposed research.

Page 29 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Section IV-B-2-b-(2). Notifying the Principal Investigator of the results of the Institutional Biosafety
Committee's review and approval.
Section IV-B-2-b-(3). Lowering containment levels for certain experiments as specified in Section III-D-2a, 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 IV-B-2-b-(4). Setting containment levels as specified in Sections III-D-4-b, Experiments Involving
Whole Animals, and III-D-5, Experiments Involving Whole Plants.
Section IV-B-2-b-(5). Periodically reviewing recombinant or synthetic nucleic acid molecule research
conducted at the institution to ensure compliance with the NIH Guidelines.
Section IV-B-2-b-(6). Adopting emergency plans covering accidental spills and personnel contamination
resulting from recombinant or synthetic nucleic acid molecule research.
Note: The Laboratory Safety Monograph describes basic elements for developing specific procedures
dealing with major spills of potentially hazardous materials in the laboratory, including information andreferences about decontamination and emergency plans. The NIH and the CDC are available to provide
consultation and direct assistance, if necessary, as posted in the Laboratory Safety Monograph. The
institution shall cooperate with the state and local public health departments by reporting any significant
research-related illness or accident that may be hazardous to the public health.
Section IV-B-2-b-(7). Reporting any significant problems with or violations of the NIH Guidelines and any
significant research-related accidents or illnesses to the appropriate institutional official and NIH OSP
within 30 days, unless the Institutional Biosafety Committee determines that a report has already been
filed by the Principal Investigator. Reports to NIH OSP shall be sent to the Office of Science Policy,
National Institutes of Health, preferably by e-mail to: NIHGuidelines@od.nih.gov; additional contact
information is also available here and on the OSP website (www.osp.od.nih.gov).
Section IV-B-2-b-(8). The Institutional Biosafety Committee may not authorize initiation of experiments
which are not explicitly covered by the NIH Guidelines until NIH establishes the containment requirement.
Section IV-B-2-b-(9). Performing such other functions as may be delegated to the Institutional Biosafety
Committee under Section IV-B-2, Institutional Biosafety Committee.
Section IV-B-3. Biological Safety Officer (BSO)
Section IV-B-3-a. The institution shall appoint a Biological Safety Officer if it engages in large-scale
research or production activities involving viable organisms containing recombinant or synthetic nucleic
acid molecules. The Biological Safety Officer shall be a member of the Institutional Biosafety Committee.
Section IV-B-3-b. The institution shall appoint a Biological Safety Officer if it engages in recombinant orsynthetic nucleic acid molecule research at BL3 or BL4. The Biological Safety Officer shall be a memberof the Institutional Biosafety Committee.
Section IV-B-3-c. The institution shall appoint a Biological Safety Officer if it engages in recombinant orsynthetic nucleic acid molecule research that involves gene drive modified organisms. The Biological
Safety Officer shall be a member of the Institutional Biosafety Committee.
Section IV-B-3-d. The Biological Safety Officer's duties include, but are not limited to:
Section IV-B-3-d-(1). Periodic inspections to ensure that laboratory standards are rigorously followed;
Section IV-B-3-d-(2). Reporting to the Institutional Biosafety Committee and the institution any significant
problems, violations of the NIH Guidelines, and any significant research-related accidents or illnesses ofPage 30 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

which the Biological Safety Officer becomes aware unless the Biological Safety Officer determines that areport has already been filed by the Principal Investigator;
Section IV-B-3-d-(3). Developing emergency plans for handling accidental spills and personnel
contamination and investigating laboratory accidents involving recombinant or synthetic nucleic acidmolecule research;
Section IV-B-3-d-(4). Providing advice on laboratory security;
Section IV-B-3-d-(5). Providing technical advice to Principal Investigators and the Institutional Biosafety
Committee on research safety procedures.
Note: See the Laboratory Safety Monograph for additional information on the duties of the Biological
Safety Officer.
Section IV-B-4. Plant, Plant Pathogen, or Plant Pest Containment Expert
When the institution conducts recombinant or synthetic nucleic acid molecule research that requires
Institutional Biosafety Committee approval in accordance with Appendix L, Physical and BiologicalContainment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Plants, theinstitution shall appoint at least one individual with expertise in plant, plant pathogen, or plant pest
containment principles (who is a member of the Institutional Biosafety Committee).
Section IV-B-5. Animal Containment Expert
When the institution conducts recombinant or synthetic nucleic acid molecule research that requires
Institutional Biosafety Committee approval in accordance with Appendix M, Physical and BiologicalContainment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Animals, theinstitution shall appoint at least one individual with expertise in animal containment principles (who is amember of the Institutional Biosafety Committee).
Section IV-B-6. Human Gene Therapy Expertise
When the institution participates in or sponsors recombinant or synthetic nucleic acid molecule research
involving human subjects, the institution must ensure that the Institutional Biosafety Committee has
adequate expertise and training (using ad hoc consultants as deemed necessary).
Section IV-B-7. Principal Investigator (PI)On behalf of the institution, the Principal Investigator is responsible for full compliance with the NIH
Guidelines in the conduct of recombinant or synthetic nucleic acid molecule research.
Section IV-B-7-a.

General Responsibilities

As part of this general responsibility, the Principal Investigator shall:
Section IV-B-7-a-(1). Initiate or modify no recombinant or synthetic nucleic acid molecule research which
requires Institutional Biosafety Committee approval prior to initiation (see Sections III-A, III-B, III-C, III-D,
and III-E, Experiments Covered by the NIH Guidelines) until that research or the proposed modification
thereof has been approved by the Institutional Biosafety Committee and has met all other requirements of
the NIH Guidelines;
Section IV-B-7-a-(2). Determine whether experiments are covered by Section III-E, Experiments that
Require Institutional Biosafety Committee Notice Simultaneous with Initiation, and ensure that theappropriate procedures are followed;

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Section IV-B-7-a-(3). Report any significant problems, violations of the NIH Guidelines, or any significant
research-related accidents and illnesses to the Biological Safety Officer (where applicable),
Greenhouse/Animal Facility Director (where applicable), Institutional Biosafety Committee, NIH OSP, andother appropriate authorities (if applicable) within 30 days. Reports to NIH OSP shall be sent to the Officeof Science Policy, National Institutes of Health, preferably by e-mail to: NIHGuidelines@od.nih.gov;
additional contact information is also available here and on the OSP website (www.osp.od.nih.gov).
Section IV-B-7-a-(4). Report any new information bearing on the NIH Guidelines to the Institutional
Biosafety Committee and to NIH OSP (reports to NIH OSP shall be sent to the Office of Science Policy,
National Institutes of Health, preferably by e-mail to: NIHGuidelines@od.nih.gov; additional contact
information is also available here and on the OSP website (www.osp.od.nih.gov).
Section IV-B-7-a-(5). Be adequately trained in good microbiological techniques;
Section IV-B-7-a-(6). Adhere to Institutional Biosafety Committee approved emergency plans forhandling accidental spills and personnel contamination; andSection IV-B-7-a-(7). Comply with shipping requirements for recombinant or synthetic nucleic acidmolecules (see Appendix H, Shipment, for shipping requirements and the Laboratory Safety Monographfor technical recommendations).
Section IV-B-7-b.

Information to Be Submitted by the Principal Investigator to NIH OSP

The Principal Investigator shall:
Section IV-B-7-b-(1). Submit information to NIH OSP for certification of new host-vector systems;
Section IV-B-7-b-(2). Petition NIH OSP, with notice to the Institutional Biosafety Committee, for
proposed exemptions to the NIH Guidelines;
Section IV-B-7-b-(3). Petition NIH OSP, with concurrence of the Institutional Biosafety Committee, forapproval to conduct experiments specified in Sections III-A-1, Major Actions Under the NIH Guidelines,
and III-B, Experiments that Require NIH OSP and Institutional Biosafety Committee Approval Before
Initiation;
Section IV-B-7-b-(4). Petition NIH OSP for determination of containment for experiments requiring caseby-case review; andSection IV-B-7-b-(5). Petition NIH OSP for determination of containment for experiments not covered by
the NIH Guidelines.
Section IV-B-7-c.
Committee

Submissions by the Principal Investigator to the Institutional Biosafety

The Principal Investigator shall:
Section IV-B-7-c-(1). Make an initial determination of the required levels of physical and biologicalcontainment in accordance with the NIH Guidelines;
Section IV-B-7-c-(2). Select appropriate microbiological practices and laboratory techniques to be used
for the research;
Section IV-B-7-c-(3). Submit the initial research protocol and any subsequent changes (e.g., changes inthe source of DNA or host-vector system), if covered under Sections III-A, III-B, III-C, III-D, or III-E(Experiments Covered by the NIH Guidelines), to the Institutional Biosafety Committee for review andPage 32 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

approval or disapproval; andSection IV-B-7-c-(4). Remain in communication with the Institutional Biosafety Committee throughout
the conduct of the project.
Section IV-B-7-d.

Responsibilities of the Principal Investigator Prior to Initiating Research

The Principal Investigator shall:
Section IV-B-7-d-(1). Make available to all laboratory staff the protocols that describe the potential
biohazards and the precautions to be taken;
Section IV-B-7-d-(2). Instruct and train laboratory staff in: (i) the practices and techniques required toensure safety, and (ii) the procedures for dealing with accidents; andSection IV-B-7-d-(3). Inform the laboratory staff of the reasons and provisions for any precautionary
medical practices advised or requested (e.g., vaccinations or serum collection).
Section IV-B-7-e.
Research

Responsibilities of the Principal Investigator During the Conduct of the

The Principal Investigator shall:
Section IV-B-7-e-(1). Supervise the safety performance of the laboratory staff to ensure that the required
safety practices and techniques are employed;
Section IV-B-7-e-(2). Investigate and report any significant problems pertaining to the operation andimplementation of containment practices and procedures in writing to the Biological Safety Officer (whereapplicable), Greenhouse/Animal Facility Director (where applicable), Institutional Biosafety Committee,
NIH OSP, and other appropriate authorities (if applicable) (reports to NIH OSP shall be sent to the Officeof Science Policy, National Institutes of Health, preferably by e-mail to: NIHGuidelines@od.nih.gov;
additional contact information is also available here and on the OSP website (www.osp.od.nih.gov).
Section IV-B-7-e-(3). Correct work errors and conditions that may result in the release of recombinant orsynthetic nucleic acid molecule materials; andSection IV-B-7-e-(4). Ensure the integrity of the physical containment (e.g., biological safety cabinets)
and the biological containment (e.g., purity and genotypic and phenotypic characteristics).
Section IV-C.

Responsibilities of the National Institutes of Health (NIH)

Section IV-C-1. NIH Director
The NIH Director is responsible for: (i) establishing the NIH Guidelines, (ii) overseeing theirimplementation, and (iii) their final interpretation. The NIH Director has responsibilities under the NIH
Guidelines that involve OSP. OSP's responsibilities under the NIH Guidelines are administrative. In
certain circumstances, there is specific opportunity for public comment with published response prior to
final action.
Section IV-C-1-a.

General Responsibilities

The NIH Director is responsible for:
Section IV-C-1-a-(1). Promulgating requirements as necessary to implement the NIH Guidelines;
Section IV-C-1-a-(2). Establishing and maintaining NIH OSP to carry out the responsibilities defined inPage 33 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Section IV-C-2, Office of Science Policy;
Section IV-C-1-a-(3). Conducting and supporting training programs in laboratory safety for Institutional
Biosafety Committee members, Biological Safety Officers and other institutional experts (if applicable),
Principal Investigators, and laboratory staff.
Section IV-C-1-b.

Specific Responsibilities

In carrying out the responsibilities set forth in this section, the NIH Director, or a designee shall weigheach proposed action through appropriate analysis and consultation to determine whether it complies with
the NIH Guidelines and presents no significant risk to health or the environment.
Section IV-C-1-b-(1).

Major Actions

To execute Major Actions, the NIH Director shall provide an opportunity for public and Federal agency
comment. The NIH Director's decision/recommendation (at his/her discretion) may be published in theFederal Register for a minimum of 15 days of comment before final action is taken. The NIH Director's
final decision/recommendation, along with responses to public comments, shall be published in theFederal Register. Institutional Biosafety Committee Chairs shall be notified of the following decisions:
Section IV-C-1-b-(1)-(a). Changing containment levels for types of experiments that are specified in the
NIH Guidelines when a Major Action is involved;
Section IV-C-1-b-(1)-(b). Assigning containment levels for types of experiments that are not explicitly
considered in the NIH Guidelines when a Major Action is involved;
Section IV-C-1-b-(1)-(c). Promulgating and amending a list of classes of recombinant or synthetic
nucleic acid molecules to be exempt from the NIH Guidelines because they consist entirely of DNA
segments from species that exchange DNA by known physiological processes or otherwise do notpresent a significant risk to health or the environment;
Section IV-C-1-b-(1)-(d). Permitting experiments specified by Section III-A, Experiments that Require
NIH Director Approval and Institutional Biosafety Committee Approval Before Initiation;
Section IV-C-1-b-(1)-(e). Certifying new host-vector systems with the exception of minor modifications(e.g., those of minimal or no consequence to the properties relevant to containment) of already certified
systems (the standards and procedures for certification are described in Appendix I-II, Certification of
Host-Vector Systems); andSection IV-C-1-b-(1)-(f). Adopting other changes in the NIH Guidelines.
Section IV-C-1-b-(2).

Minor Actions

NIH OSP shall carry out certain functions as delegated to it by the NIH Director (see Section IV-C-2,
Office of Science Policy). Minor Actions will be transmitted to Institutional Biosafety Committee Chairs:
Section IV-C-1-b-(2)-(a). Changing containment levels for experiments that are specified in Section III,
Experiments Covered by the NIH Guidelines (except when a Major Action is involved);
Section IV-C-1-b-(2)-(b). Assigning containment levels for experiments not explicitly considered in the
NIH Guidelines;
Section IV-C-1-b-(2)-(c). Revising the Classification of Etiologic Agents for the purpose of these NIH
Guidelines (see Section V-A, Footnotes and References of Sections I-IV).
Section IV-C-1-b-(2)-(d). Interpreting the NIH Guidelines for experiments to which the NIH Guidelines doPage 34 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

not specifically assign containment levels;
Section IV-C-1-b-(2)-(e). Setting containment under Sections III-D-1-d, Experiments Using Risk Group 2,
Risk Group 3, Risk Group 4, or Restricted Agents as Host-Vector Systems, and III-D-2-b, Experiments inwhich 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 IV-C-1-b-(2)-(f). Approving minor modifications of already certified host-vector systems (thestandards and procedures for such modifications are described in Appendix I-II, Certification of HostVector Systems);
Section IV-C-1-b-(2)-(g). Decertifying already certified host-vector systems;
Section IV-C-1-b-(2)-(h). Adding new entries to the list of molecules toxic for vertebrates (see Appendix
F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for
Vertebrates); andSection IV-C-1-b-(2)-(i). Determining appropriate containment conditions for experiments according tocase precedents developed under Section IV-C-1-b-(2)-(c).
Section IV-C-2. Office of Science Policy (OSP)
OSP shall serve as a focal point for information on recombinant or synthetic nucleic acid molecule
activities and provide advice to all within and outside NIH including institutions, Biological Safety Officers,
Principal Investigators, Federal agencies, state and local governments, and institutions in the private
sector. OSP shall carry out such other functions as may be delegated to it by the NIH Director. OSP's
responsibilities include (but are not limited to) the following:
Section IV-C-2-a. Reviewing and approving experiments in conjunction with ad hoc experts involving thecloning of genes encoding for toxin molecules that are lethal for vertebrates at an LD50 of less than orequal to 100 nanograms per kilogram body weight in organisms other than Escherichia coli K-12 (seeSection III-B-1, Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than 100
Nanograms Per Kilogram Body Weight, Appendix F, Containment Conditions for Cloning of Genes
Coding for the Biosynthesis of Molecules Toxic for Vertebrates);
Section IV-C-2-b. Publishing in the Federal Register, as needed.
Section IV-C-2-c. Reviewing and approving the membership of an institution's Institutional Biosafety
Committee, and where it finds the Institutional Biosafety Committee meets the requirements set forth in
Section IV-B-2, Institutional Biosafety Committee (IBC), giving its approval to the Institutional Biosafety
Committee membership.
Section IV-C-3. Other NIH Components
Other NIH components shall be responsible for certifying maximum containment (BL4) facilities,
inspecting them periodically, and inspecting other recombinant or synthetic nucleic acid molecule facilities
as deemed necessary.
Section IV-D.

Voluntary Compliance

Section IV-D-1. Basic Policy - Voluntary ComplianceIndividuals, corporations, and institutions not otherwise covered by the NIH Guidelines are encouraged tofollow the standards and procedures set forth in Sections I through IV. In order to simplify discussion,references hereafter to “institutions” are intended to encompass corporations and individuals who have noorganizational affiliation. For purposes of complying with the NIH Guidelines, an individual intending toPage 35 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

carry out research involving recombinant or synthetic nucleic acid molecules is encouraged to affiliate
with an institution that has an Institutional Biosafety Committee approved under the NIH Guidelines.
Since commercial organizations have special concerns, such as protection of proprietary data, somemodifications and explanations of the procedures are provided in Sections IV-D-2 through IV-D-5-b,
Voluntary Compliance, in order to address these concerns.
Section IV-D-2. Institutional Biosafety Committee Approval - Voluntary Compliance
It should be emphasized that employment of an Institutional Biosafety Committee member solely for
purposes of membership on the Institutional Biosafety Committee does not itself make the member an
institutionally affiliated member. Except for the unaffiliated members, a member of an Institutional
Biosafety Committee for an institution not otherwise covered by the NIH Guidelines may participate in thereview and approval of a project in which the member has a direct financial interest so long as themember has not been, and does not expect to be, engaged in the project. Section IV-B-2-a-(4),Institutional Biosafety Committee, is modified to that extent for purposes of these institutions.
Section IV-D-3. Certification of Host-Vector Systems - Voluntary Compliance
A host-vector system may be proposed for certification by the NIH Director in accordance with theprocedures set forth in Appendix I-II, Certification of Host-Vector Systems. In order to ensure protection
for proprietary data, any public notice regarding a host-vector system which is designated by theinstitution as proprietary under Section IV-D, Voluntary Compliance, will be issued only after consultation
with the institution as to the content of the notice.
Section IV-D-4. Requests for Exemptions and Approvals - Voluntary Compliance
Requests for exemptions or other approvals as required by the NIH Guidelines should be submitted
based on the procedures set forth in Sections I through IV. In order to ensure protection for proprietarydata, any public notice regarding a request for an exemption or other approval which is designated by theinstitution as proprietary under Section IV-D-5-a, Voluntary Compliance, will be issued only after
consultation with the institution as to the content of the notice.
Section IV-D-5. Protection of Proprietary Data - Voluntary Compliance
Section IV-D-5-a.

GeneralIn general, the Freedom of Information Act requires Federal agencies to make their records available tothe public upon request. However, this requirement does not apply to, among other things, “trade secrets
and commercial or financial information that is obtained from a person and that is privileged orconfidential.” Under 18 U.S.C. 1905, it is a criminal offense for an officer or employee of the U.S. or any
Federal department or agency to publish, divulge, disclose, or make known “in any manner or to any
extent not authorized by law any information coming to him in the course of his employment or official
duties or by reason of any examination or investigation made by, or return, report or record made to or
filed with, such department or agency or officer or employee thereof, which information concerns or
relates to the trade secrets, (or) processes...of any person, firm, partnership, corporation, or association.”
This provision applies to all employees of the Federal Government, including special Government
employees.
In submitting to NIH for purposes of voluntary compliance with the NIH Guidelines, an institution may
designate those items of information which the institution believes constitute trade secrets, privileged,
confidential, commercial, or financial information. If NIH receives a request under the Freedom ofInformation Act for information so designated, NIH will promptly contact the institution to secure its views
as to whether the information (or some portion) should be released. If NIH decides to release thisinformation (or some portion) in response to a Freedom of Information request or otherwise, the institution
will be advised and the actual release will be delayed in accordance with 45 Code of Federal Regulations,Page 36 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Section 5.65(d) and (e).
Section IV-D-5-b.

Pre-submission Review

Any institution not otherwise covered by the NIH Guidelines, which is considering submission of data or
information voluntarily to NIH, may request pre-submission review of the records involved to determine if
NIH will make all or part of the records available upon request under the Freedom of Information Act.
A request for pre-submission review should be submitted to NIH OSP along with the records involved to
the Office of Science Policy, National Institutes of Health, preferably by e-mail to:
NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website(www.osp.od.nih.gov). These records shall be clearly marked as being the property of the institution onloan to NIH solely for the purpose of making a determination under the Freedom of Information Act. NIH
OSP will seek a determination from the responsible official under DHHS regulations (45 CFR Part 5) as towhether the records involved, (or some portion) will be made available to members of the public under theFreedom of Information Act. Pending such a determination, the records will be kept separate from NIH
OSP files, will be considered records of the institution and not NIH OSP, and will not be received as partof NIH OSP files. No copies will be made of such records.
NIH OSP will inform the institution of the NIH Freedom of Information Officer's determination and follow
the institution's instructions as to whether some or all of the records involved are to be returned to theinstitution or to become a part of NIH OSP files. If the institution instructs NIH OSP to return the records,
no copies or summaries of the records will be made or retained by DHHS, NIH, or OSP. The NIH
Freedom of Information Officer's determination will represent that official's judgment at the time of thedetermination as to whether the records involved (or some portion) would be exempt from disclosure
under the Freedom of Information Act if at the time of the determination the records were in NIH OSP files
and a request was received for such files under the Freedom of Information Act.
***********************************************************************************************************************SECTION V.

FOOTNOTES AND REFERENCES OF SECTIONS I THROUGH IV

Section V-A. The NIH Director may revise the classification for the purposes of the NIH Guidelines (seeSection IV-C-1-b-(2)-(c), Minor Actions). The revised list of organisms in each Risk Group is reprinted inAppendix B, Classification of Human Etiologic Agents on the Basis of Hazard.
Section V-B. Section III, Experiments Covered by the NIH Guidelines, describes a number of places
where judgments are to be made. In all these cases, the Principal Investigator shall make the judgmenton these matters as part of his/her responsibility to "make the initial determination of the required levels ofphysical and biological containment in accordance with the NIH Guidelines" (see Section IV-B-7-c-(1)).
For cases falling under Sections III-A through III-E, Experiments Covered by the NIH Guidelines, thisjudgment is to be reviewed and approved by the Institutional Biosafety Committee as part of its
responsibility to make an "independent assessment of the containment levels required by the NIH
Guidelines for the proposed research" (see Section IV-B-2-b-(1), Institutional Biosafety Committee). The
Institutional Biosafety Committee may refer specific cases to NIH OSP as part of NIH OSP's functions to"provide advice to all within and outside NIH" (see Section IV-C-2).
Section V-C. U.S. Department of Health and Human Services, Public Health Service, Centers for
Disease Control and Prevention and the National Institutes of Health. Biosafety in Microbiological andBiomedical Laboratories. Copies are available from: Superintendent of Documents, U.S. Government
Printing Office, Washington, D.C. 20401-0001, Phone (202) 512-1800 [http://www.gpo.gov/].
Section V-D. Classification of Etiologic Agents on the Basis of Hazard, 4th Edition, July 1974, U.S.
Department of Health, Education, and Welfare, Public Health Service, Centers for Disease Control, Officeof Biosafety, Atlanta, Georgia 30333.

Page 37 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Section V-E. Chin, James ed., Control of Communicable Diseases Manual, 17th Edition, 2000. ISBN:087553-242-X, American Public Health Association, 800 I Street, N.W., Washington, D.C. Phone: (202)
777-2742.
Section V-F. World Health Organization Laboratory Biosafety Manual, 2nd edition. 1993. WHO Albany,
NY. Copies are available from: WHO Publication Centre, USA, (Q Corp) 49 Sheridan Avenue, Albany,
New York 12210; Phone: (518) 436-9686 (Order # 1152213).
Section V-G. A U.S. Department of Agriculture permit, required for import and interstate transport ofplant and animal pathogens, may be obtained from the U.S. Department of Agriculture, ATTN: Animal
and Plant Health Inspection Service (APHIS), Veterinary Services, National Center for Import-Export,
Products Program, 4700 River Road, Unit 40, Riverdale, Maryland 20737. Phone: (301) 734-8499; Fax:
(301) 734-8226.
Section V-H. American Type Culture Collection Catalogues of plant viruses, animal viruses, cells,
bacteria, fungi, etc. are available from American Type Culture Collection, 10801 University Boulevard,
Manassas, VA 20110-2209. Phone: (703) 365-2700.
Section V-I. U.S. Department of Labor, Occupational Safety and Health Administration, 29 CFR
1910.1030, Bloodborne Pathogens. See also, Exposure to Bloodborne Pathogens, OSHA 3127, 1996
(Revised).
Section V-J. As classified in the Virus Taxonomy: The Classification and Nomenclature of Viruses. The
Seventh Report of the International Committee on Taxonomy of Viruses, Academic Press, 2000
(0123702003) San Diego, CA.
Section V-K. i.e., the total of all genomes within a family shall not exceed two-thirds of the genome.
Section V-L. Organisms including alastrim, smallpox (variola) and whitepox may not be studied in the
United States except at specified facilities. All activities, including storage of variola and whitepox, arerestricted to the single national facility (World Health Organization Collaborating Center for Smallpox
Research, Centers for Disease Control and Prevention, Atlanta, Georgia).
Section V-M. In accordance with accepted scientific and regulatory practices of the discipline of plant
pathology, an exotic plant pathogen (e.g., virus, bacteria, or fungus) is one that is unknown to occur within
the U.S. (see Section V-G, Footnotes and References of Sections I-IV). Determination of whether apathogen has a potential for serious detrimental impact on managed (agricultural, forest, grassland) or
natural ecosystems should be made by the Principal Investigator and the Institutional Biosafety
Committee, in consultation with scientists knowledgeable of plant diseases, crops, and ecosystems in thegeographic area of the research.
Section V-N Determination of whether a gene drive modified organism has a potential for serious
detrimental impact on managed (agricultural, forest, grassland) or natural ecosystems should be made by
the Principal Investigator and the Institutional Biosafety Committee, in consultation with scientists
knowledgeable of gene drive technology, and of the environment, and ecosystems in the geographic areaof the research.
***********************************************************************************************************************
APPENDIX A.

EXEMPTIONS UNDER SECTION III-F-6--SUBLISTS OF NATURAL EXCHANGERS

Certain specified recombinant or synthetic nucleic acid molecules that consist entirely of DNA segmentsfrom different species that exchange DNA by known physiological processes, though one or more of thesegments may be a synthetic equivalent are exempt from these NIH Guidelines (see Section III-F-6,

Page 38 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

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 after
appropriate notice and opportunity for public comment (see Section IV-C-1-b-(1)-(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-F-6 Sublists of Natural Exchangers. Section
III-F-6, Exempt Experiments, describes recombinant or synthetic nucleic acid molecules that are: (1)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 Bergey’s Manual of Systematic Bacteriology; 2ndedition, Springer-Verlag; New York, NY). Although these experiments are exempt, it is recommended
that they be performed at the appropriate biosafety level for the host or recombinant/synthetic organism(see Biosafety in Microbiological and Biomedical Laboratories, U.S. DHHS, Public Health Service,
Centers for Disease Control and Prevention, and National Institutes of Health).
Appendix A-I.

Sublist A

Genus Escherichia
Genus Shigella
Genus Salmonella - including Arizona
Genus Enterobacter
Genus Citrobacter - including Levinea
Genus Klebsiella - including oxytoca
Genus Erwinia
Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas
mendocina
Serratia marcescens
Yersinia enterocolitica
Appendix A-II. Sublist B
Bacillus subtilis
Bacillus licheniformis
Bacillus pumilus
Bacillus globigii
Bacillus niger
Bacillus nato
Bacillus amyloliquefaciens
Bacillus aterrimus
Appendix A-III. Sublist C
Streptomyces aureofaciens
Streptomyces rimosus
Streptomyces coelicolor
Appendix A-IV. Sublist D
Streptomyces griseus
Streptomyces cyaneus
Streptomyces venezuelae

Page 39 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Appendix A-V. Sublist E
One way transfer of Streptococcus mutans or Streptococcus lactis DNA into Streptococcus sanguis
Appendix A-VI. Sublist F
Streptococcus sanguis
Streptococcus pneumoniae
Streptococcus faecalis
Streptococcus pyogenes
Streptococcus mutans***********************************************************************************************************************
APPENDIX B.

CLASSIFICATION OF HUMAN ETIOLOGIC AGENTS ON THE BASIS OF HAZARD

This appendix includes those biological agents known to infect humans as well as selected animal agentsthat may pose theoretical risks if inoculated into humans. Included are lists of representative genera andspecies known to be pathogenic; mutated, recombined, and non-pathogenic species and strains are notconsidered. Non-infectious life cycle stages of parasites are excluded.
This appendix reflects the current state of knowledge and should be considered a resource document.
Included are the more commonly encountered agents and is not meant to be all-inclusive. Information on
agent risk assessment may be found in the Agent Summary Statements of the CDC/NIH publication,
Biosafety in Microbiological and Biomedical Laboratories (see Sections V-C, V-D, V-E, and V-F,
Footnotes and References of Sections I through IV. Further guidance on agents not listed in Appendix B
may be obtained through: Centers for Disease Control and Prevention, Biosafety Branch, Atlanta,
Georgia 30333, Phone: (404) 639-3883, Fax: (404) 639-2294; National Institutes of Health, Division of
Safety, Bethesda, Maryland 20892, Phone: (301) 496-1357; Biosafety Manager, National Animal
Disease Center, U.S. Department of Agriculture - ARS, Ames, Iowa 50010, Phone: (515) 337-7772.
Appendix B - Table 1.

Basis for the Classification of Biohazardous Agents by Risk Group (RG)

Risk Group 1 (RG1)

Agents that are not associated with disease in healthy adult humans

Risk Group 2 (RG2)Agents that are associated with human disease which is rarely serious and forwhich preventive or therapeutic interventions are often available

Risk Group 3 (RG3)Agents that are associated with serious or lethal human disease for which
preventive or therapeutic interventions may be available (high individual risk
but low community risk)

Risk Group 4 (RG4)Agents that are likely to cause serious or lethal human disease for which
preventive or therapeutic interventions are not usually available (high individual
risk and high community risk)

Appendix B-I.

Risk Group 1 (RG1) Agents

RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents include
asporogenic Bacillus subtilis or Bacillus licheniformis (see Appendix C-IV-A, Bacillus subtilis or Bacillus
licheniformis Host-Vector Systems, Exceptions); adeno- associated virus (AAV – all serotypes); andrecombinant or synthetic AAV constructs, in which the transgene does not encode either a potentiallyPage 40 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus. A strainof Escherichia coli (see Appendix C-II-A, Escherichia coli K-12 Host Vector Systems, Exceptions) is an
RG1 agent if it (1) does not possess a complete lipopolysaccharide (i.e., lacks the O antigen); and (2)does not carry any active virulence factor (e.g., toxins) or colonization factors and does not carry any
genes encoding these factors.
Those agents not listed in Risk Groups (RGs) 2, 3 and 4 are not automatically or implicitly classified in
RG1; a risk assessment must be conducted based on the known and potential properties of the agentsand their relationship to agents that are listed.
Appendix B-II. Risk Group 2 (RG2) Agents
RG2 agents are associated with human disease which is rarely serious and for which preventive ortherapeutic interventions are often available.
Appendix B-II-A.

Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia--Acinetobacter baumannii (formerly Acinetobacter calcoaceticus)--Actinobacillus--Actinomyces pyogenes (formerly Corynebacterium pyogenes)--Aeromonas hydrophila--Amycolata autotrophica--Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum)--Arizona hinshawii - all serotypes--Bacillus anthracis--Bartonella henselae, B. quintana, B. vinsonii--Bordetella including B. pertussis--Borrelia recurrentis, B. burgdorferi--Burkholderia (formerly Pseudomonas species) except those listed in Appendix B-III-A (RG3))--Campylobacter coli, C. fetus, C. jejuni--Chlamydia psittaci, C. trachomatis, C. pneumoniae--Clostridium botulinum, C. chauvoei, C. haemolyticum, C. histolyticum, C. novyi, C. septicum, C. tetani--Coxiella burnetii – specifically the Phase II, Nine Mile strain, plaque purified, clone 4--Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale--Dermatophilus congolensis--Edwardsiella tarda--Erysipelothrix rhusiopathiae--Escherichia coli - all enteropathogenic, enterotoxigenic, enteroinvasive and strains bearing K1 antigen,including E. coli O157:H7--*Francisella tularensis specifically *F. tularensis subspecies novicida [aka F. novicida], strain Utah 112;
*F. tularensis subspecies holarctica LVS; *F. tularensis biovar tularensis strain ATCC 6223 (aka strain
B38)*For research involving high concentrations, BL3 practices should be considered (see Appendix G-IIC-2. Special Practices (BL3)).
--Haemophilus ducreyi, H. influenzae--Helicobacter pylori--Klebsiella - all species except K. oxytoca (RG1)--Legionella including L. pneumophila--Leptospira interrogans - all serotypes--Listeria--Moraxella--Mycobacterium (except those listed in Appendix B-III-A (RG3)) including M. avium complex, M.
asiaticum, M. bovis BCG vaccine strain, M. chelonae, M. fortuitum, M. kansasii, M. leprae, M.
malmoense, M. marinum, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans,
M. xenopi--Mycoplasma, except M. mycoides and M. agalactiae which are restricted animal pathogens

Page 41 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)--Neisseria gonorrhoeae, N. meningitidis--Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. transvalensis--Pseudomonas aeruginosa--Rhodococcus equi--Salmonella including S. arizonae, S. choleraesuis, S. enteritidis, S. gallinarum-pullorum, S. meleagridis,
S. paratyphi, A, B, C, S. typhi, S. typhimurium--Shigella including S. boydii, S. dysenteriae, type 1, S. flexneri, S. sonnei--Sphaerophorus necrophorus--Staphylococcus aureus--Streptobacillus moniliformis--Streptococcus including S. pneumoniae, S. pyogenes--Treponema pallidum, T. carateum--Vibrio cholerae, V. parahaemolyticus, V. vulnificus--Yersinia enterocolitica--Yersinia pestis specifically pgm(–) strains (lacking the 102 kb pigmentation locus) and lcr(–) strains(lacking the LCR plasmid)
Appendix B-II-B.

Risk Group 2 (RG2) - Fungal Agents--Blastomyces dermatitidis--Cladosporium bantianum, C. (Xylohypha) trichoides--Cryptococcus neoformans--Dactylaria galopava (Ochroconis gallopavum)--Epidermophyton--Exophiala (Wangiella) dermatitidis--Fonsecaea pedrosoi--Microsporum--Paracoccidioides braziliensis--Penicillium marneffei--Sporothrix schenckii--Trichophyton
Appendix B-II-C.

Risk Group 2 (RG2) - Parasitic Agents--Ancylostoma human hookworms including A. duodenale, A. ceylanicum--Ascaris including Ascaris lumbricoides suum--Babesia including B. divergens, B. microti--Brugia filaria worms including B. malayi, B. timori--Coccidia--Cryptosporidium including C. parvum--Cysticercus cellulosae (hydatid cyst, larva of T. solium)--Echinococcus including E. granulosis, E. multilocularis, E. vogeli--Entamoeba histolytica--Enterobius--Fasciola including F. gigantica, F. hepatica--Giardia including G. lamblia--Heterophyes--Hymenolepis including H. diminuta, H. nana--Isospora--Leishmania including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana, L. peruviana, L.
tropica--Loa loa filaria worms--Microsporidium--Naegleria fowleri--Necator human hookworms including N. americanusPage 42 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)--Onchocerca filaria worms including, O. volvulus--Plasmodium including simian species, P. cynomolgi, P. falciparum, P. malariae, P. ovale, P. vivax--Sarcocystis including S. sui hominis--Schistosoma including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi--Strongyloides including S. stercoralis--Taenia solium--Toxocara including T. canis--Toxoplasma including T. gondii--Trichinella spiralis--Trypanosoma including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cruzi--Wuchereria bancrofti filaria worms
Appendix B-II-D.

Risk Group 2 (RG2) - Viruses

Adenoviruses, human - all types
Alphaviruses (Togaviruses) - Group A Arboviruses--Chikungunya vaccine strain 181/25--Eastern equine encephalomyelitis virus--Venezuelan equine encephalomyelitis vaccine strains TC-83 and V3526--Western equine encephalomyelitis virus
Arenaviruses--Junin virus candid #1 vaccine strain--Lymphocytic choriomeningitis virus (non-neurotropic strains)--Tacaribe virus complex--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections
I through IV)
Bunyaviruses--Bunyamwera virus--Rift Valley fever virus vaccine strain MP-12--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections
I through IV)
Caliciviruses
Coronaviruses
Flaviviruses - Group B Arboviruses--Dengue virus serotypes 1, 2, 3, and 4--Japanese encephalitis virus strain SA 14-14-2--Saint Louis encephalitis virus--West Nile virus--Yellow fever virus vaccine strain 17D--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections
I through IV)
Hepatitis A, B, C, D, and E viruses
Herpesviruses - except Herpesvirus simiae (Monkey B virus) (see Appendix B-IV-D, Risk Group 4 (RG4) Viral Agents)--Cytomegalovirus--Epstein Barr virus--Herpes simplex types 1 and 2--Herpes zosterPage 43 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)--Human herpesvirus types 6 and 7
Orthomyxoviruses--Influenza viruses types A, B, and C (except those listed in Appendix B-III-D, Risk Group 3 (RG3) Viruses and Prions)--Tick-borne orthomyxoviruses
Papilloma viruses--All human papilloma viruses
Paramyxoviruses--Newcastle disease virus--Measles virus--Mumps virus--Parainfluenza viruses types 1, 2, 3, and 4--Respiratory syncytial virus
Parvoviruses--Human parvovirus (B19)
Picornaviruses--Coxsackie viruses types A and B--Echoviruses - all types--Polioviruses - all types, wild and attenuated--Rhinoviruses - all types
Poxviruses - all types except Monkeypox virus (see Appendix B-III-D, Risk Group 3 (RG3) - Viruses andPrions) and restricted poxviruses including Alastrim, Smallpox, and Whitepox (see Section V-L, Footnotes
and References of Sections I through IV)
Reoviruses - all types including Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus)
Rhabdoviruses--Rabies virus - all strains--Vesicular stomatitis virus non exotic strains: VSV-Indiana 1 serotype strains (e.g. Glasgow, MuddSummers, Orsay, San Juan) and VSV-New Jersey serotype strains (e.g. Ogden, Hazelhurst)
Rubivirus (Togaviruses)--Rubella virus
Appendix B-III. Risk Group 3 (RG3) Agents
RG3 agents are associated with serious or lethal human disease for which preventive or therapeuticinterventions may be available.
Appendix B-III-A.

Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia--Bartonella--Brucella including B. abortus, B. canis, B. suis--Burkholderia (Pseudomonas) mallei, B. pseudomallei--Coxiella burnetii (except the Phase II, Nine Mile strain listed in Appendix B-II-A, Risk Group 2 (RG2) Bacterial Agents Including Chlamydia)--Francisella tularensis (except those strains listed in Appendix B-II-A, Risk Group 2 (RG2) - Bacterial
Agents Including Chlamydia)--Mycobacterium bovis (except BCG strain, see Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents
Including Chlamydia), M. tuberculosisPage 44 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)--Orientia tsutsugamushi (was R. tsutsugamushi)--Pasteurella multocida type B -"buffalo" and other virulent strains--Rickettsia akari, R. australis, R. canada, R. conorii, R. prowazekii, R. rickettsii, R, siberica, R. typhi (R.
mooseri)--Yersinia pestis (except those strains listed in Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents
Including Chlamydia)
Appendix B-III-B.

Risk Group 3 (RG3) - Fungal Agents--Coccidioides immitis (sporulating cultures; contaminated soil)--Histoplasma capsulatum, H. capsulatum var. duboisii
Appendix B-III-C.

Risk Group 3 (RG3) - Parasitic Agents

None
Appendix B-III-D.

Risk Group 3 (RG3) - Viruses and Prions

Alphaviruses (Togaviruses) - Group A Arboviruses--Chikungunya virus (except the vaccine strain 181/25 listed in Appendix B-II-D Risk Group2 (RG2) –Viruses)--Semliki Forest virus--Venezuelan equine encephalomyelitis virus (except the vaccine strains TC-83 and V3526, see Appendix
B-II-D (RG2) – Viruses)--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections
I through IV)
Arenaviruses--Flexal--Lymphocytic choriomeningitis virus (LCM) (neurotropic strains)
Bunyaviruses--Hantaviruses including Hantaan virus--Rift Valley fever virus
Coronaviruses--SARS-associated coronavirus (SARS-CoV)--Middle East respiratory syndrome coronavirus (MERS-CoV)Flaviviruses - Group B Arboviruses--Japanese encephalitis virus (except those strains listed in Appendix B-II-D Risk Group2 (RG2) Viruses)--Yellow fever virus--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections
I through IV)
Orthomyxoviruses-- Influenza viruses 1918-1919 H1N1 (1918 H1N1), human H2N2 (1957-1968), and highly pathogenic
avian influenza H5N1 strains within the Goose/Guangdong/96-like H5 lineage (HPAI H5N1).
Poxviruses--Monkeypox virus
Prions--Transmissible spongiform encephalopathies (TSE) agents (Creutzfeldt-Jacob disease and kuru
agents)(see Section V-C, Footnotes and References of Sections I through IV, for containment

Page 45 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

instruction)
Retroviruses--Human immunodeficiency virus (HIV) types 1 and 2--Human T cell lymphotropic virus (HTLV) types 1 and 2--Simian immunodeficiency virus (SIV)
Rhabdoviruses--Vesicular stomatitis virus (except those strains listed in Appendix B-II-D Risk Group2 (RG2) - Viruses)

Appendix B-IV. Risk Group 4 (RG4) Agents

RG4 agents are likely to cause serious or lethal human disease for which preventive or therapeuticinterventions are not usually available.
Appendix B-IV-A.

Risk Group 4 (RG4) - Bacterial Agents

None
Appendix B-IV-B.

Risk Group 4 (RG4) - Fungal Agents

None
Appendix B-IV-C.

Risk Group 4 (RG4) - Parasitic Agents

None
Appendix B-IV-D.

Risk Group 4 (RG4) - Viral Agents

Arenaviruses--Guanarito virus--Lassa virus--Junin virus (except the candid #1 vaccine strain listed in Appendix B-II-D Risk Group2 (RG2) – Viruses)--Machupo virus--Sabia
Bunyaviruses (Nairovirus)--Crimean-Congo hemorrhagic fever virus
Filoviruses--Ebola viruses--Marburg viruses
Flaviruses - Group B Arboviruses--Tick-borne encephalitis virus complex including Absetterov, Central European encephalitis, Hanzalova,
Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer
encephalitis viruses
Herpesviruses (alpha)--Herpesvirus simiae (Herpes B or Monkey B virus)
Paramyxoviruses--Equine Morbillivirus (Hendra virus)
Hemorrhagic fever viruses as yet undefinedPage 46 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Appendix B-V. Animal Viral Etiologic Agents in Common Use
The following list of animal etiologic agents is appended to the list of human etiologic agents. None ofthese agents is associated with disease in healthy adult humans; they are commonly used in laboratory
experimental work.
A containment level appropriate for RG1 human agents is recommended for their use. For agents thatare infectious to human cells, e.g., amphotropic and xenotropic strains of murine leukemia virus, acontainment level appropriate for RG2 human agents is recommended.
Baculoviruses
Herpesviruses--Herpesvirus ateles--Herpesvirus saimiri--Marek's disease virus--Murine cytomegalovirus
Papilloma viruses--Bovine papilloma virus--Shope papilloma virus
Polyoma viruses--Polyoma virus--Simian virus 40 (SV40)
Retroviruses--Avian leukosis virus--Avian sarcoma virus--Bovine leukemia virus--Feline leukemia virus--Feline sarcoma virus--Gibbon leukemia virus--Mason-Pfizer monkey virus--Mouse mammary tumor virus--Murine leukemia virus--Murine sarcoma virus--Rat leukemia virus
Appendix B-V-1.

Murine Retroviral Vectors

Murine retroviral vectors to be used for human transfer experiments (less than 10 liters) that contain lessthan 50% of their respective parental viral genome and that have been demonstrated to be free ofdetectable replication competent retrovirus can be maintained, handled, and administered, under BL1
containment.
***********************************************************************************************************************
APPENDIX C.

EXEMPTIONS UNDER SECTION III-F-8

Section III-F-8 states that exempt from these NIH Guidelines are "those that do not present a significant
risk to health or the environment (see Section IV-C-1-b-(1)-(c), Major Actions), as determined by the NIH
Director following appropriate notice and opportunity for public comment. See Appendix C, Exemptions
under Sections III-F-8, for other classes of experiments which are exempt from the NIH Guidelines." ThePage 47 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

following classes of experiments are exempt under Section III-F-8:
Appendix C-I.

Recombinant or Synthetic Nucleic Acid Molecules in Tissue Culture

Recombinant or synthetic nucleic acid molecules containing less than one-half of any eukaryotic viralgenome (all viruses from a single family being considered identical -- see Appendix C-IX-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-B which require NIH OSP and Institutional Biosafety Committee approval before initiation, (ii)experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classificationof Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and Referencesof Sections I through IV) or cells known to be infected with these agents, (iii) experiments involving thedeliberate 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 (iv) whole plants regenerated from plant cells and tissue cultures are coveredby the exemption provided they remain axenic cultures even though they differentiate into embryonictissue 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 thoseexperiments 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-IX-B,
Footnotes and References of Appendix C) shall be used as vectors. However, experiments involving theinsertion into Escherichia coli K-12 of DNA from prokaryotes that exchange genetic information (seeAppendix C-IX-C, Footnotes and References of Appendix C) with Escherichia coli may be performed withany Escherichia coli K-12 vector (e.g., conjugative plasmid). When a non-conjugative vector is used, theEscherichia 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) 1
physical containment conditions are recommended. For large-scale fermentation experiments, theappropriate physical containment conditions need be no greater than those for the host organism
unmodified by recombinant or synthetic nucleic acid molecule 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-B which require NIH OSP and Institutional Biosafety Committee approval before initiation, (ii)experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classificationof Human
Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References ofSections 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, (iii)
large-scale experiments (e.g., more than 10 liters of culture), and (iv) 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).

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Appendix C-III. Saccharomyces Host-Vector Systems
Experiments involving Saccharomyces cerevisiae and Saccharomyces uvarum host-vector systems, withthe exception of experiments listed in Appendix C-III-A, are exempt from the NIH Guidelines. For these
exempt experiments, BL1 physical containment is recommended. For large-scale fermentation
experiments, the appropriate physical containment conditions need be no greater than those for theunmodified host organism; the Institutional Biosafety Committee can specify higher containment if
deemed necessary.
Appendix C-III-A.

Exceptions

The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section IIIB, which require NIH OSP and Institutional Biosafety Committee approval before initiation; (ii)experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classificationof Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and Referencesof Sections I through IV) or cells known to be infected with these agents may be conducted undercontainment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review andapproval; (iii) large-scale experiments (e.g., more than 10 liters of culture), (iv) experiments involving thedeliberate 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), and (v) experiments involving gene drive modified organisms (Section III-D-8).
Appendix C-IV. Kluyveromyces Host-Vector Systems
Experiments involving Kluyveromyces lactis host-vector systems, with the exception of experiments listed
in Appendix C-IV-A, are exempt from the NIH Guidelines provided laboratory-adapted strains are used
(i.e. strains that have been adapted to growth under optimal or defined laboratory conditions). For these
exempt experiments, BL1 physical containment is recommended. For large-scale fermentation
experiments, the appropriate physical containment conditions need be no greater than those for theunmodified host organism; the Institutional Biosafety Committee may specify higher containment if
deemed necessary.
Appendix C-IV-A

Exceptions

The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section IIIB, which require NIH OSP and Institutional Biosafety Committee approval before initiation; (ii)experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classificationof Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and Referencesof Sections I through IV) or cells known to be infected with these agents may be conducted undercontainment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review andapproval; (iii) large-scale experiments (e.g., more than 10 liters of culture), (iv) experiments involving thedeliberate 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), and (v) experiments involving gene drive modified organisms (Section III-D-8).
Appendix C-V. Bacillus subtilis or Bacillus licheniformis Host-Vector Systems
Any asporogenic Bacillus subtilis or asporogenic Bacillus licheniformis strain which does not revert to aspore-former with a frequency greater than 10-7 may be used for cloning DNA with the exception of thoseexperiments listed in Appendix C-V-A, Exceptions. For these exempt laboratory experiments, BL1
physical containment conditions are recommended. For large-scale fermentation experiments, theappropriate physical containment conditions need be no greater than those for the unmodified hostorganism; the Institutional Biosafety Committee can specify higher containment if it deems necessary.
Appendix C-V-A.

Exceptions

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The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section
III-B which require NIH OSP and Institutional Biosafety Committee approval before initiation, (ii)experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classificationof Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and Referencesof Sections I through IV) or cells known to be infected with these agents may be conducted undercontainment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review andapproval, (iii) large-scale experiments (e.g., more than 10 liters of culture), and (iv) experiments involving
the deliberate cloning of genes coding for the biosynthesis of molecules toxic for vertebrates (seeAppendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic
for Vertebrates).
Appendix C-VI. Extrachromosomal Elements of Gram Positive Organisms
Recombinant or synthetic nucleic acid molecules derived entirely from extrachromosomal elements of theorganisms 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 stearothermophilus
Bacillus subtilis
Bacillus thuringiensis
Clostridium acetobutylicum
Lactobacillus casei
Listeria grayi
Listeria monocytogenes
Listeria murrayi
Pediococcus acidilactici
Pediococcus damnosus
Pediococcus pentosaceus
Staphylococcus aureus
Staphylococcus carnosus
Staphylococcus epidermidis
Streptococcus agalactiae
Streptococcus anginosus
Streptococcus avium
Streptococcus cremoris
Streptococcus dorans
Streptococcus equisimilis
Streptococcus faecalis
Streptococcus ferus
Streptococcus lactis
Streptococcus ferns
Streptococcus mitiorStreptococcus mutans

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Streptococcus pneumoniae
Streptococcus pyogenes
Streptococcus salivarius
Streptococcus sanguis
Streptococcus sobrinus
Streptococcus thermophilus
Appendix C-VI-A.

Exceptions

The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section
III-B which require NIH OSP and Institutional Biosafety Committee approval before initiation, (ii)experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classificationof Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and Referencesof Sections I through IV) or cells known to be infected with these agents may be conducted undercontainment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review andapproval, (iii) large-scale experiments (e.g., more than 10 liters of culture), and (iv) experiments involving
the deliberate cloning of genes coding for the biosynthesis of molecules toxic for vertebrates (seeAppendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic
for Vertebrates).
Appendix C-VII. The Purchase or Transfer of Transgenic Rodents
The purchase or transfer of transgenic rodents for experiments that require BL1 containment (SeeAppendix G-III-M, Footnotes and References of Appendix G) are exempt from the NIH Guidelines.
Appendix C-VIII.

Generation of BL1 Transgenic Rodents via Breeding

The breeding of two different transgenic rodents or the breeding of a transgenic rodent and a nontransgenic rodent with the intent of creating a new strain of transgenic rodent that can be housed at BL1
containment will be exempt from the NIH Guidelines if:(1) Both parental rodents can be housed under BL1 containment; and(2) neither parental transgenic rodent contains the following genetic modifications: (i) incorporation of
more than one-half of the genome of an exogenous eukaryotic virus from a single family of viruses; or (ii)incorporation of a transgene that is under the control of a gammaretroviral long terminal repeat (LTR);and(3) the transgenic rodent that results from this breeding is not expected to contain more than one-half ofan exogenous viral genome from a single family of viruses.
Appendix C-IX. Footnotes and References of Appendix C
Appendix C-IX-A. The NIH Director may revise the classification for the purposes of these NIH
Guidelines (see Section IV-C-1-b-(2)-(b), Minor Actions). The revised list of organisms in each Risk
Group is located in Appendix B.
Appendix C-IX-B. A subset of non-conjugative plasmid vectors are poorly mobilizable (e.g., pBR322,
pBR313). Where practical, these vectors should be employed.
Appendix C-IX-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 toexempt organisms under Section III-F-6, Exempt Experiments.
Appendix C-IX-D. As classified in the Third Report of the International Committee on Taxonomy ofViruses: Classification and Nomenclature of Viruses, R. E. F. Matthews (ed.), Intervirology 12 (129-296),
1979.

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

MAJOR ACTIONS TAKEN UNDER THE NIH GUIDELINES

As noted in the subsections of Section IV-C-1-b-(1), the Director, NIH, may take certain actions withregard to the NIH Guidelines (Entries up to and including D-118 were approved using a process thatinvolved the Recombinant DNA Advisory Committee (RAC)). Some of the actions taken to date include
the following:
Appendix D-1. Permission is granted to clone foot and mouth disease virus in the EK1 host-vector
system consisting of E. coli K-12 and the vector pBR322, all work to be done at the Plum Island Animal
Disease Center.
Appendix D-2. Certain specified clones derived from segments of the foot and mouth disease virus may
be transferred from Plum Island Animal Disease Center to the facilities of Genentech, Inc., of South San
Francisco, California. Further development of the clones at Genentech, Inc., has been approved underBL1 + EK1 conditions.
Appendix D-3. The Rd strain of Hemophilus influenzae can be used as a host for the propagation of thecloned Tn 10 tet R gene derived from E. coli K-12 employing the non-conjugative Hemophilus plasmid,
pRSF0885, under BL1 conditions.
Appendix D-4. Permission is granted to clone certain subgenomic segments of foot and mouth diseasevirus in HV1 Bacillus subtilis and Saccharomyces cerevisiae host-vector systems under BL1 conditions at
Genentech, Inc., South San Francisco, California.
Appendix D-5. Permission is granted to Dr. Ronald Davis of Stanford University to field test corn plants
modified by recombinant DNA techniques under specified containment conditions.
Appendix D-6. Permission is granted to clone in E. coli K-12 under BL1 physical containment conditions
subgenomic segments of rift valley fever virus subject to conditions which have been set forth by the
Recombinant DNA Advisory Committee.
Appendix D-7. Attenuated laboratory strains of Salmonella typhimurium may be used under BL1
physical containment conditions to screen for the Saccharomyces cerevisiae pseudouridine synthetasegene. The plasmid YEp13 will be employed as the vector.
Appendix D-8. Permission is granted to transfer certain clones of subgenomic segments of foot andmouth disease virus from Plum Island Animal Disease Center to the laboratories of Molecular Genetics,
Inc., Minnetonka, Minnesota, and to work with these clones under BL1 containment conditions. Approval
is contingent upon review of data on infectivity testing of the clones by a working group of the
Recombinant DNA Advisory Committee.
Appendix D-9. Permission is granted to Dr. John Sanford of Cornell University to field test tomato andtobacco plants transformed with bacterial (E.coli K-12) and yeast DNA using pollen as a vector.
Appendix D-10. Permission is granted to Drs. Steven Lindow and Nickolas Panopoulos of the Universityof California, Berkeley, to release under specified conditions Pseudomonas syringae, pathovars (pv.)
syringae, and Erwinia herbicola carrying in vitro generated deletions of all or part of the genes involved in
ice nucleation.
Appendix D-11. Agracetus of Middleton, Wisconsin, may field test under specified conditions disease

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resistant tobacco plants prepared by recombinant DNA techniques.
Appendix D-12. Eli Lilly and Company of Indianapolis, Indiana, may conduct large-scale experiments
and production involving Cephalosporium acremonium strain LU4-79-6 under less than Biosafety Level 1- Large Scale (BL1-LS) conditions.
Appendix D-13. Drs. W. French Anderson, R. Michael Blaese, and Steven Rosenberg of the NIH,
Bethesda, Maryland, can conduct experiments in which a bacterial gene coding for neomycin
phosphotransferase will be inserted into a portion of the tumor infiltrating lymphocytes (TIL) of cancer
patients using a retroviral vector, N2. The marked TIL then will be combined with unmarked TIL, andreinfused into the patients. This experiment is an addition to an ongoing adoptive immunotherapy
protocol in which TIL are isolated from a patient's tumor, grown in culture in the presence of interleukin-2,
and reinfused into the patient. The marker gene will be used to detect TIL at various time intervals
following reinfusion.
Approval is based on the following four stipulations: (I) there will be no limitation of the number ofpatients in the continuing trial; (ii) the patients selected will have a life expectancy of about 90 days; (iii)the patients give fully informed consent to participate in the trial; and (iv) the investigators will provide
additional data before inserting a gene for therapeutic purposes. (Protocol #8810-001)
Appendix D-14. U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) may conductcertain experiments involving products of a yellow fever virus originating from the 17-D yellow fever clone
at the Biosafety Level 3 containment level using HEPA filters and vaccination of laboratory personnel.
In addition, USAMRIID may conduct certain experiments involving vaccine studies of Venezuelan equine
encephalitis virus at the Biosafety Level 3 containment level using HEPA filters and vaccination oflaboratory personnel.
Appendix D-15. Drs. R. Michael Blaese and W. French Anderson of the NIH, Bethesda, Maryland, canconduct experiments in which a gene coding for adenosine deaminase (ADA) will be inserted into T
lymphocytes of patients with severe combined immunodeficiency disease, using a retroviral vector, LASN.
Following insertion of the gene, these T lymphocytes will be reinfused into the patients. The patients willthen be followed for evidence of clinical improvement in the disease state, and measurement of multiple
parameters of immune function by laboratory testing.
Approval is based on the following two stipulations: (I) that intraperitoneal administration of transduced T
lymphocytes not be used before clearance by the Chair of the Recombinant DNA Advisory Committee;and (ii) that the number of research patients be limited to 10 at this time.
In addition to the conditions outlined in the initial approval, patients may be given a supplement of a CD34+-enriched peripheral blood lymphocytes (PBL) which have been placed in culture conditions that favor
progenitor cell growth. This enriched population of cells will be transduced with the retroviral vector,
G1NaSvAd. G1NaSvAd is similar to LASN, yet distinguishable by PCR. LASN has been used totransduce peripheral blood T lymphocytes with the ADA gene. Lymphocytes and myeloid cells will be
isolated from patients over time and assayed for the presence of the LASN or G1NaSvAd vectors. The
primary objectives of this protocol are to transduce CD 34+ peripheral blood cells with the adenosine
deaminase gene, administer these cells to patients, and determine if such cells can differentiate into
lymphoid and myeloid cells in vivo. There is a potential for benefit to the patients in that these
hematopoietic progenitor cells may survive longer, and divide to yield a broader range of gene-corrected
cells. (Protocol #9007-002)
Appendix D-16. Dr. Steven A. Rosenberg of the National Institutes of Health, Bethesda, Maryland, canconduct experiments on patients with advanced melanoma who have failed all effective therapy. Thesepatients will be treated with escalating doses of autologous tumor infiltrating lymphocytes (TIL)
transduced with a gene coding for tumor necrosis factor (TNF). Escalating numbers of transduced TIL
will be administered at three weekly intervals along with the administration of interleukin-2 (IL-2). The
objective is to evaluate the toxicity and possible therapeutic efficacy of the administration of tumor

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infiltrating lymphocytes (TIL) transduced with the gene coding for TNF. (Protocol #9007-003)
Appendix D-17. Dr. Malcolm K. Brenner of St. Jude Children's Research Hospital of Memphis,
Tennessee, can conduct experiments on patients with acute myelogenous leukemia (AML). Using the
LNL6 retroviral vector, the autologous bone marrow cells will be transduced with the gene coding forneomycin resistance. The purpose of this gene marking experiment is to determine whether the sourceof relapse after autologous bone marrow transplantation for acute myelogenous leukemia is residual
malignant cells in the harvested marrow or reoccurrence of tumor in the patient. Determining the sourceof relapse should indicate whether or not purging of the bone marrow is a necessary procedure. (Protocol#9102-004)
Appendix D-18. Dr. Malcolm K. Brenner of St. Jude Children's Research Hospital of Memphis,
Tennessee, can conduct experiments on pediatric patients with Stage D (disseminated) neuroblastoma
who are being treated with high-dose carboplatin and etoposide in either phase I/II or phase II trials. All
the patients in these studies will be subjected to bone marrow transplantation since it will allow them to be
exposed to chemoradiation that would be lethal were it not for the availability of stored autologous marrowfor rescue. The bone marrow cells of these patients will be transduced with the gene coding for neomycin
resistance using the LNL6 vector. The purpose of this gene marking study is to determine whether thesource of relapse after autologous bone marrow transplantation is residual malignant cells in theharvested marrow or residual disease in the patient. Secondly,
it is hoped to determine the contribution of marrow autographs to autologous reconstitution. (Protocol#9105-005/9105-006)
Appendix D-19. Dr. Albert B. Deisseroth of the MD Anderson Cancer Center of Houston, Texas, canconduct experiments on patients with chronic myelogenous leukemia who have been reinduced into asecond chronic phase or blast cells. The patients in these studies will receive autologous bone marrowtransplantation. Using the LNL6 vector, the bone marrow cells will be transduced with the gene coding
for neomycin resistance. The purpose of these marking studies is to determine if the origin of relapse
arises from residual leukemic cells in the patients or from viable leukemic cells remaining in the bonemarrow used for autologous transplantation. (Protocol #9105-007)
Appendix D-20. Drs. Fred D. Ledley and Savio L. C. Woo of Baylor College of Medicine of Houston,
Texas, can conduct experiments on pediatric patients with acute hepatic failure who are identified as
candidates for hepatocellular transplantation. Using the LNL6 vector, the hepatocytes will be transducedwith the gene coding for neomycin resistance. The purpose of using a genetic marker is to demonstrate
the pattern of engraftment of transplanted hepatocytes and to help determine the success or failure ofengraftment. (Protocol #9105-008)
Appendix D-21. Dr. Steven A. Rosenberg of the National Institutes of Health, Bethesda, Maryland, canconduct experiments on patients with advanced melanoma, renal cell cancer, and colon carcinoma whohave failed all effective therapy. In an attempt to increase these patients' immune responses to thetumor, the tumor necrosis factor gene or the interleukin-2 gene will be introduced into a tumor cell line
established from the patient. These gene-modified autologous tumor cells will then be injected into thethigh of the patient. To further utilize the immune system of the patient to fight the tumor, stimulated
lymphocytes will be cultured from either the draining regional lymph nodes or the injected tumor itself.
The patients will be evaluated for antitumor effects engendered by the injection of the gene modified
tumor cells themselves as well as after the infusion of the cultured lymphocytes. (Protocol #9110010/9110-011)
Appendix D-22. Dr. James M. Wilson of the University of Michigan Medical Center of Ann Arbor,
Michigan, can conduct experiments on three patients with the homozygous form of familial
hypercholesterolemia. Both children and adults will be eligible for this therapy. In an attempt to correct
the basic genetic defect in this disease, the gene coding for the low-density lipoprotein (LDL) receptor will
be introduced into liver cells taken from the patient. The gene-corrected hepatocytes will then be infused
into the portal circulation of the patient through an indwelling catheter. The patients will be evaluated forengraftment of the these treated hepatocytes through a series of metabolic studies; three months afterPage 54 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

gene therapy, a liver biopsy will be taken and analyzed for the presence of recombinant derived RNA andDNA to document the presence of the gene coding for the normal LDL receptor. (Protocol #9110-012)
Appendix D-23. Dr. Michael T. Lotze of the University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania, can conduct experiments on 20 patients with metastatic melanoma who have failed
conventional therapy. A gene transfer experiment will be performed, transducing the patients' tumor
infiltrating lymphocytes (TILs) with the gene for neomycin resistance. Through the use of this genemarking technique, it is proposed to determine how long TIL cells can be detected in vivo in the peripheral
blood of the patients, and how the administration of interleukin-2 and interleukin-4 affects localization andsurvival of TIL cells in tumor sites. (Protocol #9105-009)
Appendix D-24. Dr. Gary J. Nabel of the University of Michigan Medical School, Ann Arbor, Michigan,
can conduct gene therapy experiments on twelve patients with melanoma or adenocarcinoma. Patient
population will be limited to adults over the age of 18 and female patients must be postmenopausal or
have undergone tubal ligation or orchiectomy. The patient's immune response will be stimulated by the
introduction of a gene encoding for a Class I MHC protein, HLA-B7, in order to enhance tumor regression.
DNA/liposome-mediated transfection techniques will be used to directly transfer this foreign gene into
tumor cells. HLA-B7 expression will be confirmed in vivo, and the immune response stimulated by theexpression of this antigen will be characterized. These experiments will be analyzed for their efficacy intreating cancer. (Protocol #9202-013)
Appendix D-25. Kenneth Cornetta of Indiana University, Indianapolis, Indiana, can conduct genetransfer experiments on up to 10 patients with acute myelogenous leukemia (AML) and up to 10 patientswith acute lymphocytic leukemia (ALL). The patient population will be limited to persons between 18 and65 years of age. Using the LNL-6 vector, autologous bone marrow cells will be marked with the neomycin
resistance gene. Gene marked and untreated bone marrow cells will be reinfused at the time of bonemarrow transplantation. Patients will then be monitored for evidence of the neomycin resistance gene inperipheral blood and bone marrow cells in order to determine whether relapse of their disease is a resultof residual malignant cells remaining in the harvested marrow or inadequate ablation of the tumor cells by
chemotherapeutic agents. Determining the source of relapse may indicate whether or not purging of thebone marrow is a necessary procedure for these leukemia patients. Further studies will be performed inorder to determine the percentage of leukemic cells that contain the LNL-6 vector and the clonality of themarked cells. (Protocol #9202-014)
Appendix D-26. Dr. James S. Economou of the University of California, Los Angeles, can conduct genetransfer experiments on 20 patients with metastatic melanoma and 20 patients with renal cell carcinoma.
These patients will be treated with various combinations of tumor-infiltrating lymphocytes and peripheral
blood leukocytes, including CD8 and CD4 subsets of both types of cells. These effector cell populations
will be given in combination with interleukin-2 (IL-2) in the melanoma patients and IL-2 plus alphainterferon in the renal cell carcinoma patients. The effector cells will be transduced with the neomycin
resistance gene using either the LNL6 or G1N retroviral vectors. This "genetic marking" of the tumorinfiltrating lymphocytes and peripheral blood lymphocytes is designed to answer questions about thetrafficking of these cells, their localization to tumors, and their in vivo life span. (Protocol #9202-015)
Appendix D-27. Drs. Philip Greenberg and Stanley R. Riddell of the Fred Hutchinson Cancer ResearchCenter, Seattle, Washington, may conduct gene transfer experiments on 15 human immunodeficiency
virus (HIV) seropositive patients (18-45 years old) undergoing allogeneic bone marrow transplantation for
non-Hodgkin's lymphoma and 15 HIV-seropositive patients (18-50 years old) who do not have acquired
immunodeficiency syndrome (AIDS)-related lymphoma and who are not undergoing bone marrowtransplantation to evaluate the safety and efficacy of HIV-specific cytotoxic T lymphocyte (CTL) therapy.
CTL will be transduced with a retroviral vector (HyTK) encoding a gene that is a fusion product of thehygromycin phosphotransferase gene (HPH) and the herpes simplex virus thymidine kinase (HSV-TK)
gene. This vector will deliver both a marker gene and an ablatable gene in these T cell clones in theevent that patients develop side effects as a consequence of CTL therapy. Data will be correlated overtime, looking at multiple parameters of HIV disease activity. The objectives of these studies include
evaluating the safety and toxicity of CTL therapy, determining the duration of in vivo survival of HIV-

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specific CTL clones, and determining if ganciclovir therapy can eradicate genetically modified, adoptivelytransferred CTL cells. (Protocol #9202-017)
Appendix D-28. Dr. Malcolm Brenner of St. Jude Children's Research Hospital, Memphis, Tennessee,
can conduct gene therapy experiments on twelve patients with relapsed/refractory neuroblastoma whohave relapsed after receiving autologous bone marrow transplant. In an attempt to stimulate the patient's
immune response, the gene coding for Interleukin-2 (IL-2) will be used to transduce tumor cells, andthese gene-modified cells will be injected subcutaneously in a Phase 1 dose escalation trial. Patients will
be evaluated for an anti-tumor response. (Protocol #9206-018)
Appendix D-29. Drs. Edward Oldfield, Kenneth Culver, Zvi Ram, and R. Michael Blaese of the National
Institutes of Health, Bethesda, Maryland, can conduct gene therapy experiments on ten patients withprimary malignant brain tumors and ten patients with lung cancer, breast cancer, malignant melanoma, or
renal cell carcinoma who have brain metastases. The patient population will be limited to adults over theage of 18.
Patients will be divided into two groups based on the surgical accessibility of their lesions. Both surgicallyaccessible and surgically inaccessible lesions will receive intra-tumoral injections of the retroviral Herpes
simplex thymidine kinase (HS-tk) vector-producer cell line, G1TkSvNa, using a guided stereotaxic
approach. Surgically accessible lesions will be excised seven days after stereotaxic injection, and thetumor bed will be infiltrated with the HS-tk producer cells. The removed tumor will be evaluated for theefficiency of transduction. Ganciclovir (GCV) will be administered beginning on the fifth postoperativeday. In the case of surgically inaccessible lesions, the patients will receive intravenous therapy with GCVseven days after receiving the intra-tumoral injections of the retroviral HS-tk vector-producer cells.
(Protocol #9206-019)
Appendix D-30. Dr. Albert D. Deisseroth of MD Anderson Cancer Center, Houston, Texas, can conductgene transfer experiments on ten patients who have developed blast crisis or accelerated phase chronic
myelogenous leukemia (CML). The retroviral vectors G1N and LNL6 which code for neomycin resistancewill be used to transduce autologous peripheral blood and bone marrow cells that have been removed
and stored at the time of cytogenetic remission or re-induction of chronic phase in Philadelphia
chromosome positive CML patients. Following reinduction of the chronic phase of CML and preparative
chemotherapy, patients will be infused with the transduced autologous cells.
This protocol is designed to determine the cause of relapse of CML. If polyclonal CML neomycin marked
blastic cells appear at the time of relapse, their presence will indicate that relapse arises from theleukemic CML blast cells present in the autologous cells infused following chemotherapy. If residual
systemic disease contributes to relapse, the neomycin resistance gene will not be detected in the CML
leukemic blasts at the time of relapse.
This study will compare the relative contributions of the peripheral blood and bone marrow to generate
hematopoietic recovery after bone marrow transplantation and evaluate purging and selection of
peripheral blood or bone marrow as a source of stem cells for transplant. The percentage of neomycin
resistant CML cells which are leukemic will be determined by PCR analysis and detection of bcr-abl
mRNA. (Protocol #9206-020)
Appendix D-31. Dr. Cynthia Dunbar of the National Institutes of Health, Bethesda, Maryland, canconduct gene transfer experiments on up to 48 patients with multiple myeloma, breast cancer, or chronic
myelogenous leukemia. The retroviral vectors G1N and LNL6 will be used to transfer the neomycin
resistance marker gene into autologous bone marrow and peripheral blood stem cells in the presence ofgrowth factors to examine hematopoietic reconstitution after bone marrow transplantation. The efficiencyof transduction of both short and long term autologous bone marrow reconstituting cells will be examined.
Autologous bone marrow and CD34+ peripheral blood stem cells will be enriched prior to transduction.
Myeloma and CML patients will receive both autologous bone marrow and peripheral blood stem celltransplantation. These separate populations will be marked with both the G1N and LNL6 retroviral
vectors. If short and long term marking experiments are successful, important information may bePage 56 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

obtained regarding the biology of autologous reconstitution, the feasibility of retroviral gene transfer into
hematopoietic cells, and the contribution of viable tumor cells within the autograft to disease relapse.
(Protocol #9206-023/9206-024/9206-025)
Appendix D-32. Dr. Bernd Gansbacher of the Memorial Sloan-Kettering Cancer Center, New York, New
York, can conduct gene therapy experiments on twelve patients over 18 years of age with metastaticmelanoma who are HLA-A2 positive and who have failed conventional therapy. This is a phase I study to
examine whether allogeneic HLA-A2 matched melanoma cells expressing recombinant humanInterleukin-2 (IL-2) can be injected subcutaneously and used to create a potent tumor specific immune
response without producing toxicity. By allowing the tumor cells to present the MHC Class I molecule aswell as the secreted IL-2, a clonal expansion of tumor specific effector cells is expected. These effector
populations may access residual tumor at distant sites via the systemic circulation. (Protocol #9206-021)
Appendix D-33. Dr. Bernd Gansbacher of the Memorial Sloan-Kettering Cancer Center, New York, New
York, can conduct gene therapy experiments on twelve patients over 18 years of age with renal cell
carcinoma who are HLA-A2 positive and who have failed conventional therapy. This Phase I study will
examine whether allogeneic HLA-A2 matched renal cell carcinoma cells expressing recombinant humanInterleukin-2 (IL-2) can be injected subcutaneously and used to create a potent tumor specific immune
response without producing toxicity. By allowing the tumor cells to present the MHC Class I molecule aswell as the secreted IL-2, a clonal expansion of tumor specific effector cells is expected. These effector
populations may access residual tumor at distant sites via the systemic circulation. (Protocol #9206-022)
Appendix D-34. Dr. Michael T. Lotze, University of Pittsburgh, Pittsburgh, Pennsylvania, can conduct
experiments on twenty patients with metastatic, and/or unresectable, locally advanced melanoma, renal
cell carcinoma, breast cancer, or colon cancer who have failed standard therapy. Patients will receive
multiple subcutaneous injections of autologous tumor cells combined with an autologous fibroblast cellline that has been transduced in vitro with the gene coding for Interleukin-4 (IL-4) to augment the in vivo
antitumor effect. Patients will be monitored for antitumor effect by PCR analysis and multiple biopsy of
the injection site. (Protocol #9209-033)
Appendix D-35. Dr. Friedrich G. Schuening, Fred Hutchinson Cancer Research Center, Seattle,
Washington, can conduct human gene transfer experiments on patients ≥ 18 years of age with breast
cancer, Hodgkin's disease, or non-Hodgkin's lymphoma. A total of 10 patients per year will be enrolled in
the studies over a period of four years. Patients will undergo autologous bone marrow transplantation
with a selected population of Interleukin-3 (IL-3) or granulocyte colony-stimulating factor (G-CSF)
stimulated CD34(+) peripheral blood repopulating cells (PBRC) that have been transduced with the genecoding for neomycin resistance (neoR) using the retroviral vector, LN. Patients will be continuously
monitored for neoR to determine the relative contributionof autologous PBRCs to long-term hematopoietic reconstitution. Demonstration of long-term contributionof autologous PBRC to hematopoiesis will enable the use of PBRC alone for autologous transplants andsuggest the use of PBRC as long-term carriers of therapeutically relevant genes. (Protocol #9209027/9209-028)
Appendix D-36. Dr. Friedrich G. Schuening, Fred Hutchinson Cancer Research Center, Seattle,
Washington, can conduct human gene transfer experiments on patients ≥ 18 years of age with breast
cancer, Hodgkin's disease, or non-Hodgkin's lymphoma. A total of 5 patients per year will be enrolled in
the study over a period of four years. Patients will undergo allogeneic bone marrow transplant with
granulocyte colony-stimulating factor (G-CSF) stimulated CD34(+) PBRC harvested from an identical twinthat have been transduced with neoR using the retroviral vector, LN. Patients will be continuously
monitored for neoR to determine the relative contribution of G-CSF stimulated allogeneic PBRCs to longterm bone marrow engraftment. Demonstration of long-term contribution of allogeneic PBRC tohematopoiesis will enable the use of PBRC alone for allogeneic transplants and suggest the use of PBRC
as long-term carriers of therapeutically relevant genes. (Protocol #9209-029)

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Appendix D-37. Dr. Malcolm K. Brenner of St. Jude Children's Hospital, Memphis, Tennessee, and Dr.
Bonnie J. Mills of Baxter Healthcare Corporation, Santa Ana, California, can conduct a multicenter
uncontrolled human gene transfer experiment on 12 patients ≤ 21 years of age with Stage D
Neuroblastoma in first or second marrow remission. Autologous bone marrow cells will be separated into
two fractions, purged and unpurged. Each fraction will be transduced with the neoR gene by either LNL6
or G1Na. Patients will be monitored by the polymerase chain reaction (PCR) for the presence of neoR.
The protocol is designed to evaluate the safety and efficacy of the Neuroblastoma Bone Marrow Purging
System following high dose chemotherapy. (Protocol #9209-032)
Appendix D-38. Drs. Carolyn Keierleber and Ann Progulske-Fox of the University of Florida, Gainesville,
Florida, can conduct experiments involving the introduction of a gene coding for tetracycline resistanceinto Porphyromonas gingivalis at a physical containment level of Biosafety Level-2 (BL-2).
Appendix D-39. Dr. Scott M. Freeman of Tulane University Medical Center, New Orleans, Louisiana,
can conduct experiments on patients with epithelial ovarian carcinoma who have clinical evidence ofrecurrent, progressive, or residual disease who have no other therapy available to prolong survival.
Patients will be injected intraperitoneally with the irradiated PA-1 ovarian carcinoma cell line which has
been transduced with the herpes simplex thymidine kinase (HSV-TK) gene. The patients will then receive
ganciclovir therapy. Previous, data indicates that HSV-TK+ tumor cells exhibit a killing effect on HSV-TKcells when exposed to ganciclovir therapy. Patients will be evaluated for safety and side effects of thistreatment. (Protocol #9206-016)
Appendix D-40. Dr. Michael J. Welsh, Howard Hughes Medical Institute Research Laboratories,
University of Iowa College of Medicine, Iowa City, Iowa, may conduct experiments on 3 cystic fibrosis(CF) patients ≥ 18 years of age with mild to moderate disease. This Phase I study will determine the: (1)in vivo safety and efficacy of the administration of the replication-deficient type 2 adenovirus vector,
Ad2/CFTR-1, to the nasal epithelium; (2) efficacy in correcting the CF chloride transport defect in vivo;and (3) effect of adenovirus vector dosage on safety and efficacy. (Protocol #9212-036)
Appendix D-41. Dr. Ronald G. Crystal, National Institutes of Health, Bethesda, Maryland, may conduct
experiments on 10 cystic fibrosis (CF) patients ≥ 21 years of age. Patients will receive an initialadministration of the replication-deficient type 5 adenovirus vector, AdCFTR, to their left nares. If no
toxicity is observed from intranasal administration, patients will receive a single administration of AdCFTR
to the respiratory epithelium of their left large bronchi. Five groups of patients (2 patients per group) will
be studied based on increased dosage administration of AdCFTR. This study will determine the: (1) invivo safety and efficacy of the administration of AdCFTR into the respiratory epithelium; (2) efficacy of thecorrection of the biologic abnormalities of CF in the respiratory epithelium; (3) duration of the biologic
correction; (4) efficacy of the correction of the abnormal electrical potential difference of the airway
epithelial sheet; (5) clinical parameters relevant to the disease process; and (6) if humoral immunity
develops against AdCFTR sufficient to prevent repeat administration. (Protocol #9212-034)
Appendix D-42. Dr. Kenneth Culver, Iowa Methodist Medical Center, Des Moines, Iowa, and Dr. John
Van Gilder, University of Iowa, Iowa City, Iowa, may conduct experiments on 15 patients ≥ 18 years ofage with recurrent malignant primary brain tumors or lung, melanoma, renal cell carcinoma, or breast
carcinoma brain metastases who have failed standard therapy for their disease. Patient eligibility will belimited to those patients who have measurable residual tumor immediately following the post-operative
procedure as demonstrated by imaging studies. The number of patients treated will be equally divided
between the Iowa Methodist Medical Center and the University of Iowa. If a positive response isobserved in any of the first 15 patients, the investigators may submit a request to treat an additional 15
patients.
Following surgical debulking, patients will receive a maximum of 3 intralesional injections of the
G1TkSvNa vector- producing cell line (VPC) to induce regression of residual tumor cells by ganciclovir(GCV) therapy. Patients who demonstrate stable disease for a minimum of 6 months following thistreatment will be eligible for additional VPC injections and subsequent GCV therapy. (Protocol #9303037)

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Appendix D-43. Drs. Malcolm Brenner, Robert Krance, Helen E. Heslop, Victor Santana, and James
Ihle, St. Jude Children's Research Hospital, Memphis, Tennessee, may conduct experiments on 35
patients ≥ 1 year and ≤ 21 years of age at the time of initial diagnosis of acute myelogenous leukemia(AML). The investigators will use the two retroviral vectors, LNL6 and G1Na, to determine the efficacy ofthe bone marrow purging techniques: 4-hydroxyperoxicyclophosphamide and interleukin-2 (IL-2)
activation of endogenous cytotoxic effector cells, in preventing relapse from the reinfusion of autologous
bone marrow cells. (Protocol #9303-039)
Appendix D-44. Drs. Helen E. Heslop, Malcolm Brenner, and Cliona Rooney, St Jude Children's
Research Hospital, Memphis, Tennessee, may conduct experiments of 35 patients ≤ 21 years of age whowill be recipients of mismatched-related or phenotypically similar unrelated donor marrow grafts forleukemia. In this Phase I dose escalation study, spontaneous lymphoblastoid cell lines will beestablished that express the same range of Epstein-Barr Virus (EBV) encoded proteins as the recipient.
These EBV-specific cell lines will be transduced with LNL6 or G1Na and readministered at the time ofbone marrow transplant. This study will determine: (1) survival and expansion of these EBV-specific celllines in vivo, (2) the ability of these adoptively transferred cells to confer protection against EBV infection,and (3) appropriate dosage and administration schedules. (Protocol #9303-038)
Appendix D-45. Drs. Robert W. Wilmott and Jeffrey Whitsett, Children's Hospital Medical Center,
Cincinnati, Ohio, and Dr. Bruce Trapnell, Genetic Therapy, Inc., Gaithersburg, Maryland, may conduct
experiments on 15 cystic fibrosis (CF) patients who have mild to moderate disease ≥ 21 years of age.
The replication-deficient type 5 adenovirus vector, Av1CF2, will be administered to the nasal and lobar
bronchial respiratory tract of patients. This study will demonstrate the: (1) expression of normal cystic
fibrosis transmembrane conductance regulator (CFTR) mRNA in vivo, (2) synthesis of CFTR protein, and(3) correction of epithelial cell cAMP dependent Cl- permeability. The pharmacokinetics of CFTR
expression and ability to re-infect the respiratory tract with AvCF2 will be determined. Systemic and local
immunologic consequences of Av1CF2 infection, the time of viral survival, and potential for recombinationor complementation of the virus will be monitored. (Protocol #9303-041)
Appendix D-46. Dr. James M. Wilson of the University of Pennsylvania Medical Center, Philadelphia,
Pennsylvania, may conduct experiments on 20 adult patients with advanced cystic fibrosis lung disease.
An isolated segment of the patients' lung will be transduced with the E1 deleted, replication-incompetentadenovirus vector, Ad.CΒ-CFTR using a bronchoscope for gene delivery. Ad.CB-CFTR contains thehuman gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein.
Pulmonary biopsies will be obtained by bronchoscopy at 4 days, 6 weeks, and 3 months following
treatment. Patients will be monitored for evidence of CFTR gene transfer and expression, immunologicalresponses to CFTR or adenovirus proteins, and toxicity. (Protocol #9212-035)
Appendix D-47. Dr. Hilliard F. Seigler of Duke University Medical Center, Durham, North Carolina, may
conduct experiments on 20 patients with disseminated malignant melanoma. Autologous tumor cells willbe transduced with a retroviral vector, pHuγ-IFN, that contains the gene encoding human γ-IFN.
Following lethal irradiation, the transduced cells will be readministered to patients for the purpose ofgenerating cytotoxic T cells that are tumor specific along with the up-regulation of Class I major
histocompatibility antigens. Patients will be monitored for clinical regression of tumors and generation oftumor-specific cytotoxic T lymphocytes. (Protocol #9306-043)
Appendix D-48. Drs. Stefan Karlsson and Cynthia Dunbar of the National Institutes of Health, Bethesda,
Maryland, and Dr. Donald B. Kohn of the Children’s Hospital of Los Angeles, Los Angeles, California,
may conduct experiments on 10 patients with Gaucher disease. CD34(+) hematopoietic stem cells will be
isolated from bone marrow or from peripheral blood treated with granulocyte-colony stimulating factor.
CD34(+) cells will be transduced with a retrovirus vector, G1Gc, containing cDNA encoding humanglucocerebrosidase and administered intravenously. Patients will be monitored for toxicity andglucocerebrosidase expression. (Protocol #9306-047)
Appendix D-49. Dr. Gary J. Nabel of the University of Michigan Medical Center, Ann Arbor, Michigan,
may conduct experiments on 12 patients with AIDS to be divided into 4 experimental groups. CD4(+)

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lymphocytes will be isolated from peripheral blood and transduced with Rev M10, a transdominant
inhibitory mutant of the rev gene of the human immunodeficiency virus (HIV). Transduction of the rev
mutant will be mediated either by the retrovirus vector, PLJ-cREV M10, or by particle-mediated genetransfer of plasmid DNA. Patients will be monitored for survival of the transduced CD4(+) cells bypolymerase chain reaction and whether Rev M10 can confer protection against HIV infection to CD4(+)
cells. (Protocol #9306-049)
Appendix D-50. Dr. Gary J. Nabel of the University of Michigan Medical Center, Ann Arbor, Michigan,
may conduct experiments on 24 patients with advanced cancer. Patients will undergo in vivotransduction with DNA/liposome complexes containing genes encoding the HLA-B7 histocompatibility
antigen and beta-2 microglobulin in a non-viral plasmid. These DNA/liposome complexes will beadministered either by intratumoral injection or catheter delivery. Patients will be monitored for enhanced
immune responses against tumor cells, and safe and effective doses will be determined. (Protocol #9306045)
Appendix D-51. Dr. John A. Barranger of the University of Pittsburgh, Pittsburgh, Pennsylvania, may
conduct experiments on 5 patients with Gaucher disease. The CD34(+) hematopoietic stem cells will be
isolated from peripheral blood and transduced in vitro with the retrovirus vector, N2-Sv-GC, encoding the
glucocerebrosidase (GC) enzyme. Following reinfusion of the transduced cells, patients will be monitoredby PCR analysis for GC expression in peripheral blood leukocytes. Patients currently receiving GC
replacement therapy and who demonstrate clinical responsiveness will be withdrawn from exogenous GC
therapy. Patients not previously treated with exogenous GC, will be monitored for clinical reversal of lipid
storage symptoms. (Protocol #9306-046)
Appendix D-52. Dr. Robert Walker of the National Institutes of Health, Bethesda, Maryland, may conduct
experiments on 12 HIV-infected patients who have a seronegative identical twin. CD4(+) and CD8(+)
cells will be isolated from the seronegative twin and induced to polyclonal proliferation with anti-CD3 andinterleukin-2. The enriched population of cells will be transduced with either LNL6 or G1Na, whichcontain the neoR gene. The transduced cells will be expanded in tissue culture and administered to theHIV-infected twin. Patients will be monitored for immune function and the presence of marked cells.
(Protocol #9209-026)
Appendix D-53. Dr. Corey Raffel of the Children’s Hospital Los Angeles, Los Angeles, California, and
Dr. Kenneth Culver of Iowa Methodist Medical Center, Des Moines, Iowa, may conduct experiments on
30 patients between 2 and 18 years of age with recurrent malignant astrocytoma. Fifteen patients will be
accrued into this study initially. If at least one patient responds to therapy, an additional 14 patients willbe treated. Patients with either surgically accessible or non-accessible tumors will be treated with thevector producing cell line (PA317) carrying the retrovirus vector, G1TkSvNa. This vector will transduce
tumor cells in vivo with the Herpes simplex thymidine kinase (HS-tk) gene that renders the cells sensitive
to killing by ganciclovir. Surgically accessible patients will undergo surgical debulking of their tumor
followed by repeated administration of the HS-tk vector producer cells into the tumor bed. Children withunresectable tumors will undergo stereotaxic injection of vector producer cells into tumors. (Protocol#9306-050)
Appendix D-54. Dr. Jeffrey E. Galpin of the University of Southern California, Los Angeles, California,
and Dr. Dennis A. Casciato of the University of California, Los Angeles, California, may conduct
experiments on 15 HIV(+) asymptomatic patients. Patients will receive 3 monthly intramuscular injectionsof the retrovirus vector (N2IIIBenv) encoding the HIV-1 IIIB envelope protein. Patients will be monitored
for acute toxicity, CD4 levels, HIV-specific CTL responses, and viral burdens. (Protocol #9306-048)
Appendix D-55. Drs. Charles Hesdorffer and Karen Antman of Columbia University College ofPhysicians and Surgeons, New York, New York, may conduct experiments on 20 patients with advanced
breast, ovary, and brain cancer. CD34(+) hematopoietic stem cells will be isolated from bone marrow,transduced with the retrovirus vector, PHaMDR1/A, and readministered to patients. Patients will bemonitored for the presence and expression of the MDR-1 gene. The investigators will determine whether
MDR-1 expression increases following chemotherapy. (Protocol #9306-051)

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Appendix D-56. Dr. Enzo Paoletti of Virogenetics Corporation, Troy, New York, may conduct
experiments with poxvirus vectors NYVAC, ALVAC, and TROVAC at Biosafety Level 1.
Appendix D-57. Drs. Richard C. Boucher and Michael R. Knowles of the University of North Carolina,
Chapel Hill, North Carolina, may conduct experiments on 9 patients (18 years old or greater) with cystic
fibrosis to test for the safety and efficacy of an E1-deleted recombinant adenovirus containing the cystic
fibrosis transmembrane conductance regulator (CFTR) cDNA, Ad.CB-CFTR. A single dose of 108, 3 x
109 or 1011 pfu/ml will be administered to the nasal cavity of 3 patients in each dose group. Patients will
be monitored by nasal lavage and biopsy to assess safety and restoration of normal epithelial function.
(Protocol #9303-042)
Appendix D-58. Dr. Joyce A. O'Shaughnessy of the National Institutes of Health, Bethesda, Maryland,
may conduct experiments on 18 patients (18-60 years old) with Stage IV breast cancer who haveachieved a partial or complete response to induction chemotherapy. This study will determine thefeasibility of obtaining engraftment of CD34(+) hematopoietic stem cells transduced by a retroviral vector,
G1MD, and expressing a cDNA for the human multi-drug resistance-1 (MDR-1) gene following high dose
chemotherapy, and whether the transduced MDR-1 gene confers drug resistance to hematopoietic cells
and functions as an in vivo dominant selectable marker. Patients will be monitored for evidence of
myeloprotection and presence of the transduced MDR-1 gene." (Protocol #9309-054)
Appendix D-59. Drs. Larry E. Kun, R. A. Sanford, Malcolm Brenner, and Richard L. Heideman of St.
Jude Children's Research Hospital, Memphis, Tennessee, and Dr. Edward H. Oldfield of the National
Institutes of Health, Bethesda, Maryland, may conduct experiments on 6 patients (3-21 years old) with
progressive or recurrent malignant supratentorial tumors resistant to standard therapies. Mouse cells
producing the retroviral vector containing the herpes simplex thymidine kinase gene (G1TKSVNa) will beinstilled into the tumor areas via multiple stereotactically placed cannulas. Patients will be treated with
ganciclovir to eliminate cells expressing the transduced gene. Patients will be monitored for central
nervous system, hematologic, renal or other toxicities, and for anti-tumor responses by magnetic
resonance imaging studies. (Protocol #9309-055)
Appendix D-60. The physical containment level may be reduced from Biosafety Level 3 to Biosafety
Level 2 for a Semliki Forest Virus (SFV) vector expression system of Life Technologies, Inc.,
Gaithersburg, Maryland.
Appendix D-61. Dr. Albert B. Deisseroth of the University of Texas MD Anderson Cancer Center,
Houston, Texas, may conduct experiments on 10 patients (≥ 16 to ≤ 60 years of age) with chronic
lymphocytic leukemia. Autologous peripheral blood and bone marrow cells will be removed from patients
following chemotherapy and marked by transduction with two distinguishable retroviral vectors, G1Na and
LNL6, containing the neomycin resistance gene. The gene marked cells will be reinfused into patients todetermine the efficiency of bone marrow purging and the origin of relapse following autologous bonemarrow transplantation. (Protocol #9209-030)
Appendix D-62. Dr. Jonathan Simons of the Johns Hopkins Oncology Center, Baltimore, Maryland, may
conduct experiments on 26 patients (≥ 18 years of age) with metastatic renal cell carcinoma to evaluate
the safety and tolerability of intradermally injected autologous irradiated tumor cells transduced with theretrovirus vector, MFG, containing the human granulocyte-macrophage colony stimulating factor gene.
Acute and long-term clinical toxicities and in vitro and in vivo induction of specific anti-tumor immune
responses will be monitored. (Protocol #9303-040)
Appendix D-63. Dr. Albert B. Deisseroth of the University of Texas MD Anderson Cancer Center,
Houston, Texas, may conduct experiments on 20 patients (≥ 18 and ≤ 60 years old) with ovarian cancer.
A murine viral vector was constructed from the third generation of L series retroviruses with the insert of
the human multi-drug resistance-1 (MDR-1) transduced gene. The investigators will assess the safety
and feasibility of administering CD34 (+) autologous peripheral blood and bone marrow cells. Patients

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will be monitored for the presence of the MDR-1 gene and for the effect of gene transfer on hematopoietic
function following the transplantation. (Protocol #9306-044)
Appendix D-64. Dr. Joseph Ilan of the Case Western Reserve University School of Medicine andUniversity Hospital of Cleveland, Cleveland, Ohio, may conduct experiments on 12 patients (≥ 18 years ofage) with advanced brain cancer. Human malignant glioma tumor cells will be cultured, transfected with
Epstein-Barr virus (EBV)-based vector, anti-Insulin growth factor-I, lethally irradiated, and injectedsubcutaneously into patients in an attempt to express antisense Insulin growth factor-1. Patients will bemonitored for toxicity and immunologic responses to the vector. (Protocol #9306-052)
Appendix D-65. Drs. James S. Economou and John Glaspy of the University of California, Los Angeles,
California, may conduct experiments on 30 patients (≥ 18 to ≤ 70 years of age) with metastaticmelanoma. A human melanoma cell line (M-24) will be transduced with the retroviral vector, G1NaCvi2,
expressing the human interleukin-2 (IL-2) gene. The IL-2 producing cells will be mixed with the patient's
autologous tumor cells, irradiated, and injected subcutaneously in an attempt to enhance the tumorspecific immunologic response. Patients will be monitored for toxicity, in vitro and in vivo immunologic
responses, and clinical anti-tumor effects. (Protocol #9309-058)
Appendix D-66. Drs. Peter Cassileth, Eckhard R. Podack, and Kasi Sridhar of the University of Miami,
and Niramol Savaraj of the Miami Veterans Administration Hospital, Miami, Florida, may conduct
experiments on 12 patients (≥ 18 years of age) with limited stage small cell lung cancer. Autologous
tumor cells will be removed, expanded in culture, and transduced by lipofection with the BMG-Neo-hIL2vector (derived from bovine papilloma virus). The objective of this protocol is to demonstrate the safety
and efficacy of administering IL-2 transduced autologous tumor cells in an attempt to stimulate a tumorspecific cytotoxic T lymphocyte (CTL) response, and to determine the quantity and characteristics of the
CTL that have been generated. (Protocol #9309-053)
Appendix D-67. Drs. Edward H. Oldfield and Zvi Ram of the National Institutes of Health, Bethesda,
Maryland, may conduct experiments on 20 patients (≥ 18 years of age) with leptomeningeal
carcinomatosis. The patients will receive intraventricular or subarachnoid injection of murine vector
producing cells containing the retroviral vector, G1Tk1SvNa. Tumor cells expressing the herpes simplex
thymidine kinase gene will be rendered sensitive to killing by subsequent administration of ganciclovir.
Patients will be monitored for safety and anti-tumor response by magnetic resonance imaging (MRI) andcerebral spinal fluid cytological analysis. (Protocol #9312-059)
Appendix D-68. Drs. Tapas K. Das Gupta and Edward P. Cohen of the University of Illinois College ofMedicine, Chicago, Illinois, may conduct experiments on 12 subjects who differ in at least 3 out of 6
alleles at the Class I histocompatibility locus (≥ 18 years of age) with Stage IV malignant melanoma. The
subjects will be immunized with a lethally irradiated allogeneic human melanoma cell line transduced withthe human interleukin-2 expressing retroviral vector, pZipNeoSv-IL-2. Subjects will be monitored for
toxicity, induction of B and T cell antitumor responses in vitro and in vivo, and any clinical evidence ofantitumor effect. (Protocol #9309-056)
Appendix D-69A. Dr. Michael J. Welsh of the Howard Hughes Medical Institute, Iowa City, Iowa, may
conduct experiments on 20 patients (≥ 18 years of age) with cystic fibrosis. The investigator willadminister increasing doses of either of the two adenovirus vectors, AD2/CFTR-1 or AD2-ORF6/PGKCFTR, to the nasal epithelium of 10 patients (1 nostril) or maxillary sinus epithelium of 10 patients (1maxillary sinus). Patients will be isolated for a period of 24 hours following vector administration;
however, if 1 patient demonstrates secreted virus at 24 hours, the investigator will notify the Recombinant
DNA Advisory Committee for reconsideration of the isolated period. Patients will be assessed for thesafety and efficacy of multiply administration of adenovirus vectors encoding the cystic fibrosis
transmembrane conductance regulator (CFTR) gene. (Protocol #9312-067)
Appendix D-69B. Dr. Richard Haubrich of the University of California at San Diego Treatment Center,
San Diego, California, may conduct experiments on 25 human immunodeficiency virus (HIV)-infected,
seropositive, asymptomatic subjects (≥ 18 to ≤ 65 years of age). Subjects will receive 3 monthly

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intramuscular injections of the retroviral vector, N2/IIIB env/rev, which encodes for HIV-1 IIIB env/rev
proteins. The objective of the study is to induce HIV-1- specific CD8(+) cytotoxic T lymphocyte andantibody responses in order to eliminate HIV-infected cells and residual virus. This Phase I/II study will
evaluate acute toxicity, identify long-term treatment effects, and evaluate the disease progression.
(Protocol #9312-062)
Appendix D-70. Dr. Mario Sznol of the National Institutes of Health, Frederick, Maryland, may conduct
experiments on 50 subjects (≥ 18 years of age) with advanced stage melanoma. Subjects will receive
subcutaneous injections of lethally irradiated allogeneic melanoma cells that have been transduced bylipofection with the plasmid DNA vector, CMV-B7, derived from bovine papilloma virus to express thehuman B7 antigen. The B7 antigen, which binds to the CD28 receptor of T cells, will serve as a costimulatory signal to elicit an antitumor immune response. Subjects will be monitored for induction ofcytotoxic T lymphocyte, antitumor responses in vitro and in vivo and any clinical evidence of antitumor
effect. (Protocol #9312-063)
Appendix D-71. Dr. Joseph Rubin of the Mayo Clinic, Rochester, Minnesota, may conduct experiments
on 15 subjects with hepatic metastases from advanced colorectal cancer (≥ 18 years of age). Subjects
will receive intratumoral hepatic injections of the plasmid DNA/lipid complex, pHLA-B7/β-2 microglobulin,
expressing a heterodimeric cell surface protein consisting of the HLA-B7 histocompatibility antigen and β2 microglobulin. Subjects must be HLA-B7 negative. The objective of this study is to determine a safeand effective dose of the DNA/lipid complex. Subjects will be monitored for antigen-specific immune
responses and in vivo HLA-B7 expression. (Protocol #9312-064)
Appendix D-72. Dr. Nicholas J. Vogelzang of the University of Chicago Medical Center, Chicago, Illinois,
may conduct experiments on 15 subjects with metastatic renal cell carcinoma ≥ 18 years of age.
Subjects will receive intratumoral injections of the plasmid DNA/liposome vector pHLA-B7/β-2
microglobulin, expressing a heterodimeric cell surface protein consisting of the HLA-B7 histocompatibility
antigen and β-2 microglobulin. Subjects must be HLA-B7 negative. Subjects will be monitored for
antigen-specific immune responses and in vivo HLA-B7 expression. (Protocol #9403-071)
Appendix D-73. Dr. Evan M. Hersh of the Arizona Cancer Center and Drs. Emmanuel Akporiaye, David
Harris, Alison T. Stopeck, Evan C. Unger, and James A. Warneke of the University of Arizona, Tucson,
Arizona, may conduct experiments on 15 subjects with metastatic malignant melanoma ≥ 18 years of age.
Subjects will receive intratumoral injections of the plasmid DNA/liposome vector, pHLA-B7/β-2
microglobulin, expressing a heterodimeric cell surface protein consisting of the HLA-B7 histocompatibility
antigen and β-2 microglobulin. Subjects must be HLA-B7 negative. Subjects will be monitored for
antigen-specific immune responses and in vivo HLA-B7 expression. (Protocol #9403-072)
Appendix D-74. Dr. Ralph Freedman of MD Anderson Cancer Center, Houston, Texas, may conductgene marking experiments on 9 subjects with ovarian carcinoma or peritoneal carcinomatosis (≥ 16 yearsof age). Autologous CD3(+)/CD8(+) tumor infiltrating lymphocyte derived T cells will be transduced withthe retroviral vector G1Na that encodes for neoR. Subjects will receive intraperitoneal administration of
bulk expanded transduced and nontransduced T cells and recombinant interleukin-2. Previously
documented tumor sites and normal tissues will be monitored for neoR and the proportion of
CD3(+)/CD8(+) T cells will be determined. (Protocol #9406-075)
Appendix D-75. Drs. Helen Heslop, Malcolm Brenner, and Robert Krance of St. Jude Children’s
Research Hospital, Memphis, Tennessee, may conduct gene marking experiments on 20 subjects
undergoing autologous bone marrow transplantation for therapy of leukemia or solid tumor (< 21 years ofage). The distinguishable retroviral vectors, LNL6 and G1Na (both encoding neoR), will be used todetermine the rate of reconstitution of untreated versus cytokine expanded CD34(+) selected autologous
bone marrow cells. (Protocol #9406-076)
Appendix D-76. Drs. Albert Deisseroth, Gabriel Hortobagyi, Richard Champlin, and Frankie Holmes of
MD Anderson Cancer Center, Houston, Texas, may conduct experiments on 10 fully evaluable subjects(maximum of 20 entered) with Stage III or IV breast cancer (≥ 18 and ≤ 60 years of age). Subjects will

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receive autologous CD34(+) peripheral blood cells that have been transduced with the retroviral vector,
pVMDR-1, which encodes the multi-drug resistance gene. The objective of this study is to evaluate thesafety and feasibility of transducing early hematopoietic progenitor cells with pVMDR-1 and to determine
in vivo selection of chemotherapy resistant hematopoietic cells. (Protocol #9406-077)
Appendix D-77. Drs. Johnson M. Liu and Neal S. Young of the National Institutes of Health, Bethesda,
Maryland, may conduct experiments on 6 patients with Fanconi anemia (≥ 5 years of age). Subjects will
receive autologous CD34(+) cells that have been transduced with the retroviral vector, FACC, which
encodes the normal Fanconi anemia complementation group C gene. The objective of this study is todetermine whether autologous FACC transduced hematopoietic progenitor cells can be safely
administered to subjects, the extent of engraftment, and correction of cell phenotype. (Protocol #9406078)
Appendix D-78. Drs. Robert E. Sobol and Ivor Royston of the San Diego Regional Cancer Center, San
Diego, California, may conduct experiments on 15 subjects with recurrent residual glioblastoma multiforme (≥ 18 years of age). Subjects will receive subcutaneous injections of autologous tumor cells thathave been lethally irradiated and transduced with the retroviral vector, G1NaCvi2.23, which encodes forinterleukin-2. Subjects will be monitored in vitro for cellular and humoral antitumor responses and in vivo
for antitumor activity. (Protocol #9406-080)
Appendix D-79. Dr. Alfred E. Chang of the University of Michigan Medical Center, Ann Arbor Michigan,
may conduct gene marking experiments on 15 subjects with metastatic melanoma (≥ 18 years of age).
Subjects will undergo adoptive immunotherapy of anti-CD3/interleukin-2 activated lymph node cells thathave been primed in vivo with tumor cells that have been transduced with the retrovirus vector, GBAH4,
encoding the gene for interleukin-4. The investigator will evaluate the antitumor efficacy and in vivo
immunological reactivity in subjects receiving adoptively transferred T cells, and the in vitro immunologicalreactivities of the activated T cells that might correlate with their in vivo antitumor function. (Protocol#9312-065)
Appendix D-80. Dr. Robert Walker of the National Institutes of Health, Bethesda, Maryland, may conductgene marking experiments on 40 HIV(+) subjects (≥ 18 years of age). The investigator may also enter an
additional number of subjects (to be determined by the investigator) who will receive a single
administration of 1 x 107 HIV-specific CD8(+) cells. The investigator will: (1) Assess the safety andtolerance of the adoptive transfer of anti-HIV cytotoxic, syngeneic, CD8(+) peripheral blood lymphocytesthat have been transduced with the retrovirus
vector, rkat4svgF3e-, that encodes for a universal chimeric T cell receptor. (2) Determine the longevity of
the genetically marked CD8(+) lymphocytes in the subject's peripheral blood. (Protocol #9403-069)
Appendix D-81. Dr. Joseph Rosenblatt of the University of California, Los Angeles, California, and Dr.
Robert Seeger of Children’s Hospital, Los Angeles, California, may conduct gene transfer experiments on
18 subjects with neuroblastoma (≤ 21 years of age). Patients at high risk of relapse with minimal or no
detectable disease following myeloablative therapy and autologous bone marrow transplantation, or
patients with progressive or persistent disease despite conventional therapy will be serially immunizedwith autologous or allogeneic neuroblastoma cells transduced to express γ interferon. Cells will betransduced with the retroviral vector, pHuγ-IFN, encoding the human gene for γ interferon and lethally
irradiated prior to use as an immunogen. The objectives of the study are: (1) to determine the maximum
tolerable dose of transduced cells; (2) to determine the local, regional, and systemic toxicities of injected
cells; and (3) to determine the antitumor response in vivo as measured by standard clinical tests andimmunocytologic evaluation of marrow metastases. (Protocol #9403-068)
Appendix D-82. Dr. Kenneth L. Brigham of Vanderbilt University, Nashville, Tennessee, may conductgene transfer experiments on 10 subjects (≤ 21 years of age) in two different patient protocols (5 for each
protocol). Both protocols will use the same DNA/liposome preparations to deliver a plasmid DNA
construct, pCMV4-AAT, encoding human alpha-1 antitrypsin gene driven by a cytomegalovirus promoter.
In patients scheduled for elective pulmonary resection, the DNA/liposome complexes will be instilled
through a fiber optic bronchoscope into a subsegment of the lung. Tissues of the lung will be obtained at

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the time of surgery. Transgene expression will be assessed by immunohistochemistry, in situ
hybridization, and Western and Northern blot analyses. The effect of DNA/liposome complex
administration on the histological appearance of the lung will also be evaluated. In patients with alpha-1
antitrypsin deficiency, the DNA/liposome complexes will be instilled into the nostril. Transgene
expression will be determined in cells obtained by nasal lavage and nasal scraping, and the time courseof transgene expression will be measured. The secretion of the alpha-1 antitrypsin protein in nasal fluid
will be determined. Histological appearance of nasal mucosa will also be examined. The study willassess safety and feasibility of gene delivery to the human respiratory tract. (Protocol #9403-070)
Appendix D-83. Dr. H. Kim Lyerly of Duke University Medical Center, Durham, North Carolina, may
conduct gene transfer experiments on 20 subjects with refractory or recurrent metastatic breast cancer (≥18 years of age). Autologous breast cancer cells will be transduced with the DNA/liposome complex,
pMP6-IL2, containing a plasmid DNA vector derived from adeno-associated virus (AAV) that expresses
the gene for human interleukin-2. Subjects will receive 4 subcutaneous injections of lethally irradiatedtumor cells transduced with the DNA/liposome complex prior to injection. The objective of this study is to:(1) evaluate the safety and toxicity of the treatment, (2) determine the immunological effects, (3)determine the duration of clinical responses, and (4) measure patient survival. (Protocol #9409-086)
Appendix D-84. Drs. Flossie Wong-Staal, Eric Poeschla, and David Looney of the University ofCalifornia at San Diego, La Jolla, California, may conduct gene transfer experiments on 6 subjects (≥ 18
and ≤ 65 years of age) infected with human immunodeficiency virus-1 (HIV-1). Autologous CD4(+) T
lymphocytes will be transduced ex vivo with the retroviral vector, pMJT, expressing a hairpin ribozymethat cleaves the HIV-1 RNA in the 5' leader sequence. The transduced cells will be expanded andreinfused into the patients. The objectives of the study are: (1) to evaluate safety of reinfusing thetransduced lymphocytes, (2) to compare (in vivo) the kinetics and survival of ribozyme-transduced cellswith that of cells transduced with a control vector, (3) to determine in vivo expression of the ribozyme
sequences in transduced lymphocytes, (4) to determine whether host immune responses directed against
the transduced cells will occur in vivo, and (5) to obtain preliminary data on the effects of ribozyme genetherapy on in vivo HIV mRNA expression, viral burden and CD4(+) lymphocyte levels. (Protocol #9309057)
Appendix D-85. Dr. Friedrich Schuening of the Fred Hutchinson Cancer Research Center, Seattle,
Washington, may conduct gene transfer experiments on 10 subjects (≥ 18 years of age) with Type I
Gaucher's disease. The peripheral blood repopulating cells (mobilized by patient pretreatment withrecombinant granulocyte colony-stimulating factor) will be harvested and CD34(+) cells selected.
CD34(+) cells will be transduced ex vivo with the retroviral vector, LgGC, that encodes humanglucocerebrosidase cDNA. Following transduction, the transduced cells will be infused into the patient
without myeloablative treatment. The primary endpoint of this study is to examine the safety of infusing
CD34(+) cells transduced with the human glucocerebrosidase cDNA. Patients will be monitored for
persistence and expression of the glucocerebrosidase gene in hematopoietic cells. (Protocol #9312-061)
Appendix D-86. Dr. Terence R. Flotte of the Johns Hopkins Children's Center, Baltimore, Maryland, may
conduct gene transfer experiments on 16 subjects (≥ 18 years of age) with mild cystic fibrosis (CF). An
adeno-associated virus (AAV) derived vector, encoding cystic fibrosis transmembrane conductanceregulator (CFTR) gene, (tgAAVCF), will be administered to nasal (direct) and airway (bronchoscope)epithelial cells. This is a dose escalation study involving 8 cohorts. Each subject will receive both
intranasal and bronchial administration of the adenoviral vector at 4 escalating doses. Nasal doses will
range between 1 x 106 and 1 x 109 pfu. Lung administration will range between 1 x 107 and 1 x 1010 pfu.
The primary goal of the study is to assess the safety of vector administration. Respiratory and nasal
epithelial cells will be evaluated for gene transfer, expression, and physiologic correction. (Protocol#9409-083)
Appendix D-87. Drs. Jeffrey M. Isner and Kenneth Walsh of St. Elizabeth's Medical Center, Tufts
University, Boston, Massachusetts, may conduct gene transfer experiments on 12 subjects (≥ 40 years ofage) with peripheral artery disease (PAD). A plasmid DNA vector, phVEGF165, encoding the humangene for vascular endothelial growth factor (VEGF) will be used to express VEGF to induce collateral

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neovascularization. Percutaneous arterial gene transfer will be achieved using an angioplasty catheterwith a hydrogel coated balloon to deliver the plasmid DNA vector to the artery. The objectives of thestudy are: (1) to determine the efficacy of arterial gene therapy to relieve rest pain and/or heal ischemic
ulcers of the lower extremities in patients with PAD; and (2) to document the safety of the phVEGF arterial
gene therapy for therapeutic angiogenesis. Subjects will undergo anatomic and physiologic examination
to determine the extent of collateral artery development following phVEGF arterial gene therapy.
(Protocol #9409-088)
Appendix D-88A. Dr. Ronald G. Crystal of New York Hospital-Cornell Medical Center, New York, New
York, may conduct gene transfer experiments on 26 patients (≥ 15 years of age) with cystic fibrosis (CF).
A replication deficient recombinant adenovirus vector will be used to transduce epithelial cells of the large
bronchi with the E1/E3 deleted type 5 adenovirus vector, AdGVCFTR.10, which encodes the human cystic
fibrosis transmembrane conductance regulator (CFTR) gene. The objective of this study is to define thesafety and pharmacodynamics of CFTR gene expression in airway epithelial cells following single
administration of
escalating doses to the vector. If single administration is determined to be safe, subjects will undergo
repeat administration to localized areas of the bronchi. (Protocol #9409-085)
Appendix D-88B. Drs. Eric J. Sorscher and James L. Logan of the University of Alabama, Birmingham,
Alabama, may conduct gene transfer experiments on 9 subjects (≥18 years of age) with cystic fibrosis(CF). The normal human cystic fibrosis transmembrane conductance regulator (CFTR) gene will be
expressed by a plasmid DNA vector, pKCTR, driven by the simian virus-40 (SV40) early gene promoter.
The CFTR DNA construct will be delivered by cationic liposome-based gene transfer to nasal epithelial
cells. The objectives of the study are to: (1) evaluate the safety of lipid-mediated gene transfer to nasal
epithelial cells (including local inflammation and mucosal tissue); and (2) evaluate efficacy as determinedby correction of the chloride ion transport defect, and wild-type CFTR mRNA and protein expression.
(Protocol #9312-066)
Appendix D-89. Dr. Steven M. Albelda of the University of Pennsylvania Medical Center, Philadelphia,
Pennsylvania, may conduct gene transfer experiments on 12 subjects with advanced mesothelioma. The
adenovirus vector encoding the Herpes simplex virus thymidine kinase (HSV-TK) gene, H5.020RSVTK,
will be administered through a chest tube to the pleural cavity. Tumor biopsies will be assayed for genetransfer and expression. Subjects will be monitored for immunological responses to the adenovirus
vector. Ganciclovir will be administered intravenously 14 days following vector administration. The
primary objective of this Phase I study is to evaluate the safety of direct adenovirus vector gene delivery
to the pleural cavity of patients with malignant melanoma. (Protocol #9409-090)
Appendix D-90. Drs. Jeffrey Holt and Carlos B. Arteaga of the Vanderbilt University, Nashville,
Tennessee, may conduct gene transfer experiments on 10 female patients (over 18 years of age) withmetastatic breast cancer. Patient effusions from pleura or peritoneum will be drained and the fluid will be
replaced with supernatant containing the retroviral vectors, XM6:antimyc or XM6:antifos, which express cmyc and c-fos antisense sequences, respectively, under the control of a mouse mammary tumor virus
promoter. The objectives of this study are to: (1) assess uptake and expression of the vector sequencesin breast cancer cells present in pleural and peritoneal fluids, and determine if this expression is tumor
specific, (2) assess the safety of localized administration of antisense retroviruses, and (3) monitor
subjects for clinical evidence of antitumor response. (Protocol #9409-084)
Appendix D-91. Dr. Jack A. Roth of MD Anderson Cancer Center, Houston, Texas, may conduct genetransfer experiments on 14 non-small cell lung cancer subjects (≥ 18 and ≤ 80 years of age) who havefailed conventional therapy and who have bronchial obstruction. LNSX-based retroviral vectors
containing the β-actin promoter will be used to express: (1) the antisense RNA of the K-ras oncogene(LN-K-rasB), and (2) the wildtype p53 tumor suppressor gene (LNp53B). Tumor biopsies will be obtained
to characterized K-ras and p53 mutations. Relative to their specific mutation, subjects will undergo partial
endoscopic resection of the tumor bed followed by bronchoscopic administration of the appropriate
retrovirus construct. The objective of this study is to evaluate the safety and efficacy of intralesional

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administration of LN-K-rasB and LNp53 retrovirus constructs. (Protocol #9403-031)
Appendix D-92. Drs. Robert E. Sobol and Ivor Royston of the San Diego Regional Cancer Center, San
Diego, California, may conduct gene transfer experiments on 12 subjects (≥ 18 years of age) withmetastatic colon carcinoma. The autologous skin fibroblasts will be transduced with the retroviral vector,
LNCX/IL-2, which encodes the gene for human interleukin-2 (IL-2). In this dose-escalation study,
subjects will receive subcutaneous injections of lethally irradiated autologous tumor cells. The objectivesof the study are to: (1) evaluate the safety of subcutaneous administration of LNCX/IL-2 transduced
fibroblasts, (2) determine in vivo antitumor activity, and (3) monitor cellular and humoral antitumor
responses. (Protocol #9312-060)
Appendix D-93. Dr. Michael Lotze of the University of Pittsburgh, Pittsburgh, Pennsylvania, may conductgene transfer experiments on 18 subjects (≥ 18 years of age) with advanced melanoma, 6 with T-cell
lymphoma, breast cancer, or head and neck cancer. Subjects should have accessible cutaneous tumors,and have failed standard therapy. Over 4 weeks, subjects will receive a total of 4 intratumoral injectionsof autologous fibroblasts transduced with the retrovirus vector, TFG-hIL-12-Neo. This vector, which
consists of the murine MFG backbone, expresses both the p35 and p40 subunits of interleukin-12 (IL-12)
and the neoR selection marker. The objectives of the study are to: (1) define the local and systemic
toxicity associated with peritumoral injections of gene-modified fibroblasts, (2) examine the local andsystemic immunomodulatory effects of these injections, and (3) evaluate clinical antitumor efficacy.
(Protocol #9406-081)
Appendix D-94. Drs. Evan Hersh, Emmanuel Akporiaye, David Harris, Alison Stopeck, Evan Unger,
James Warneke, of the Arizona Cancer Center, Tucson, Arizona, may conduct gene transfer experiments
on 25 subjects (≥ 18 years of age) with solid malignant tumors or lymphomas. A plasmid DNA/lipid
complex designated as VCL-1102 (IL-2 Plasmid DNA/DMRIE/DOPE) will be used to transduce the humangene for interleukin-2 (IL-2). Patients with advanced cancer who have failed conventional therapy will
undergo a procedure in which VCL-1102 is injected directly into the tumor mass to induce tumor-specificimmunity. The objectives of the study are to: (1) determine safety and toxicity associated with escalating
doses of VCL-1102; (2) confirm IL-2 expression in target cells; (3) determine biological activity andpharmacokinetics; and (4) determine whether IL-2 expression stimulates tumor regression in subjects withmetastatic malignancies. (Protocol #9412-095)
Appendix D-95. Drs. Richard Morgan and Robert Walker of the National Institutes of Health, Bethesda,
Maryland, may conduct gene transfer experiments on 48 human immunodeficiency virus (HIV)seropositive subjects (≥ 18 years of age). This Phase I/II study involves identical twins (one HIV
seropositive and the other HIV seronegative). CD4(+) T cells will be enriched following apheresis of the
HIV seronegative twin, induced to polyclonal proliferation with anti-CD3 and recombinant IL-2, transducedwith either the LNL6/NeoR or G1Na/NeoR, and transduced with up to 2 additional retroviral vectors(G1RevTdSN and/or GCRTdSN(TAR)) containing potentially therapeutic genes (antisense TAR and/ortransdominant Rev). These T cell populations will be expanded 10 to 1,000 fold in culture for 1 to 2
weeks and reinfused into the HIV seropositive twin. Subjects will receive up to 4 cycles of treatmentusing identical or different combinations of control and anti-HIV retrovirus vectors. The relative survival ofthese transduced T cell populations will be monitored by vector-specific polymerase chain reaction, while
the subjects' functional immune status is monitored by standard in vitro and in vivo assays. (Protocol#9503-103)
Appendix D-96. Dr. Harry L. Malech of the National Institutes of Health, Bethesda, Maryland, may
conduct gene transfer experiments on 2 subjects ≥ 18 years of age (with or without concurrent serious
infection), and 3 subjects ≥ 18 years of age (with or without concurrent serious infection) or minors 13-17
years of age who have concurrent serious infection who have chronic granulomatous disease (CGD).
CGD is an inherited immune deficiency disorder in which blood neutrophils and monocytes fail to produce
antimicrobial oxidants (p47phox mutation) resulting in recurrent life-threatening infections. Subjects will
undergo CD34(+) mobilization with granulocyte colony stimulating factor (G-CSF). These CD34(+) cells
will be transduced with the retrovirus vector, MFG-S-p47phox, which encodes the gene for normal p47phox.
The objectives of this study are to: (1) determine the safety of administering MFG-S-p47phox transduced

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CD34(+) cells, and (2) demonstrate increased functional oxidase activity in circulating neutrophils.
(Protocol #9503-104)
Appendix D-97. Drs. Chris Evans and Paul Robbins of the University of Pittsburgh, Pittsburgh,
Pennsylvania, may conduct gene transfer experiments on 6 subjects (≥ 18 and ≤ 76 years of age) withrheumatoid arthritis. Rheumatoid arthritis is a chronic, progressive disease thought to be of autoimmune
origin. A gene encoding an interleukin-1 receptor antagonist protein (IRAP) will be delivered to therheumatoid metacarpal-phalangeal joints to determine the autoimmune reactions can be interrupted. The
vector construct, DFG-IRAP, is based on the MFG murine retrovirus vector backbone, and encodes thehuman IRAP gene. Synovial fibroblasts will be generated from the rheumatoid arthritic joint tissue
obtained from patients who are scheduled to undergo surgery. The fibroblasts will be transduced with the
DFG-IRAP vector, and the transduced cells injected into the synovial space. The synovial fluid and jointmaterial will be collected 7 days later to determine the presence and location of the transduced synovial
fibroblasts and the level of IRAP in the joint fluid. (Protocol 9406-074)
Appendix D-98. Dr. R. Scott McIvor of the University of Minnesota, Minneapolis, Minnesota, may
conduct gene transfer experiments on 2 children with purine nucleoside phosphorylase (PNP) deficiency.
PNP deficiency results in severe T-cell immunodeficiency, an autosomal recessive inherited disease
which is usually fatal in the first decade of life. Autologous peripheral blood lymphocytes will be cultured
in an artificial capillary cartridge in the presence of anti-CD3 monoclonal antibody and interleukin-2 andtransduced with the retroviral vector, LPNSN-2, encoding human PNP. Subjects will undergo bimonthly
intravenous administration of transduced T cells for a maximum of 1 year. The objectives of the study are
to determine: (1) the safety of intravenous administration of transduced T cells in children with PNP
deficiency, (2) the efficiency of PNP gene transfer and duration of gene expression in vivo, and (3) the
effect of PNP gene transfer on immune function. (Protocol #9506-110)
Appendix D-99. Drs. Nikhil C. Munshi and Bart Barlogie of the University of Arkansas School for Medical
Sciences, Little Rock, Arkansas, may conduct gene transfer experiments on 21 subjects (>18 and <65
years of age) with relapsed or persistent multiple myeloma who are undergoing T cell depleted allogeneic
bone marrow transplantation. Donor peripheral blood lymphocytes will be cultured in vitro withinterleukin-2 and anti-CD3 monoclonal antibody. T cell depleted lymphocytes will be transduced with theretroviral construct, G1Tk1SvNa.7, which encodes the Herpes simplex virus thymidine kinase (HSV-TK)
gene. The transduced cells will be reinfused. In this dose escalation study, 3 subjects will undergo cellmediated gene transfer per cohort (maximum of 5 cohorts) until Grade III or IV Graft versus Host Disease(GVHD) is observed. A maximum of 6 additional patients may be entered at that maximum tolerated
dose. The objectives of this study are to determine the: (1) safety of transduced donor cell infusions, (2)effectiveness of donor cell infusions in decreasing the effects of severe GVHD, (3) effectiveness of donor
cell infusions in prolonging multiple myeloma remission, and (4) effectiveness of ganciclovir in eliminating
donor cells for the purpose of preventing the depletion of erythrocytes. (Protocol #9506-107)
Appendix D-100. Dr. Wayne A. Marasco of Dana-Farber Cancer Institute, Boston, Massachusetts, may
conduct gene transfer experiments on 6 subjects (≥ 18 and ≤ 65 years of age) with humanimmunodeficiency virus type-1 (HIV-1). Autologous lymphocytes from asymptomatic subjects will betransduced ex vivo with a retroviral vector, LNCs105, encoding the sFv105 antibody specific for the HIV-1
envelope protein. An identical aliquot will be simultaneously transduced with a control retroviral vector
lacking the sFv105 cassette. Transduced cells will be reinfused into patients and the differential survivalof both populations of CD4+ lymphocytes compared. The objective of the study is to determine whether
the intracellular expression of a human single chain antibody against HIV-1 envelope glycoprotein gp160
that blocks gp160 processing and the production of infectious virions can safely prolong the survival of
CD4(+) lymphocytes in HIV-1-infected subjects. (Protocol #9506-111)
Appendix D-101. Dr. Henry Dorkin of the New England Medical Center, Boston, Massachusetts, and Dr.
Allen Lapey of Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts,
propose to conduct gene transfer experiments on 16 subjects (≥ 18 years of age). An E1/partial E4deleted, replication-deficient, type 2 adenovirus vector, AD2/CFTR-2, will be used to deliver the humancystic fibrosis transmembrane conductance regulator (CFTR) gene by aerosol administration

Page 68 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)(nebulization) to the lung of CF patients. Aerosol administration will be initiated only after initial safety
data has been obtained from the lobar administration protocol (#9409-091). This is a single
administration dose-escalation study in which subjects will receive between 8 x 106 and 2.5 x 1010 pfu.
Subjects will be assessed for evidence of adverse, systemic, immune, inflammatory, or respiratory effects
in response to AD2/CFTR-2. Subjects will be monitored for virus shedding and transgene expression.
Health care workers present in the facility will be required to sign an Informed Consent document
regarding the possibility of virus transmission. (Protocol #9412-074)
Appendix D-102. Drs. Charles J. Link and Donald Moorman of the Human Gene Therapy ResearchInstitute, Des Moines, Iowa, may conduct gene transfer experiments on 24 female subjects (≥ 18 years ofage) with refractory or recurrent ovarian cancer. Subjects will undergo intraperitoneal delivery (viaTenkhoff catheter) of the vector producing cells (VPC), PA317/LTKOSN.2. These VPC express the
Herpes simplex virus thymidine kinase (HSV-TK) gene which confers sensitivity to killing by the antiviraldrug, ganciclovir (GCV). The LTKOSN.2 retrovirus vector is based on the LXSN backbone. Two weeks
following intraperitoneal delivery of the VPC, subjects will receive 5 mg/kg intravenous GCV twice daily for
14 days. Subjects will receive between 1 x 105 and 1 x 108 VPC/kg in this dose escalation study.
Subjects will be evaluated by X-ray and peritoneoscopy of the abdomen for evidence of clinical response.
The objectives of this study are to determine the safety of intraperitoneal VPC administration. (Protocol#9503-100)
Appendix D-103. Dr. David T. Curiel of the University of Alabama, Birmingham, Alabama, may conductgene transfer experiment of 15 subjects (≥ 18 years of age) with metastatic colorectal cancer. Subjects
will receive intramuscular injection of the polynucleotide vaccine, pGT63, which is a plasmid DNA vector
expressing carcinoembryonic antigen (CEA) and hepatitis B surface antigen (HBsAg). The objectives ofthe study are to: (1) characterize the immune response to CEA and HBsAg following a single
intramuscular injection and following 3 consecutive intramuscular injections, and (2) determine the safety
of intramuscular injection of the plasmid DNA vector at doses ranging between 0.1 to 1.0 milligrams(single dose) and 0.9 to 3.0 milligrams (total multidose). (Protocol #9506-073)
Appendix D-104. Dr. Chester B. Whitley of the University of Minnesota, Minneapolis, Minnesota, may
conduct gene transfer experiments on two adult subjects (18 years of age or older) with mild Hunter
syndrome (Mucopolysaccharidosis Type II). The autologous peripheral blood lymphocytes will betransduced ex vivo with the retroviral vector, L2SN, encoding the human cDNA for iduronate-2-sulfatase(IDS). The transduced lymphocytes will be reinfused into the patients on a monthly basis. The study will
determine the frequency of peripheral blood lymphocyte transduction and the half-life of the infused cells.
Evaluation of patients will include measurement of blood levels of IDS enzyme, assessment of metabolic
correction by urinary glycosaminoglycan levels, clinical response of the disease, and monitoring forpotential toxicity. This Phase I study is to demonstrate the safety of the L2SN-mediated gene therapy
and to provide a preliminary evaluation of clinical efficacy. (Protocol #9409-087)
Appendix D-105. Drs. James Economou, John Glaspy, and William McBride of the University ofCalifornia, Los Angeles, California, may conduct gene transfer experiments on 25 subjects (≥ 18 years ofage) with metastatic melanoma. The protocol is an open label, Phase I trial to evaluate the safety andimmunological effects of administering lethally irradiated allogeneic and autologous melanoma cells
transduced with the retroviral vector, IL-7/HyTK, which encodes the gene for human interleukin-7 (IL-7).
Subjects will receive 1 x 107 irradiated unmodified autologous tumor cells in combination with escalating
doses of IL-7/HyTK transduced allogeneic melanoma cells (M24 cell line). The number of M24 cells
administered will be adjusted based on the level of IL-7 expression. Subjects will receive 3 biweekly
subcutaneous injections of M24 cells expressing 10, 100, or 1000 nanograms of IL-7/hour in vivo. A final
cohort of 5 subjects will receive IL-7/HyTK transduced autologous cells. Subjects will be monitored for
antitumor activity by skin tests, biopsy analysis, tumor-specific antibody activity, and cytotoxic T
lymphocyte precursor evaluation. Non-immunologic parameters will also be monitored. (Protocol #9503101)
Appendix D-106. Dr. Jack A. Roth, MD Anderson Cancer Center, may conduct gene transfer
experiments on 42 subjects (≥ 18 years of age) with refractory non-small cell lung cancer (NSCLC).

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Subjects will receive direct intratumoral injection of a replication-defective type 5 adenovirus vector,
AD5CMV-p53, to deliver the normal human p53 tumor suppressor gene. The E1 region of AD5CMV-p53
has been replaced with a p53 expression cassette containing the human cytomegalovirus promoter(CMV). Subjects will be divided into 2 treatment groups: (1) 21 subjects will receive Ad5CMV-p53 alone,and (2) 21 subjects will receive Ad5CMV-p53 in combination with cisplatin. Following vector
administration, subjects will be isolated for 96 hours during which time, assays will be conducted todemonstrate the lack of shedding of adenovirus vector. The objectives of this study are determine: (1)the maximum tolerated dose of AD5CMV-p53, (2) qualitative and quantitative toxicity related to vector
administration, and (3) biologic activity.
Prior to administration, adenovirus vector stocks will be screened for p53 mutants using the SAOS
osteosarcoma cell assay that was submitted by Dr. Roth on June 23, 1995. This biologic assay
compares the activity of a standard stock of Adp53 vector to the activity of newly produced stocks. The
standard stock of Adp53 will be defined as mediating cell death in 100% of SAOS cells (humanosteosarcoma cell line with homozygous p53 deletion) at an MOI of 50:1 (titer > 5 x 1010) on day 5 ofculture. The sensitivity of the assay for detecting inactive (presumed mutant) Adp53 vector will be
determined by adding increasing amounts of Adluc (control adenovirus vector containing the luciferasegene) to the Adp53 stock to determine the percentage of inactive vector required to decrease growth
inhibition of SAOS cells mediated by Adp53. The test lot of Adp53 will be tested for its ability to inhibit
SAOS in a 5 day assay. Significant loss of inhibitory activity compared with the standard would indicate
an unacceptable level of inactive (presumed mutant) vector. (Protocol #9406-079)
Appendix D-107A. Dr. Gary Clayman. M.D. Anderson Cancer Center, Houston, Texas, may conductgene transfer experiments on 21 subjects (≥ 18 years of age) with refractory squamous cell carcinoma of
the head and neck. Subjects will receive direct intratumoral injection of a replication-defective type 5
adenovirus vector, AD5CMV-p53, to deliver the normal human p53 tumor suppressor gene. The E1
region of AD5CMV-p53 has been replaced with a p53 expression cassette containing the humancytomegalovirus promoter (CMV). Subjects will be divided into 2 treatment groups: (1) those with nonresectable tumors, and (2) those with surgically accessible tumors. Subjects will receive multiple
injections of vector in each dose-escalation cohort. Following vector administration, subjects will be
isolated for 48 hours during which time, assays will be conducted to demonstrate the lack of shedding of
adenovirus vector. The objectives of the study are to determine: (1) the maximum tolerated dose of
AD5CMV-p53, (2) qualitative and quantitative toxicity related to vector administration, and (3) biologic
activity.
Prior to administration, adenovirus vector stocks will be screened for p53 mutants using the SAOS
osteosarcoma cell assay that was submitted by Dr. Roth on June 23, 1995. This biologic assay
compares the activity of a standard stock of Adp53 vector to the activity of newly produced stocks. The
standard stock of Adp53 will be defined as mediating cell death in 100% of SAOS cells (humanosteosarcoma cell line with homozygous p53 deletion) at an MOI of 50:1 (titer > 5x1010) on day 5 ofculture. The sensitivity of the assay for detecting inactive (presumed mutant) Adp53 vector will be
determined by adding increasing amounts of Adluc to the Adp53 stock to determine the percentage ofinactive vector required to decrease growth inhibition of SAOS cells mediated by Adp53. The test lot of
Adp53 will be tested for its ability to inhibit SAOS in a 5 day assay. Significant loss of inhibitory activity
compared with the standard would indicate an unacceptable level of inactive (presumed mutant) vector.
(Protocol #9412-096)
Appendix D-107B. Drs. Bernard A. Fox and Walter J. Urba of Earle A. Chiles Research Institute,
Providence Medical Center, Portland, Oregon, may conduct gene transfer experiments on 18 subjects (≥18 years of age) with metastatic renal cell carcinoma or melanoma. Autologous tumor cells will besurgically removed, transduced in vitro with the cationic liposome plasmid vector, VCL-1005, which
encodes human leukocyte antigen (HLA)-B7 and beta-2 microglobulin. Subjects will receive
subcutaneous injection of lethally irradiated transduced cells in one limb. The contralateral limb will be
injected with lethally irradiated untransduced tumor cells in combination with Bacille Calmette-Guerin(BCG). Approximately 21 days following tumor cell injection, subjects will undergo lymphadenectomy for
subsequent in vitro expansion of anti-CD3 activated lymphocytes. Activated lymphocytes will bePage 70 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

adoptively transferred on approximately day 35 in combination with a 5-day course of interleukin-2 (IL-2).
On approximately day 45, subjects will receive a second cycle of IL-2. The objectives of this study are to
determine: (1) the safety of administering anti-CD3 activated antitumor effector T cells in draining lymph
nodes, and (2) whether HLA-B7/β-2 gene transfer augments the sensitization of anti-tumor effector Tcells in draining lymph nodes. (Protocol 9506-108)
Appendix D-108. Dr. Mitchell S. Steiner, University of Tennessee, Memphis, Tennessee, and Dr. Jeffrey
T. Holt, Vanderbilt University School of Medicine, Nashville, Tennessee, may conduct gene transfer
experiments on 15 male subjects (35 to 75 years of age) with metastatic prostate cancer. Malignant cells
obtained from advanced prostate cancer subjects have been demonstrated to express high levels of the
protooncogene c-myc in vivo. The mouse mammary tumor virus (MMTV) long terminal repeat (LTR) is
expressed at high levels in prostate tissue. Following removal of malignant cells via biopsy, subjects will
receive a single transrectal ultrasound-guided intraprostate quadrant injection of the retrovirus vector,
XM6:MMTV-antisense c-myc, for 4 consecutive days at the site of the original biopsy. The objectives ofthis Phase I study are to: (1) quantitatively assess the uptake and expression of XM6:MMTV-antisense cmyc by prostate cancer cells in vivo, (2) determine whether c-myc gene expression is prostate tumorspecific, (3) assess safety of intraprostate injection of XM6:MMTV-antisense c-myc, and (4) biologic
efficacy (antisense inhibition of tumor growth). (Protocol #9509-123)
Appendix D-109. Drs. Ronald G. Crystal, Edward Hershowitz, and Michael Lieberman, New York
Hospital-Cornell Medical Center, New York, New York, may conduct gene transfer experiments on 18
subjects (18 to 70 years of age) with metastatic colon carcinoma with liver metastases. In this Phase I
dose-escalation study, subjects will receive computed tomography (CT)-guided intratumoral injections of
the adenovirus vector, AdGVCD.10, into the same hepatic metastasis in 4 equal volumes (100 microliters),
each with a separate entry into the liver. This dosage schedule will be performed on Days 1 and 7. 5fluorocytosine (200 milligrams/ kilogram/24 hours) will be administered orally in 4 equal doses starting on
day 2 and continuing through the time of laparotomy. The objectives of this study are to: (1) determine
the dose-dependent toxicity of direct administration of AdGVCD.10 to hepatic metastases combined withoral administration of 5-fluorocytosine, (2) quantitatively assess transfer and expression of the cytosine
deaminase gene in target cells, and (3) determine the biologic effects of direct ADGVCD.10 administration
to hepatic metastases. (Protocol #9509-125)
Appendix D-110. Drs. Andres Berchuck and H. Kim Lyerly of Duke University Medical Center, Durham,
North Carolina, may conduct gene transfer experiments on 18 subjects (≥ 18 years of age) with refractory
metastatic ovarian cancer. Autologous tumor cells obtained from ascites or surgically removed tumor willbe transduced with the cationic liposome vector, PMP6A-IL2, that contains an adeno-associated virus
derived plasmid DNA, a cytomegalovirus (CMV) promoter, and interleukin-2 (IL-2) complementary DNA(cDNA). In this dose-escalation study, subjects will undergo 4 cycles of intradermal injections (thigh orabdomen) of ex vivo transduced, lethally irradiated tumor cells in an attempt to induce an antitumor
response. The objectives of the study are to evaluate: (1) the safety of intradermally injected transduced
cells, and (2) antitumor response following therapy. (Protocol #9506-110)
Appendix D-111. Drs. Stephen L. Eck and Jane B. Alavi of the University of Pennsylvania Medical
Center, Philadelphia, Pennsylvania, may conduct gene transfer experiments on 18 subjects (>18 years ofage) with malignant glioma. The adenovirus vector encoding the Herpes simplex virus thymidine kinase(HSV-TK) gene, H5.020RSVTK, will be injected by a stereotactic guided technique into brain tumors.
Afterwards, the patients will receive systemic ganciclovir (GCV) treatment. Patients eligible to undergo apalliative debulking procedure will receive the same treatment followed by resection on day 7, and asecond dose of the vector intra-operatively. Brain tissues removed by resection will be analyzed foradenovirus infection, transgene expression, and signs of inflammation. The size and metabolic activity oftumors will be monitored by scanning with magnetic resonance imaging and positron emission
tomography. The objective of the study is to evaluate the overall safety of this treatment and to gain
insight into the parameters that may limit the general applicability of this approach. (Protocol #9409-089)
Appendix D-112. Drs. Robert Grossman and Savio Woo of the Baylor College of Medicine & Methodist
Hospital, Houston, Texas, may conduct gene transfer experiments on 20 subjects (≥ 18 years of age) with

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refractive central nervous system malignancies. Subjects will receive stereotaxic injections of a
replication-defective, type 5 E1/E3-deleted adenovirus vector, ADV/RSV-tk, to deliver the Herpes simplex
virus thymidine kinase (HSV-TK) gene to tumor cells. Expression of the HSV-TK gene is driven by aRous sarcoma virus long terminal repeat (RSV-LTR). Subjects will receive a single time-course of
intravenous ganciclovir (GCV) (14 consecutive days) following vector administration. Following
demonstration of safety with the initial starting dose of 1 x 108 particles in 5 subjects, additional cohorts
will receive between 5 x 108 and 1.5 x 109 particles. Each cohort will be monitored for toxicity for one
month before administration of the next higher dose to subsequent cohorts. Subjects will be monitored
for evidence of clinical efficacy by magnetic resonance imaging and/or computer tomography scans. The
primary objective of this Phase I study is to determine the safety of vector administration. (Protocol#9412-098)
Appendix D-113. Drs. Gabriel N. Hortobagyi, Gabriel Lopez-Berestein, and Mien-Chie Hung, of the
University of Texas MD Anderson Cancer Center, Houston, Texas, may conduct gene transfer
experiments on a maximum of 24 adult patients (12 for each cancer) with metastatic breast or ovariancarcinoma. Overexpression of the HER-2/neu oncogene occurs in 30% of ovarian and breast cancers,
and it is associated with enhanced metastatic potential, drug resistance, and poor survival. The E1A
gene of the adenovirus type 5 functions as a tumor suppressor gene when transfected into cancer cells
which overexpress the HER-2/neu oncogene. E1A expression induces down regulation of the level of theHER-2/neu oncoprotein by a transcriptional control mechanism. A plasmid, pE1A, encoding the
adenovirus E1A gene with its own promoter will be administered as a DNA/lipid complex via theintraperitoneal or intrapleural route. The objectives of the study are: (1) to determine E1A genetransduction into malignant cells after the administration of E1A/lipid complex by intrapleural or
intraperitoneal administration, (2) to determine whether E1A gene therapy can down-regulate HER-2/neu
expression after intrapleural or intraperitoneal administration, (3) to determine the maximum biologically
active dose (MBAD), or the maximum tolerated dose (MTD) of E1A/lipid complex, (4) to determine thetoxicity and tolerance of E1A/lipid complex administered into the pleural or peritoneal space, and toassess the reversibility of such toxicity, and (5) to evaluate tumor response. (Protocol #9512-137)
Appendix D-114. Drs. Keith L. Black and Habib Fakhrai of the University of California, Los Angeles,
California, may conduct gene transfer experiments on 12 subjects (≥ 18 years of age) with glioblastoma
multiform. An Epstein-Barr virus (EBV) based plasmid vector, pCEP-4/TGF- β2 antisense, encoding
antisense RNA will be used to inhibit TGF- β2 production. Tumor samples obtained from the patients atthe time of clinically indicated surgery will be grown in culture to establish a cell line for each patient. The
patients' tumor cells will be genetically altered with the pCEP-4/TGF-β2 vector to inhibit their secretion of
TGF-β. Following completion of the traditional post surgical radiation therapy, the first cohort of patients
will receive, at 3 week intervals, 4 injections of 5 x 106 irradiated gene modified autologous tumor cells.
Subsequently, in dose escalation studies, the second cohort will receive 1 x 107 cells, and the thirdcohort, 2 x 107 cells. The results of this Phase I trial will be used to assess the safety of this form of genetherapy and may provide preliminary data to evaluate the potential utility of TGF- β2 antisense genetherapy in the management of gliomas. (Protocol #9512-138)
Appendix D-115. Dr. Ronald G. Crystal of New York Hospital-Cornell Medical Center, New York, New
York, may conduct gene transfer experiments on a total of 21 (with an option for an additional 5) normal
males and female subjects, age ≥ 18 years. Replication-deficient adenovirus (Ad) vector previously has
been used in a number of human gene therapy strategies to transfer genes in vivo for therapeutic
purposes. The purpose of this protocol is to characterize the local (skin), systemic (blood), and distantcompartment (lung) immunity in normal individuals after intradermal administration of a replication
deficient Ad5-based vector, named AdGVCD.10, carrying the gene coding for the E. coli enzyme, cytosine
deaminase (CD). Following intradermal administration of the vector to normal individuals, the skin, blood,and lung immune responses to the Ad vector and CD transgene will be evaluated over time. This vector
has been safety administered intrahepatically ten times to five individuals with colon carcinoma. No
adverse effects in Protocol #9509-125 have been observed. The present protocol will yield insights into
normal human immune responses to both the Ad vector, as well as to a heterologous (i.e., non-human)gene product (CD). Note: This study is designed to answer basic biological questions regarding
characterization of the immune responses to such vectors that have been previously documented.

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Appendix D-116. Dr. Daniel Rockey at Oregon State University and Dr. Walter Stamm at the Universityof Washington may conduct experiments to deliberately transfer a gene encoding tetracycline resistancefrom Chlamydia suis (a swine pathogen) into C. trachomatis (a human pathogen). This approval isspecific to Drs. Rockey and Stamm and research with these resistant organisms may only occur under
the conditions as specified by the NIH Director (72 FR 61661). This approval was effective as of
September 24, 2007.
Appendix D-117. Dr. David Walker at the University of Texas Medical Branch may conduct experiments
to deliberately introduce a gene encoding chloramphenicol resistance into Rickettsia conorii. Thisapproval is specific to Dr. Walker and research with these resistant organisms may only occur under theconditions as specified by the NIH Director (73 FR 32719). This approval was effective as of April 7,
2008.
Appendix D-118. Dr. Harlan Caldwell at the Rocky Mountain Laboratories may conduct experiments todeliberately introduce a gene encoding tetracycline resistance into Chlamydia trachomatis serovar L2.
This approval is specific to Dr. Caldwell and research with this resistant organism may only occur under
the conditions as specified by the NIH Director (76 FR 27653). This approval was effective as of April 26,
2010.
***********************************************************************************************************************
APPENDIX E.

CERTIFIED HOST-VECTOR SYSTEMS (See Appendix I, Biological Containment)

While many experiments using Escherichia coli K-12, Saccharomyces cerevisiae, and Bacillus subtilis are
currently exempt from the NIH Guidelines under Section III-F, Exempt Experiments, some derivatives ofthese host-vector systems were previously classified as Host-Vector 1 Systems or Host-Vector 2
Systems. A listing of those systems follows:
Appendix E-I.

Bacillus subtilis

Appendix E-I-A. Bacillus subtilis Host-Vector 1 Systems
The following plasmids are accepted as the vector components of certified B. subtilis systems: pUB110,
pC194, pS194, pSA2100, pE194, pT127, pUB112, pC221, pC223, and pAB124. B. subtilis strains RUB
331 and BGSC 1S53 have been certified as the host component of Host-Vector 1 systems based onthese plasmids.
Appendix E-I-B. Bacillus subtilis Host-Vector 2 Systems
The asporogenic mutant derivative of Bacillus subtilis, ASB 298, with the following plasmids as the vector
component: pUB110, pC194, pS194, pSA2100, pE194, pT127, pUB112, pC221, pC223, and pAB124.
Appendix E-II. Saccharomyces cerevisiae
Appendix E-II-A.

Saccharomyces cerevisiae Host-Vector 2 Systems

The following sterile strains of Saccharomyces cerevisiae, all of which have the ste-VC9 mutation, SHY1,
SHY2, SHY3, and SHY4. The following plasmids are certified for use: YIp1, YEp2, YEp4, YIp5, YEp6,
YRp7, YEp20, YEp21, YEp24, YIp25, YIp26, YIp27, YIp28, YIp29, YIp30, YIp31, YIp32, and YIp33.
Appendix E-III. Escherichia coli

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Appendix E-III-A.

(April 2024)

Escherichia coli (EK2) Plasmid Systems

The Escherichia coli K-12 strain chi-1776. The following plasmids are certified for use: pSC101, pMB9,
pBR313, pBR322, pDH24, pBR325, pBR327, pGL101, and pHB1. The following Escherichia coli/S.
cerevisiae hybrid plasmids are certified as EK2 vectors when used in Escherichia coli chi-1776 or in thesterile yeast strains, SHY1, SHY2, SHY3, and SHY4: YIpI, YEp2, YEp4, YIp5, YEp6, YRp7, YEp20,
YEp21, YEP24, YIp25, YIp26, YIp27, YIp28, YIp29, YIp30, YIp31, YIp32, and YIp33.
Appendix E-III-B.

Escherichia coli (EK2) Bacteriophage Systems

The following are certified EK2 systems based on bacteriophage lambda:
Vectorλgt WESλB'
λgt WESλB*
λgt ZJ virλB'
λgtALO·λB
Charon 3A
Charon 4A
Charon 16A
Charon 21A
Charon 23A
Charon 24A

Host
DP50supF
DP50supF
Escherichia coli K-12
DP50supF
DP50 or DP50supF
DP50 or DP50supF
DP50 or DP50supF
DP50supF
DP50 or DP50supF
DP50 or DP50supF

Escherichia coli K-12 strains chi-2447 and chi-2281 are certified for use with lambda vectors that are
certified for use with strain DP50 or DP50supF provided that the su-strain not be used as a propagation
host.
Appendix E-IV. Neurospora crassa
Appendix E-IV-A.

Neurospora crassa Host-Vector 1 Systems

The following specified strains of Neurospora crassa which have been modified to prevent aerial
dispersion:
In1 (inositol-less) strains 37102, 37401, 46316, 64001, and 89601. Csp-1 strain UCLA37 and csp-2
strains FS 590, UCLA101 (these are conidial separation mutants).
Eas strain UCLA191 (an "easily wettable" mutant).
Appendix E-V. Streptomyces
Appendix E-V-A.

Streptomyces Host-Vector 1 Systems

The following Streptomyces species: Streptomyces coelicolor, S. lividans, S. parvulus, and S. griseus.
The following are accepted as vector components of certified Streptomyces Host-Vector 1 systems:
Streptomyces plasmids SCP2, SLP1.2, pIJ101, actinophage phi C31, and their derivatives.
Appendix E-VI. Pseudomonas putida
Appendix E-VI-A.

Pseudomonas putida Host-Vector 1 Systems

Pseudomonas putida strains KT2440 with plasmid vectors pKT262, pKT263, and pKT264.
**********************************************************************************************************************

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APPENDIX F. CONTAINMENT CONDITIONS FOR CLONING OF GENES CODING FOR THE
BIOSYNTHESIS OF MOLECULES TOXIC FOR VERTEBRATESAppendix F-I.

General Information

Appendix F specifies the containment to be used for the deliberate cloning of genes coding for thebiosynthesis of molecules toxic for vertebrates. The cloning of genes coding for molecules toxic forvertebrates that have an LD50 of < 100 nanograms per kilograms body weight (e.g., microbial toxins suchas the botulinum toxins, tetanus toxin, diphtheria toxin, Shigella dysenteriae neurotoxin) are covered
under Section III-B-1 (Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than 100
Nanograms Per Kilogram Body Weight) and require Institutional Biosafety Committee and NIH OSPapproval before initiation. No specific restrictions shall apply to the cloning of genes if the protein
specified by the gene has an LD50 ≥ 100 micrograms per kilograms of body weight. Experiments
involving genes coding for toxin molecules with an LD50 of < 100 micrograms per kilograms and > 100
nanograms per kilograms body weight require Institutional Biosafety Committee approval and registration
with NIH OSP prior to initiating the experiments. A list of toxin molecules classified as to LD50 is available
from NIH OSP. Testing procedures for determining toxicity of toxin molecules not on the list are available
from the Office of Science Policy, National Institutes of Health, preferably by submitting a request for thisinformation to: NIHGuidelines@od.nih.gov; additional contact information is also available here and on
the OSP website (www.osp.od.nih.gov). The results of such tests shall be forwarded to NIH OSP, which
will consult with ad hoc experts, prior to inclusion of the molecules on the list (see Section IV-C-1-b-(2)(c), Minor Actions).
Appendix F-II.

Cloning of Toxin Molecule Genes in Escherichia coli K-12

Appendix F-II-A. Cloning of genes coding for molecules toxic for vertebrates that have an LD50 of >100
nanograms per kilograms and <1000 nanograms per kilograms body weight (e.g., abrin, Clostridium
perfringens epsilon toxin) may proceed under Biosafety Level (BL) 2 + EK2 or BL3 + EK1 containment
conditions.
Appendix F-II-B. Cloning of genes for the biosynthesis of molecules toxic for vertebrates that have an
LD50 of >1 microgram per kilogram and <100 microgram per kilogram body weight may proceed underBL1 + EK1 containment conditions (e.g., Staphylococcus aureus alpha toxin, Staphylococcus aureus beta
toxin, ricin, Pseudomonas aeruginosa exotoxin A, Bordetella pertussis toxin, the lethal factor of Bacillus
anthracis, the Pasteurella pestis murine toxins, the oxygen-labile hemolysins such as streptolysin O, andcertain neurotoxins present in snake venoms and other venoms).
Appendix F-II-C. Some enterotoxins are substantially more toxic when administered enterally thanparenterally. The following enterotoxins shall be subject to BL1 + EK1 containment conditions: cholera
toxin, the heat labile toxins of Escherichia coli, Klebsiella, and other related proteins that may be identified
by neutralization with an antiserum monospecific for cholera toxin, and the heat stable toxins ofEscherichia coli and of Yersinia enterocolitica.
Appendix F-III. Cloning of Toxic Molecule Genes in Organisms Other Than Escherichia coli K-12
Requests involving the cloning of genes coding for toxin molecules for vertebrates at an LD50 of <100
nanograms per kilogram body weight in host-vector systems other than Escherichia coli K-12 will be
evaluated by NIH OSP in consultation with ad hoc toxin experts (see Sections III-B-1, Experiments
Involving the Cloning of Toxin Molecules with LD50 of Less than 100 Nanograms Per Kilogram Body
Weight, and IV-C-1-b-(2)-(c), Minor Actions).
Appendix F-IV. Specific Approvals
An updated list of experiments involving the deliberate formation of recombinant or synthetic nucleic acidmolecules containing genes coding for toxins lethal for vertebrates at an LD50 of <100 nanograms per

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kilogram body weight is available from the Office of Science Policy, National Institutes of Health,preferably by submitting a request for this information to: NIHGuidelines@od.nih.gov; additional contact
information is also available here and on the OSP website (www.osp.od.nih.gov).
***********************************************************************************************************************
APPENDIX G.

PHYSICAL CONTAINMENT

Appendix G specifies physical containment for standard laboratory experiments and defines Biosafety
Level 1 through Biosafety Level 4. For large-scale (over 10 liters) research or production, Appendix K(Physical Containment for Large Scale Uses of Organisms Containing Recombinant or Synthetic Nucleic
Acid Molecules) supersedes Appendix G. Appendix K defines Good Large Scale Practice throughBiosafety Level 3 - Large Scale. For certain work with plants, Appendix L (Physical and BiologicalContainment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Plants) supersedes
Appendix G. Appendix L defines Biosafety Levels 1 through 4 - Plants. For certain work with animals,
Appendix M (Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule
Research Involving Animals) supersedes Appendix G. Appendix M defines Biosafety Levels 1 through 4 Animals.
Appendix G-I.

Standard Practices and Training

The first principle of containment is strict adherence to good microbiological practices (see Appendices GIII-A through G-III-J, Footnotes and References of Appendix G). Consequently, all personnel directly or
indirectly involved in experiments using recombinant or synthetic nucleic acid molecules shall receive
adequate instruction (see Sections IV-B-1-h, Responsibilities of the Institution--General Information, andIV-B-7-d, Responsibilities of the Principal Investigator Prior to Initiating Research). At a minimum, these
instructions include training in aseptic techniques and in the biology of the organisms used in theexperiments so that the potential biohazards can be understood and appreciated.
Any research group working with agents that are known or potential biohazards shall have an emergency
plan that describes the procedures to be followed if an accident contaminates personnel or theenvironment. The Principal Investigator shall ensure that everyone in the laboratory is familiar with boththe potential hazards of the work and the emergency plan (see Sections IV-B-7-d, Responsibilities of the
Principal Investigator Prior to Initiating Research and IV-B-7-e, Responsibilities of the Principal
Investigator During the Conduct of the Research). If a research group is working with a known pathogen
for which there is an effective vaccine, the vaccine should be made available to all workers. Serologicalmonitoring, when clearly appropriate, will be provided (see Section IV-B-1-f, Responsibilities of theInstitution--General Information).
The Laboratory Safety Monograph (see Appendix G-III-O, Footnotes and References of Appendix G) andBiosafety in Microbiological and Biomedical Laboratories (see Appendix G-III-A, Footnotes andReferences of Appendix G) describe practices, equipment, and facilities in detail.
Appendix G-II. Physical Containment Levels
The objective of physical containment is to confine organisms containing recombinant or synthetic nucleic
acid molecules and to reduce the potential for exposure of the laboratory worker, persons outside of thelaboratory, and the environment to organisms containing recombinant or synthetic nucleic acid molecules.
Physical containment is achieved through the use of laboratory practices, containment equipment, andspecial laboratory design. Emphasis is placed on primary means of physical containment which areprovided by laboratory practices and containment equipment. Special laboratory design provides asecondary means of protection against the accidental release of organisms outside the laboratory or to
the environment. Special laboratory design is used primarily in facilities in which experiments ofmoderate to high potential hazard are performed.

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Combinations of laboratory practices, containment equipment, and special laboratory design can bemade to achieve different levels of physical containment. Four levels of physical containment, which aredesignated as BL1, BL2, BL3, and BL4 are described. It should be emphasized that the descriptions andassignments of physical containment detailed below are based on existing approaches to containment ofpathogenic organisms (see Appendix G-III-A, Footnotes and References of Appendix G). The National
Cancer Institute describes three levels for research on oncogenic viruses which roughly correspond to ourBL2, BL3, and BL4 levels (see Appendix G-III-C, Footnotes and References of Appendix G).
It is recognized that several different combinations of laboratory practices, containment equipment, andspecial laboratory design may be appropriate for containment of specific research activities. The NIH
Guidelines, therefore, allow alternative selections of primary containment equipment within facilities thathave been designed to provide BL3 and BL4 levels of physical containment. The selection of alternative
methods of primary containment is dependent, however, on the level of biological containment provided
by the host-vector system used in the experiment. Consideration will be given to other combinations
which achieve an equivalent level of containment (see Sections IV-C-1-b-(1), Major Actions and IV-C-1-b(2), Minor Actions).
Appendix G-II-A.
Appendix G)

Biosafety Level 1 (BL1) (See Appendix G-III-M, Footnotes and References ofAppendix G-II-A-1.

Standard Microbiological Practices (BL1)

Appendix G-II-A-1-a. Access to the laboratory is limited or restricted at the discretion of the Principal
Investigator when experiments are in progress.
Appendix G-II-A-1-b. Work surfaces are decontaminated once a day and after any spill of viablematerial.
Appendix G-II-A-1-c. All contaminated liquid or solid wastes are decontaminated before disposal.
Appendix G-II-A-1-d. Mechanical pipetting devices are used; mouth pipetting is prohibited.
Appendix G-II-A-1-e. Eating, drinking, smoking, and applying cosmetics are not permitted in the workarea. Food may be stored in cabinets or refrigerators designated and used for this purpose only.
Appendix G-II-A-1-f. Persons wash their hands: (i) after they handle materials involving organisms
containing recombinant or synthetic nucleic acid molecules and animals, and (ii) before exiting thelaboratory.
Appendix G-II-A-1-g. All procedures are performed carefully to minimize the creation of aerosols.
Appendix G-II-A-1-h. In the interest of good personal hygiene, facilities (e.g., hand washing sink,shower, changing room) and protective clothing (e.g., uniforms, laboratory coats) shall be provided thatare appropriate for the risk of exposure to viable organisms containing recombinant or synthetic nucleic
acid molecules.
Appendix G-II-A-2.

Special Practices (BL1)

Appendix G-II-A-2-a. Contaminated materials that are to be decontaminated at a site away from thelaboratory are placed in a durable leak-proof container which is closed before being removed from thelaboratory.
Appendix G-II-A-2-b. An insect and rodent control program is in effect.
Appendix G-II-A-3.

Containment Equipment (BL1)Page 77 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Appendix G-II-A-3-a. Special containment equipment is generally not required for manipulations ofagents assigned to BL1.
Appendix G-II-A-4.

Laboratory Facilities (BL1)

Appendix G-II-A-4-a. The laboratory is designed so that it can be easily cleaned.
Appendix G-II-A-4-b. Bench tops are impervious to water and resistant to acids, alkalis, organic
solvents, and moderate heat.
Appendix G-II-A-4-c. Laboratory furniture is sturdy. Spaces between benches, cabinets, and equipment
are accessible for cleaning.
Appendix G-II-A-4-d. Each laboratory contains a sink for hand washing.
Appendix G-II-A-4-e. If the laboratory has windows that open, they are fitted with fly screens.
Appendix G-II-B.
Appendix G)

Biosafety Level 2 (BL2) (See Appendix G-III-N, Footnotes and References ofAppendix G-II-B-1.

Standard Microbiological Practices (BL2)

Appendix G-II-B-1-a. Access to the laboratory is limited or restricted by the Principal Investigator whenwork with organisms containing recombinant or synthetic nucleic acid molecules is in progress.
Appendix G-II-B-1-b. Work surfaces are decontaminated at least once a day and after any spill of viablematerial.
Appendix G-II-B-1-c. All contaminated liquid or solid wastes are decontaminated before disposal.
Appendix G-II-B-1-d. Mechanical pipetting devices are used; mouth pipetting is prohibited.
Appendix G-II-B-1-e. Eating, drinking, smoking, and applying cosmetics are not permitted in the workarea. Food may be stored in cabinets or refrigerators designated and used for this purpose only.
Appendix G-II-B-1-f. Persons wash their hands: (i) after handling materials involving organisms
containing recombinant or synthetic nucleic acid molecules and animals, and (ii) when exiting thelaboratory.
Appendix G-II-B-1-g. All procedures are performed carefully to minimize the creation of aerosols.
Appendix G-II-B-1-h. Experiments of lesser biohazard potential can be conducted concurrently in
carefully demarcated areas of the same laboratory.
Appendix G-II-B-2.

Special Practices (BL2)

Appendix G-II-B-2-a. Contaminated materials that are to be decontaminated at a site away from thelaboratory are placed in a durable leak-proof container which is closed before being removed from thelaboratory.
Appendix G-II-B-2-b. The Principal Investigator limits access to the laboratory. The Principal
Investigator has the final responsibility for assessing each circumstance and determining who may enteror work in the laboratory.
Appendix G-II-B-2-c. The Principal Investigator establishes policies and procedures whereby only
persons who have been advised of the potential hazard and meet any specific entry requirements (e.g.,

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immunization) may enter the laboratory or animal rooms.
Appendix G-II-B-2-d. When the organisms containing recombinant or synthetic nucleic acid molecules in
use in the laboratory require special provisions for entry (e.g., vaccination), a hazard warning sign
incorporating the universal biosafety symbol is posted on the access door to the laboratory work area.
The hazard warning sign identifies the agent, lists the name and telephone number of the Principal
Investigator or other responsible person(s), and indicates the special requirement(s) for entering thelaboratory.
Appendix G-II-B-2-e. An insect and rodent control program is in effect.
Appendix G-II-B-2-f. Laboratory coats, gowns, smocks, or uniforms are worn while in the laboratory.
Before exiting the laboratory for non-laboratory areas (e.g., cafeteria, library, administrative offices), thisprotective clothing is removed and left in the laboratory or covered with a clean coat not used in thelaboratory.
Appendix G-II-B-2-g. Animals not involved in the work being performed are not permitted in thelaboratory.
Appendix G-II-B-2-h. Special care is taken to avoid skin contamination with organisms containing
recombinant or synthetic nucleic acid molecules; gloves should be worn when handling experimental
animals and when skin contact with the agent is unavoidable.
Appendix G-II-B-2-i. All wastes from laboratories and animal rooms are appropriately decontaminatedbefore disposal.
Appendix G-II-B-2-j. Hypodermic needles and syringes are used only for parenteral injection andaspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes ordisposable syringe-needle units (i.e., needle is integral to the syringe) are used for the injection or
aspiration of fluids containing organisms that contain recombinant or synthetic nucleic acid molecules.
Extreme caution should be used when handling needles and syringes to avoid autoinoculation and thegeneration of aerosols during use and disposal. Needles should not be bent, sheared, replaced in theneedle sheath or guard, or removed from the syringe following use. The needle and syringe should be
promptly placed in a puncture-resistant container and decontaminated, preferably autoclaved, before
discard or reuse.
Appendix G-II-B-2-k. Spills and accidents which result in overt exposures to organisms containing
recombinant or synthetic nucleic acid molecules are immediately reported to the Institutional Biosafety
Committee and NIH OSP. Reports to NIH OSP shall be sent to the Office of Science Policy, National
Institutes of Health, preferably by e-mail to: NIHGuidelines@od.nih.gov; additional contact information is
also available here and on the OSP website (www.osp.od.nih.gov). Medical evaluation, surveillance, andtreatment are provided as appropriate and written records are maintained.
Appendix G-II-B-2-l. When appropriate, considering the agent(s) handled, baseline serum samples forlaboratory and other at-risk personnel are collected and stored. Additional serum specimens may becollected periodically depending on the agents handled or the function of the facility.
Appendix G-II-B-2-m. A biosafety manual is prepared or adopted. Personnel are advised of special
hazards and are required to read and follow instructions on practices and procedures.
Appendix G-II-B-3.

Containment Equipment (BL2)

Appendix G-II-B-3-a. Biological safety cabinets (Class I or II) (see Appendix G-III-L, Footnotes andReferences of Appendix G) or other appropriate personal protective or physical containment devices areused whenever:Page 79 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Appendix G-II-B-3-a-(1). Procedures with a high potential for creating aerosols are conducted (seeAppendix G-III-O, Footnotes and References of Appendix G). These may include centrifuging, grinding,blending, vigorous shaking or mixing, sonic disruption, opening containers of materials whose internalpressures may be different from ambient pressures, intranasal inoculation of animals, and harvesting
infected tissues from animals or eggs.
Appendix G-II-B-3-a-(2). High concentrations or large volumes of organisms containing recombinant orsynthetic nucleic acid molecules are used. Such materials may be centrifuged in the open laboratory ifsealed beads or centrifuge safety cups are used and if they are opened only in a biological safety cabinet.
Appendix G-II-B-4.

Laboratory Facilities (BL2)

Appendix G-II-B-4-a. The laboratory is designed so that it can be easily cleaned.
Appendix G-II-B-4-b. Bench tops are impervious to water and resistant to acids, alkalis, organic
solvents, and moderate heat.
Appendix G-II-B-4-c. Laboratory furniture is sturdy and spaces between benches, cabinets, andequipment are accessible for cleaning.
Appendix G-II-B-4-d. Each laboratory contains a sink for hand washing.
Appendix G-II-B-4-e. If the laboratory has windows that open, they are fitted with fly screens.
Appendix G-II-B-4-f. An autoclave for decontaminating laboratory wastes is available.
Appendix G-II-C.
Appendix G)

Biosafety Level 3 (BL3) (See Appendix G-III-P, Footnotes and References ofAppendix G-II-C-1.

Standard Microbiological Practices (BL3)

Appendix G-II-C-1-a. Work surfaces are decontaminated at least once a day and after any spill of viablematerial.
Appendix G-II-C-1-b. All contaminated liquid or solid wastes are decontaminated before disposal.
Appendix G-II-C-1-c. Mechanical pipetting devices are used; mouth pipetting is prohibited.
Appendix G-II-C-1-d. Eating, drinking, smoking, storing food, and applying cosmetics are not permitted
in the work area.
Appendix G-II-C-1-e. Persons wash their hands: (i) after handling materials involving organisms
containing recombinant or synthetic nucleic acid molecules, and handling animals, and (ii) when exiting
the laboratory.
Appendix G-II-C-1-f. All procedures are performed carefully to minimize the creation of aerosols.
Appendix G-II-C-1-g. Persons under 16 years of age shall not enter the laboratory.
Appendix G-II-C-1-h. If experiments involving other organisms which require lower levels of containment
are to be conducted in the same laboratory concurrently with experiments requiring BL3 level physicalcontainment, they shall be conducted in accordance with all BL3 level laboratory practices.
Appendix G-II-C-2.

Special Practices (BL3)

Appendix G-II-C-2-a. Laboratory doors are kept closed when experiments are in progress.

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Appendix G-II-C-2-b. Contaminated materials that are to be decontaminated at a site away from thelaboratory are placed in a durable leak-proof container which is closed before being removed from thelaboratory.
Appendix G-II-C-2-c. The Principal Investigator controls access to the laboratory and restricts access topersons whose presence is required for program or support purposes. The Principal Investigator has the
final responsibility for assessing each circumstance and determining who may enter or work in thelaboratory.
Appendix G-II-C-2-d. The Principal Investigator establishes policies and procedures whereby only
persons who have been advised of the potential biohazard, who meet any specific entry requirements(e.g., immunization), and who comply with all entry and exit procedures entering the laboratory or animalrooms.
Appendix G-II-C-2-e. When organisms containing recombinant or synthetic nucleic acid molecules orexperimental animals are present in the laboratory or containment module, a hazard warning sign
incorporating the universal biosafety symbol is posted on all laboratory and animal room access doors.
The hazard warning sign identifies the agent, lists the name and telephone number of the Principal
Investigator or other responsible person(s), and indicates any special requirements for entering thelaboratory such as the need for immunizations, respirators, or other personal protective measures.
Appendix G-II-C-2-f. All activities involving organisms containing recombinant or synthetic nucleic acidmolecules are conducted in biological safety cabinets or other physical containment devices within thecontainment module. No work in open vessels is conducted on the open bench.
Appendix G-II-C-2-g. The work surfaces of biological safety cabinets and other containment equipment
are decontaminated when work with organisms containing recombinant or synthetic nucleic acidmolecules is finished. Plastic-backed paper toweling used on non-perforated work surfaces withinbiological safety cabinets facilitates clean-up.
Appendix G-II-C-2-h. An insect and rodent program is in effect.
Appendix G-II-C-2-i. Laboratory clothing that protects street clothing (e.g., solid front or wrap-aroundgowns, scrub suits, coveralls) is worn in the laboratory. Laboratory clothing is not worn outside thelaboratory, and it is decontaminated prior to laundering or disposal.
Appendix G-II-C-2-j. Special care is taken to avoid skin contamination with contaminated materials;
gloves should be worn when handling infected animals and when skin contact with infectious materials is
unavoidable.
Appendix G-II-C-2-k. Molded surgical masks or respirators are worn in rooms containing experimental
animals.
Appendix G-II-C-2-l. Animals and plants not related to the work being conducted are not permitted in thelaboratory.
Appendix G-II-C-2-m. Laboratory animals held in a BL3 area shall be housed in partial-containment
caging systems, such as Horsfall units (see Appendix G-III-K, Footnotes and References of Appendix G),
open cages placed in ventilated enclosures, solid-wall and -bottom cages covered by filter bonnets orsolid-wall and -bottom cages placed on holding racks equipped with ultraviolet in radiation lamps andreflectors.
Note: Conventional caging systems may be used provided that all personnel wear appropriate personal
protective devices. These protective devices shall include at a minimum wrap-around gowns, head
covers, gloves, shoe covers, and respirators. All personnel shall shower on exit from areas where thesePage 81 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

devices are required.
Appendix G-II-C-2-n. All wastes from laboratories and animal rooms are appropriately decontaminatedbefore disposal. For research involving mammalian-transmissible HPAI H5N1 virus, liquid effluentsshould be chemically disinfected or heat-treated, or collected and processed in a central effluent
decontamination system. Decontamination of shower and toilet effluents is not required, provided
appropriate practices and procedures are in place for primary containment of mammaliantransmissible HPAI H5N1 virus. Animal tissues, carcasses, and bedding originating from the animalroom must be decontaminated by an effective and validated method (e.g., use of an autoclave)
preferably before leaving the containment barrier. If waste must be transported, special practices
should be developed for transport of infectious materials to designated alternate location(s) within thefacility.
Appendix G-II-C-2-o. Vacuum lines are protected with high efficiency particulate air/HEPA filters andliquid disinfectant traps.
Appendix G-II-C-2-p. Hypodermic needles and syringes are used only for parenteral injection andaspiration of fluids from laboratory animals and diaphragm bottles. Only needle locking syringes ordisposable syringe-needle units (i.e., needle is integral to the syringe) are used for the injection or
aspiration of fluids containing organisms that contain recombinant or synthetic nucleic acid molecules.
Extreme caution should be used when handling needles and syringes to avoid autoinoculation and thegeneration of aerosols during use and disposal. Needles should not be bent, sheared, replaced in theneedle sheath or guard, or removed from the syringe following use. The needle and syringe should be
promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before
discard or reuse.
Appendix G-II-C-2-q. Spills and accidents which result in overt or potential exposures to organisms
containing recombinant or synthetic nucleic acid molecules are immediately reported to the Biological
Safety Officer, Institutional Biosafety Committee, and NIH OSP. Reports to NIH OSP shall be sent to the
Office of Science Policy, National Institutes of Health, preferably by e-mail to:
NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website(www.osp.od.nih.gov). Appropriate medical evaluation, surveillance, and treatment are provided andwritten records are maintained.
Appendix G-II-C-2-r. Baseline serum samples for all laboratory and other at-risk personnel should becollected and stored in accordance with institutional policy and at least for the time period in which thepersonnel continues to work with the agent at biosafety level 3 containment. Such samples must becollected and stored for laboratory and other at-risk personnel who will work with mammaliantransmissible HPAI H5N1 virus. Additional serum specimens may be collected periodically depending on
the agents handled or the function of the laboratory.
Appendix G-II-C-2-s. A biosafety manual is prepared or adopted. Personnel are advised of special
hazards and are required to read and follow the instructions on practices and procedures.
Appendix G-II-C-2-t.

Alternative Selection of Containment Equipment (BL3)

Experimental procedures involving a host-vector system that provides a one-step higher level of biologicalcontainment than that specified may be conducted in the BL3 laboratory using containment equipment
specified for the BL2 level of physical containment. Experimental procedures involving a host-vector
system that provides a one-step lower level of biological containment than that specified may be
conducted in the BL3 laboratory using containment equipment specified for the BL4 level of physicalcontainment. Alternative combination of containment safeguards are shown in Appendix G-Table 1,
Possible Alternate Combinations of Physical and Biological Containment Safeguards.

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Appendix G-II-C-3.

(April 2024)

Containment Equipment (BL3)

Appendix G-II-C-3-a. Biological safety cabinets (Class I, II, or III) (see Appendix G-III-L, Footnotes andReferences of Appendix G) or other appropriate combinations of personal protective or physicalcontainment devices (e.g., special protective clothing, masks, gloves, respirators, centrifuge safety cups,
sealed centrifuge rotors, and containment caging for animals) are used for all activities with organisms
containing recombinant or synthetic nucleic acid molecules which pose a threat of aerosol exposure.
These include: manipulation of cultures and of those clinical or environmental materials which may be asource of aerosols; the aerosol challenge of experimental animals; the harvesting of infected tissues or
fluids from experimental animals and embryonate eggs; and the necropsy of experimental animals.
Appendix G-II-C-4.

Laboratory Facilities (BL3)

Appendix G-II-C-4-a. The laboratory is separated from areas which are open to unrestricted traffic flow
within the building. Passage through two sets of doors is the basic requirement for entry into thelaboratory from access corridors or other contiguous areas. Physical separation of the high containment
laboratory from access corridors or other laboratories or activities may be provided by a double-door
clothes change room (showers may be included), airlock, or other access facility which requires passage
through two sets of doors before entering the laboratory.
Appendix G-II-C-4-b. The interior surfaces of walls, floors, and ceilings are water resistant so that they
can be easily cleaned. Penetrations in these surfaces are sealed or capable of being sealed to facilitate
decontaminating the area.
Appendix G-II-C-4-c. Bench tops are impervious to water and resistant to acids, alkalis, organic
solvents, and moderate heat.
Appendix G-II-C-4-d. Laboratory furniture is sturdy and spaces between benches, cabinets, andequipment are accessible for cleaning.
Appendix G-II-C-4-e. Each laboratory contains a sink for hand washing. The sink is foot, elbow, or
automatically operated and is located near the laboratory exit door.
Appendix G-II-C-4-f. Windows in the laboratory are closed and sealed.
Appendix G-II-C-4-g. Access doors to the laboratory or containment module are self-closing.
Appendix G-II-C-4-h. An autoclave for decontaminating laboratory wastes is available preferably within
the laboratory.
Appendix G-II-C-4-i. A ducted exhaust air ventilation system is provided. This system creates
directional airflow that draws air into the laboratory from uncontaminated spaces surrounding thelaboratory. The exhaust air is not recirculated to any other area of the building, is discharged to theoutside, and is dispersed away from the occupied areas and air intakes. Personnel shall verify that thedirection of the airflow (into the laboratory) is proper. The exhaust air from the laboratory room may bedischarged to the outside without being filtered or otherwise treated unless research is being conductedwith mammaliantransmissible HPAI H5N1 virus. For research with mammalian-transmissible HPAI
H5N1 virus, exhaust air must be HEPA filtered and there must be sealed ductwork from thecontainment barrier to the filter. In addition, the air handling system shall be designed such that
under failure conditions, the airflow will not be reversed and periodic verification, with annual
verification of the HEPA filters, shall be performed. Finally, backup power shall be available for criticalcontrols and instrumentation necessary to maintain containment.
Appendix G-II-C-4-j. The high efficiency particulate air/HEPA filtered exhaust air from Class I or Class II
biological safety cabinets is discharged directly to the outside or through the building exhaust system.

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Exhaust air from Class I or II biological safety cabinets may be recirculated within the laboratory if thecabinet is tested and certified at least every twelve months. If the HEPA-filtered exhaust air from Class I
or II biological safety cabinets is to be discharged to the outside through the building exhaust air system,
it is connected to this system in a manner (e.g., thimble unit connection (see Appendix G-III-L, Footnotes
and References of Appendix G)) that avoids any interference with the air balance of the cabinets orbuilding exhaust system.
Appendix G-II-C-5.
Biosafety Level 3 Enhanced for Research Involving Risk Group 3 Influenza
Viruses. (See Appendices G-II-C-2-n, G-II-C-2-r, and G-II-C-4-i for additional guidance for facilities,
waste handling, and serum collection for research involving mammaliantransmissible HPAI H5N1
virus.
Appendix G-II-C-5-a. Containment, Practices, and Training for Research with Risk Group 3
Influenza Viruses (BL3 Enhanced).
Appendix G-II-C-5-a-(1). In addition to standard BL3 practices, the following additional personal
protective equipment and practices shall be used: (1) Powered Air-purifying Respirators (PAPR) areworn. (2) Street clothes are changed to protective suit (e.g., wrap-back disposable gown, olefin protectivesuit). (3) Double gloves (disposable) are worn. For research with mammalian-transmissible HPAI H5N1
viruses, protective sleeves shall be worn over the gown while working in a biosafety cabinet. (4)Appropriate shoe coverings are worn (e.g., double disposable shoe coverings, single disposable shoecoverings if worn with footwear dedicated to BL3 enhanced laboratory use, or impervious boots or shoesof rubber or other suitable material that can be decontaminated). (5) Showers prior to exiting thelaboratory should be considered depending on risk assessment of research activities, with the exceptionthat showers prior to exiting the laboratory are required for all research with mammalian-transmissible
HPAI H5N1 virus, including care of animals infected with mammalian-transmissible HPAI H5N1 virus.
(6) For research with mammalian-transmissible HPAI H5N1 virus, prior to leaving containment,
personal protective equipment shall be sprayed or wiped down with a disinfectant that has activity
against influenza viruses. (7) In order to promote adherence to proper practices, including proper
removal of personal protective equipment, and reporting of any loss of containment or exposures, atleast two individuals should be in the laboratory at all times when research with mammaliantransmissible HPAI H5N1 virus involves experimental procedures with animals or sharps, or whenprocedures are being conducted whereby the generation of aerosols is reasonably anticipated.
Removal of personal protective equipment should be observed.
Appendix G-II-C-5-a-(2). As proper training of laboratory workers is an essential component of biosafety,
retraining and periodic reassessments (at least annually) in BL3 enhanced practices, especially theproper use of respiratory equipment, such as PAPRs, and clothing changes are required. For researchwith mammalian-transmissible HPAI H5N1 virus, laboratory workers shall be required to sign adocument acknowledging their understanding of and intent to adhere to biosafety, biosecurity, andoccupational health requirements. This document shall include a statement that the laboratory worker
agrees to report any exposures or accidents, including those by other individuals in the lab.
Appendix G-II-C-5-a-(3). Reporting of all spills and accidents, even if relatively minor, is required as
described in Appendix G-II-C-2-q.
Appendix G-II-C-5-a-(4). To avoid inadvertent cross contamination of 1918 H1N1, HPAI H5N1 or human
H2N2 (1957-1968): (1) Containment facilities and practices appropriate for highest Risk Group virus shallbe used at all times with lower Risk Group viruses, when studied in the same laboratory room. (2) Tissue
cultures with these viruses shall be conducted at separate times (temporal spacing) in the same room.
(3) Separate reagents shall be used to minimize risk of cross contamination. (4) A laboratory worker shallnot perform concurrent influenza virus experiments that carry the risk of unintended reassortment among1918 H1N1, human H2N2 (1957-1968), HPAI H5N1 and other human influenza viruses. (5) Two or morelaboratory workers shall not perform within the same work area simultaneous influenza virus experimentsthat carry the risk of unintended segment reassortment between 1918 H1N1, or HPAI H5N1, or human
H2N2 (1957-1968) and other human influenza viruses. (6) Between experiments good biosafety

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decontamination practices (e.g., surface and biosafety cabinet surface decontamination according tostandard BL3 procedures) shall be used and there shall be a thirty minute wait period after
decontamination before equipment is used for experiments with any other influenza A viruses. (7)Between experiments, in addition to decontamination of the work area, clothing changes and PAPR
disinfection shall be performed prior to handling a different influenza virus in the same work area.
(Shower-out capability may be required by USDA/APHIS for certain experiments with HPAI H5N1.)
Appendix G-II-C-5-a-(5). Continued susceptibility of the reassortant influenza viruses containing genes
and/or segments from 1918 H1N1, HPAI H5N1, and human H2N2 (1957-1968) to antiviral agents shall beestablished by sequence analysis or suitable biological assays. After manipulation of genes that
influence sensitivity to antiviral agents, susceptibility to these agents shall be reconfirmed. If susceptibility
to neuraminidase inhibitors or other effective antiviral agents is lost as a result of genetic modificationor serial passage of a mammalian-transmissible HPAI H5N1 virus, then any research with this antiviral
agent resistant virus shall be stopped and research shall only proceed after review by the NIH (asoutlined in Section III-A-l-a) or the appropriate federal regulatory agency.
Appendix G-II-C-5-b. Containment for Animal Research.
Guidance provided in Appendix G-II-C and Appendix M-II-C is applicable with the following emphasis on
standard BL3 or BL3-N containment or additional enhancements.
Appendix G-II-C-5-b-(1). Research with small animals shall be conducted in a class II biosafety cabinet.
Small animals such as rodents (e.g. mice, hamsters, rats, guinea pigs) can be housed within a negative
pressure BL3 animal suite using high-density individually vented caging (IVC) systems that independently
supply high efficiency particulate air/HEPA-filtered and directional air circulation. Other animals (e.g.
rabbits, ferrets) that are of a size or have growth or caging requirements that preclude the use of highdensity IVC systems are to be housed in negative pressure bioisolators.
Appendix G-II-C-5-b-(2). Large animals such as non-human primates shall be housed in primary barrier
environments according to BL3-N containment requirements (see Appendix M-II-C).
Appendix G-II-C-5-b-(3). Specialized training and proven competency in all assigned practices andprocedures shall be required for laboratory staff, including staff involved in animal care.
Appendix G-II-C-5-b-(4). For HPAI H5N1 research, the NIH Guidelines defer to USDA/APHIS
recommendations for biocontainment practices for loose housed animals.
Appendix G-II-C-5-c. Occupational HealthA detailed occupational health plan shall be developed in advance of working with these agents in
consultation, as needed, with individuals with the appropriate clinical expertise. In addition, theappropriate public health authority shall be consulted (e.g. local public health officials) on the plan and amock drill of this plan shall be undertaken periodically. The plan shall include a description of theincident reporting system in place for incidents, which include any loss of containment, spills,
accidents, or potential exposures. The plan must specify that all incidents must be reported
immediately to the appropriate institutional authorities, and no later than 24 hours to the appropriate
public health authorities (e.g., the USDA, the CDC, NIH, local and state health authorities).
Appendix G-II-C-5-c-(1). Laboratory workers shall be provided with medical cards which include, at aminimum, the following information: characterization of the influenza virus to which they have beenpotentially exposed, and 24-hour contact numbers for the Principal Investigator and institution’s
occupational health care provider(s).
Appendix G-II-C-5-c-(2). A detailed occupational health plan shall include: (1) Unless there is a medical
contraindication to vaccination (e.g. severe egg allergy) annual seasonal influenza vaccination as
prerequisite for research to reduce risk of influenza like illness that would require isolation and testing torule out infection with experimental viruses and raise the risk for possible co-infection with circulating
influenza strains. (2) Virus specific vaccination, if available, should be offered and if a licensed HPAI

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H5N1 vaccine is available, and there are no medical contraindications, laboratory workers performing
research with mammalian-transmissible HPAI H5N1 virus should be vaccinated. A post-vaccination
serum sample shall be collected, assessed for immune response, and stored in accordance withinstitutional policy, at least for the time in which the laboratory worker continues to conduct HPAI
H5N1 virus research. (3) Reporting of all respiratory symptoms and/or fever (i.e. influenza-like illnesses).
For research involving mammalian-transmissible HPAI H5N1 virus, laboratory workers shall be actively
monitored for influenza-like illness (i.e., fever and respiratory symptoms). (4) 24-hour access to a
medical facility that is prepared to implement appropriate respiratory isolation to prevent transmission andis able to provide appropriate antiviral agents. Real-time reverse transcription-polymerase chain reaction(RT-PCR) assays should be used for virus detection and to discriminate these viruses from currently
circulating human influenza viruses. For exposures to viruses containing genes from 1918 H1N1 or the
HA gene from human H2N2 (1957-1968), specimens shall be sent to the CDC for testing (RT-PCR andconfirmatory sequencing).
Appendix G-II-C-5-c-(3). In preparing to perform research with 1918 H1N1, human H2N2 (1957-1968), or
HPAI H5N1, Principal Investigators should develop a clear plan specifying who will be contacted in theevent of a potential exposure (during and after work hours) to conduct a risk assessment and makedecisions as to the required response, including the need for and extent of isolation of the exposed
worker. After any kind of potential exposure, a rapid risk assessment shall be performed by the Principal
Investigator, health and biosafety officials and subsequent actions should depend on the appraised levelof risk of respiratory infection for the individual and potential for transmission to others. A laboratory
worker performing research with either an influenza virus containing the HA gene from human H2N2 oran influenza virus containing genes and/or segments from 1918 H1N1 or mammalian-transmissible HPAI
H5N1 viruses, shall be informed in advance that, in the case of a known laboratory exposure with a highrisk for infection, e.g., involving the upper or lower respiratory tract or mucous membranes, the laboratory
worker will need to be isolated in a predetermined facility, rather than home isolation, until infection canbe ruled out by testing (e.g., negative RT-PCR for 1918 H1N1 or human H2N2 (1957-1968), or HPAI
H5N1) of appropriately timed specimens. Laboratory workers with a known laboratory exposure withhigh risk for infection during research with HPAI H5N1 virus strains that are not transmissible among
mammals should be prepared to self-isolate (for example at home) until infection can be ruled out by
testing (e.g., negative RT-PCR for HPAI H5N1) of appropriately timed specimens. The action taken forother types of exposures should be based on the risk assessment. In addition, based on the risk
assessment: (1) treatment with appropriate antiviral agents shall be initiated, and (2) the appropriate
public health authorities shall be notified.
Appendix G-II-C-5-c-(4). Influenza-like illness. If an individual has entered (within ten days) alaboratory conducting research with influenza viruses containing the human H2N2 HA gene or any
gene from the 1918 H1N1 or HPAI H5N1 viruses, or housing animals exposed to such viruses, and theindividual demonstrates symptoms and/or signs of influenza infection (e.g., fever/chills, cough, myalgias,
headache), then he/she shall report by phone to the supervisor/Principal Investigator and otherindividuals identified in the occupational health plan. If needed, the person with influenza-like illness
shall be transported, under the appropriate isolation conditions, to a healthcare facility that can provide
adequate respiratory isolation, appropriate medical therapy, and testing to determine whether the
infection is due to a recombinant or synthetic influenza virus. The appropriate public health authorities
shall be informed whenever a suspected case is isolated.
Appendix G-II-C-5-c-(5). For 1918 H1N1 research, the use of antiviral agents (e.g., oseltamivir) for preexposure prophylaxis shall be discussed with laboratory workers in advance including a discussion of thedata on the safety of long term exposure to these agents and their ability to reduce the risk of clinicaldisease and the limits of the data regarding protection of close contacts and the community.
Appendix G-II-C-5-c-(6). Antiviral agents for postexposure prophylaxis shall be provided only after
medical evaluation. Home supplies shall not be provided in advance for research with 1918 H1N1,
mammaliantransmissible HPAI H5N1 or influenza viruses containing the HA gene from human H2N2.

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Appendix G-II-D.

Biosafety Level 4 (BL4)

Appendix G-II-D-1.

Standard Microbiological Practices (BL4)(April 2024)

Appendix G-II-D-1-a. Work surfaces are decontaminated at least once a day and immediately after any
spill of viable material.
Appendix G-II-D-1-b. Only mechanical pipetting devices are used.
Appendix G-II-D-1-c. Eating, drinking, smoking, storing food, and applying cosmetics are not permitted
in the laboratory.
Appendix G-II-D-1-d. All procedures are performed carefully to minimize the creation of aerosols.
Appendix G-II-D-2.

Special Practices (BL4)

Appendix G-II-D-2-a. Biological materials to be removed from the Class III cabinets or from the
maximum containment laboratory in a viable or intact state are transferred to a non-breakable, sealed
primary container and then enclosed in a non-breakable, sealed secondary container which is removed
from the facility through a disinfectant dunk tank, fumigation chamber, or an airlock designed for thispurpose.
Appendix G-II-D-2-b. No materials, except for biological materials that are to remain in a viable or intact
state, are removed from the maximum containment laboratory unless they have been autoclaved or
decontaminated before exiting the facility. Equipment or material which might be damaged by hightemperatures or steam is decontaminated by gaseous or vapor methods in an airlock or chamber
designed for this purpose.
Appendix G-II-D-2-c. Only persons whose presence in the facility or individual laboratory rooms isrequired for program or support purposes are authorized to enter. The supervisor has the final
responsibility for assessing each circumstance and determining who may enter or work in the laboratory.
Access to the facility is limited by means of secure, locked doors; accessibility is managed by the
Principal Investigator, Biological Safety Officer, or other person responsible for the physical security of the
facility. Before entering, persons are advised of the potential biohazards and instructed as to appropriate
safeguards for ensuring their safety. Authorized persons comply with the instructions and all otherapplicable entry and exit procedures. A logbook signed by all personnel indicates the date and time ofeach entry and exit. Practical and effective protocols for emergency situations are established.
Appendix G-II-D-2-d. Personnel enter and exit the facility only through the clothing change and shower
rooms. Personnel shower each time they exit the facility. Personnel use the air locks to enter or exit thelaboratory only in an emergency.
Appendix G-II-D-2-e. Street clothing is removed in the outer clothing change room and kept there.
Complete laboratory clothing (may be disposable), including undergarments, pants and shirts or jumpsuits, shoes, and gloves, is provided and used by all personnel entering the facility. Head covers areprovided for personnel who do not wash their hair during the exit shower. When exiting the laboratory
and before proceeding into the shower area, personnel remove their laboratory clothing and store it in alocker or hamper in the inner change room. Protective clothing shall be decontaminated prior tolaundering or disposal.
Appendix G-II-D-2-f. When materials that contain organisms containing recombinant or synthetic nucleic
acid molecules or experimental animals are present in the laboratory or animal rooms, a hazard warning
sign incorporating the universal biosafety symbol is posted on all access doors. The sign identifies theagent, lists the name of the Principal Investigator or other responsible person(s), and indicates any
special requirements for entering the area (e.g., the need for immunizations or respirators).

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Appendix G-II-D-2-g. Supplies and materials needed in the facility are brought in by way of the doubledoored autoclave, fumigation chamber, or airlock which is appropriately decontaminated between eachuse. After securing the outer doors, personnel within the facility retrieve the materials by opening theinterior doors or the autoclave, fumigation chamber, or airlock. These doors are secured after materials
are brought into the facility.
Appendix G-II-D-2-h. An insect and rodent control program is in effect.
Appendix G-II-D-2-i. Materials (e.g., plants, animals, and clothing) not related to the experiment being
conducted are not permitted in the facility.
Appendix G-II-D-2-j. Hypodermic needles and syringes are used only for parenteral injection andaspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes ordisposable syringe-needle units (i.e., needle is integral part of unit) are used for the injection or aspirationof fluids containing organisms that contain recombinant or synthetic nucleic acid molecules. Needles
should not be bent, sheared, replaced in the needle sheath or guard, or removed from the syringe
following use. The needle and syringe should be placed in a puncture-resistant container anddecontaminated, preferably by autoclaving before discard or reuse. Whenever possible, cannulas areused instead of sharp needles (e.g., gavage).
Appendix G-II-D-2-k. A system is set up for reporting laboratory accidents, exposures, employee
absenteeism, and for the medical surveillance of potential laboratory-associated illnesses. Spills andaccidents which result in overt exposures to organisms containing recombinant or synthetic nucleic acidmolecules are immediately reported to the Biological Safety Officer, Institutional Biosafety Committee,
and NIH OSP. Reports to the NIH OSP shall be sent to the Office of Science Policy, National Institutes of
Health, preferably by e-mail to: NIHGuidelines@od.nih.gov; additional contact information is alsoavailable here and on the OSP website (www.osp.od.nih.gov). Written records are prepared andmaintained. An essential adjunct to such a reporting-surveillance system is the availability of a facility forquarantine, isolation, and medical care of personnel with potential or known laboratory associatedillnesses.
Appendix G-II-D-2-l.

Containment for Animal Research

Appendix G-II-D-2-l-(1). Laboratory animals involved in experiments requiring BL4 level physicalcontainment shall be housed either in cages contained in Class III cabinets or in partial containment
caging systems, such as Horsfall units (see Appendix G-III-K, Footnotes and References of Appendix G),
open cages placed in ventilated enclosures, or solid-wall and -bottom cages placed on holding racks
equipped with ultraviolet irradiation lamps and reflectors that are located in a specially designed area inwhich all personnel are required to wear one-piece positive pressure suits.
Appendix G-II-D-2-l-(2). Non-human primates (NHP) may be housed (1) under the containment
conditions described in Appendix G-II-D-2-l-(1) above, or (2) in open cages within a dedicated animal
holding room that serves as the primary barrier and in which all personnel are required to wear one-piece
positive pressure suits. A room serving as a primary barrier must be air-tight and capable of being
decontaminated using fumigation.
If NHPs are to be contained in a dedicated animal holding room serving as the primary barrier, thefollowing conditions shall be met:(i) Access to the animal holding room from service corridors outside of the BL4 containment space shallrequire passage through two sets of doors, and the inner most door must be an air pressure resistant(APR) door;(ii) For any animal holding room considered to be a primary barrier, APR door(s) providing direct ingressfrom the exterior service corridor shall be fitted with appropriate and redundant lock-out mechanisms toprevent access when the animal holding room is contaminated and in use. There should be more than
one mechanism to ensure that this primary barrier door cannot be opened when the animal room iscontaminated and the APR door shall not serve as an emergency exit from the BL4 laboratory. The APR
door shall be appropriately tested to demonstrate that in the closed, locked-out mode, the door provides

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an air-tight barrier proven by pressure decay testing or other equivalent method;(iii) Any door(s) allowing access into a corridor from which there is direct ingress to an animal holding
room must be fitted with either (1) an APR door or (2) a non-APR door, provided directional airflow ismaintained from the laboratory corridor space into the animal room. For the purpose of fumigation, animalrooms equipped with non-APR doors opening into the adjacent interior corridors shall be considered one
space (i.e., areas between air-tight doors shall be fumigated together);(iv) Any door(s) used for access to the service corridor (the secondary barrier) shall be self-closing and ofsolid construction, designed not to corrode, split or warp;(v) Access to the service corridor inside the secondary barrier shall be restricted and strictly controlled
when animal rooms are in use. Whenever possible, the secondary barrier door(s) should be fitted withsafety interlock switches designed to prevent it from opening when an animal holding room door (theprimary barrier) is opened following room decontamination; if interlock devices cannot be used, specificadministrative procedures shall be implemented to control access to the service corridor;(vi) The service corridor shall maintain a negative pressure (inward directional airflow) relative to adjoining
traffic corridors;(vii) Prior to fumigation of the animal holding room, cages should be removed for autoclaving or chemical
decontamination;(viii) Caging should be chosen to reduce the amount of animal detritus that can be thrown out of the cageand onto the floor of the animal holding room;(ix) The flow of personnel, material and equipment should be directed in order to minimize the spread ofcontamination from the animal holding room into adjacent areas of the laboratory;(x) Following animal room decontamination, safeguards involving the use of personal protective
equipment and appropriate administrative controls shall be implemented for the safe retrieval of biologicalindicators in order to prevent the spread of infectious agents in the event of a decontamination failure.
Appendix G-II-D-2-m. Alternative Selection of Containment Equipment (BL4)Experimental procedures involving a host-vector system that provides a one-step higher level of biologicalcontainment than that specified may be conducted in the BL4 facility using containment equipment
requirements specified for the BL3 level of physical containment. Alternative combinations ofcontainment safeguards are shown in Appendix G-Table 1, Possible Alternate Combinations of Physicaland Biological Containment Safeguards.
Appendix G-II-D-3.

Containment Equipment (BL4)

Appendix G-II-D-3-a. All procedures within the facility with agents assigned to Biosafety Level 4 areconducted in the Class III biological safety cabinet or in Class I or II biological safety cabinets used inconjunction with one-piece positive pressure personnel suits ventilated by a life-support system.
Appendix G-II-D-4.

Laboratory Facilities (BL4)

Appendix G-II-D-4-a. The maximum containment facility consists of either a separate building or aclearly demarcated and isolated zone within a building. Outer and inner change rooms separated by ashower are provided for personnel entering and exiting the facility. A double-doored autoclave,
fumigation chamber, or ventilated airlock is provided for passage of those materials, supplies, or
equipment which are not brought into the facility through the change room.
Appendix G-II-D-4-b. Walls, floors, and ceilings of the facility are constructed to form a sealed internal
shell which facilitates fumigation and is animal and insect proof. The internal surfaces of this shell are
resistant to liquids and chemicals, thus facilitating cleaning and decontamination of the area. All
penetrations in these structures and surfaces are sealed. Any drains in the floors contain traps filled witha chemical disinfectant of demonstrated efficacy against the target agent, and they are connected directly
to the liquid waste decontamination system. Sewer and other ventilation lines contain high efficiency
particulate air/HEPA filters.

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Appendix G-II-D-4-c. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes,
are arranged to minimize the horizontal surface area on which dust can settle.
Appendix G-II-D-4-d. Bench tops have seamless surfaces which are impervious to water and resistantto acids, alkalis, organic solvents, and moderate heat.
Appendix G-II-D-4-e. Laboratory furniture is simple and of sturdy construction; and spaces between
benches, cabinets, and equipment are accessible for cleaning.
Appendix G-II-D-4-f. A foot, elbow, or automatically operated hand washing sink is provided near thedoor of each laboratory room in the facility.
Appendix G-II-D-4-g. If there is a central vacuum system, it does not serve areas outside the facility. Inline high efficiency particulate air/HEPA filters are placed as near as practicable to each use point orservice cock. Filters are installed to permit in-place decontamination and replacement. Other liquid andgas services to the facility are protected by devices that prevent back-flow.
Appendix G-II-D-4-h. If water fountains are provided, they are foot operated and are located in thefacility corridors outside the laboratory. The water service to the fountain is not connected to the backflow protected distribution system supplying water to the laboratory areas.
Appendix G-II-D-4-i. Access doors to the laboratory are self-closing and locking.
Appendix G-II-D-4-j. Any windows are breakage resistant.
Appendix G-II-D-4-k. A double-door autoclave is provided for decontaminating materials passing out of
the facility. The autoclave door which opens to the area external to the facility is sealed to the outer wall
and automatically controlled so that the outside door can only be opened after the autoclave "sterilization"
cycle has been completed.
Appendix G-II-D-4-l. A pass-through dunk tank, fumigation chamber, or an equivalent decontamination
method is provided so that materials and equipment that cannot be decontaminated in the autoclave canbe safely removed from the facility.
Appendix G-II-D-4-m. Liquid effluent from laboratory sinks, biological safety cabinets, floors, andautoclave chambers are decontaminated by heat treatment before being released from the maximum
containment facility. Liquid wastes from shower rooms and toilets may be decontaminated with chemical
disinfectants or by heat in the liquid waste decontamination system. The procedure used for heatdecontamination of liquid wastes is evaluated mechanically and biologically by using a recording
thermometer and an indicator microorganism with a defined heat susceptibility pattern. If liquid wastes
from the shower room are decontaminated with chemical disinfectants, the chemical used is ofdemonstrated efficacy against the target or indicator microorganisms.
Appendix G-II-D-4-n. An individual supply and exhaust air ventilation system is provided. The system
maintains pressure differentials and directional airflow as required to assure flows inward from areas
outside of the facility toward areas of highest potential risk within the facility. Manometers are used tosense pressure differentials between adjacent areas maintained at different pressure levels. If a system
malfunctions, the manometers sound an alarm. The supply and exhaust airflow is interlocked to assure
inward (or zero) airflow at all times.
Appendix G-II-D-4-o. The exhaust air from the facility is filtered through high efficiency particulate
air/HEPA filters and discharged to the outside so that it is dispersed away from occupied buildings and air
intakes. Within the facility, the filters are located as near the laboratories as practicable in order to reduce
the length of potentially contaminated air ducts. The filter chambers are designed to allow in situ
decontamination before filters are removed and to facilitate certification testing after they are replaced.
Coarse filters and HEPA filters are provided to treat air supplied to the facility in order to increase thePage 90 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

lifetime of the exhaust HEPA filters and to protect the supply air system should air pressures become
unbalanced in the laboratory.
Appendix G-II-D-4-p. The treated exhaust air from Class I and II biological safety cabinets may bedischarged into the laboratory room environment or the outside through the facility air exhaust system. If
exhaust air from Class I or II biological safety cabinets is discharged into the laboratory the cabinets aretested and certified at minimum on a yearly basis. More frequent testing and certification, based on theamount of use or other safety factors, shall be left to the discretion of the IBC. The exhaust air from Class
III biological safety cabinets is discharged, without recirculation through two sets of high efficiency
particulate air/HEPA filters in series, via the facility exhaust air system. If the treated exhaust air from anyof these cabinets is discharged to the outside through the facility exhaust air system, it is connected tothis system in a manner (e.g., thimble unit connection (see Appendix G-III-L, Footnotes and Referencesof Appendix G)) that avoids any interference with the air balance of the cabinets or the facility exhaust air
system.
Appendix G-II-D-4-q. A specially designed suit area may be provided in the facility. Personnel whoenter this area shall wear a one-piece positive pressure suit that is ventilated by a life-support system.
The life-support system includes alarms and emergency backup breathing air tanks. Entry to this area isthrough an airlock fitted with airtight doors. A chemical shower is provided to decontaminate the surfaceof the suit before the worker exits the area. The exhaust air from the suit area is filtered by two sets ofhigh efficiency particulate air/HEPA filters installed in series. A duplicate filtration unit, exhaust fan, andan automatically starting emergency power source are provided. The air pressure within the suit area isgreater than that of any adjacent area. Emergency lighting and communication systems are provided. All
penetrations into the internal shell of the suit are sealed. A double-doored autoclave is provided fordecontaminating waste materials to be removed from the suit areas.
Appendix G - Table 1. Possible Alternate Combinations Of Physical And Biological Containment
Safeguards

Classificationof Physical &BiologicalContainment

Alternate Physical Containment

Alternate
BiologicalContainment

Laboratory
Facilities

Laboratory
Practices

Laboratory
Equipment

BL3/HV2

BL3
BL3

BL3
BL3

BL3
BL4

HV2
HV1

BL3/HV1

BL3
BL3

BL3
BL3

BL3
BL2

HV1
HV2

BL4/HV1

BL4
BL4

BL4
BL4

BL4
BL3

HV1
HV2

BL - Biosafety Level
HV - Host-Vector System
Appendix G-III. Footnotes and References of Appendix G
Appendix G-III-A. Biosafety in Microbiological and Biomedical Laboratories, U.S. Department of Healthand Human Services, Public Health Service, Centers for Disease Control and Prevention, Atlanta,
Georgia, and National Institutes of Health, Bethesda, Maryland.

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Appendix G-III-B. Arthropod Containment Guidelines, Version 3.2, 2019, and Addendum 1 Containment
Practices for Arthropods Modified with Engineered Transgenes Capable of Gene Drive, 2022, American
Committee of Medical Entomology, American Society of Tropical Medicine and Hygiene, Arlington,
Virginia.
Appendix G-III-C. National Cancer Institute Safety Standards for Research Involving Oncogenic Viruses,
U.S. Department of Health, Education, and Welfare Publication No. (NIH) 75-790, October 1974.
Appendix G-III-D. National Institutes of Health Biohazards Safety Guide, U.S. Department of Health,
Education, and Welfare, Public Health Service, NIH, U.S. Government Printing Office, Stock No. 174000383, 1974.
Appendix G-III-E. A. Hellman, M. N. Oxman, and R. Pollack (eds.), Biohazards in Biological Research,
Cold Spring Harbor Laboratory 1973.
Appendix G-III-F. N. V. Steere (ed.), Handbook of Laboratory Safety, 2nd edition, The Chemical RubberCo., Cleveland, Ohio, 1971.
Appendix G-III-G. Bodily, J. L, "General Administration of the Laboratory," H. L. Bodily, E. L. Updyke,
and J. O. Mason (eds.), Diagnostic Procedures for Bacterial, Mycotic, and Parasitic Infections, American
Public Health Association, New York, 1970, pp. 11-28.
Appendix G-III-H. Darlow, H. M. (1969). "Safety in the Microbiological Laboratory," in J. R. Norris and D.
W. Robbins (eds.), Methods in Microbiology, Academic Press, Inc., New York, pp. 169-204.
Appendix G-III-I. The Prevention of Laboratory Acquired Infection, C. H. Collins, E. G. Hartley, and R.
Pilsworth, Public Health Laboratory Service, Monograph Series No. 6, 1974.
Appendix G-III-J. Chatigny, M. A., "Protection Against Infection in the Microbiological Laboratory:
Devices and Procedures," in W. W. Umbreit (ed.), Advances in Applied Microbiology, Academic Press,
New York, New York, 1961, 3:131-192.
Appendix G-III-K. Horsfall, F. L. Jr., and J. H. Baner, Individual Isolation of Infected Animals in a Single
Room, J. Bact., 1940, 40, 569-580.
Appendix G-III-L. Biological safety cabinets referred to in this section are classified as Class I, Class II,
or Class III cabinets. A Class I is a ventilated cabinet for personnel protection having an inward flow of airaway from the operator. The exhaust air from this cabinet is filtered through a high efficiency particulate
air/HEPA filter. This cabinet is used in three operational modes: (i) with a full-width open front, (ii) with an
installed front closure panel (having four 6-inch diameter openings) without gloves, and (iii) with an
installed front closure panel equipped with arm-length rubber gloves. The face velocity of the inward flowof air through the full-width open front is 75 feet per minute or greater. A Class II cabinet is a ventilated
cabinet for personnel and product protection having an open front with inward air flow for personnel
protection, and HEPA filtered mass recirculated air flow for product protection. The cabinet exhaust air isfiltered through a HEPA filter. The face velocity of the inward flow of air through the full-width open front
is 75 feet per minute or greater. Design and performance specifications for Class II cabinets have been
adopted by the National Sanitation Foundation, Ann Arbor, Michigan. A Class III cabinet is a closed-front
ventilated cabinet of gas tight construction which provides the highest level of personnel protection of all
biosafety safety cabinets. The interior of the cabinet is protected from contaminants exterior to thecabinet. The cabinet is fitted with arm-length rubber gloves and is operated under a negative pressure ofat least 0.5 inches water gauge. All supply air is filtered through HEPA filters. Exhaust air is filteredthrough two HEPA filters or one HEPA filter and incinerator before being discharged to the outsideenvironment. National Sanitation Foundation Standard 49. 1976. Class II (Laminar Flow) Biohazard
Cabinetry, Ann Arbor, Michigan.
Appendix G-III-M. Biosafety Level 1 is suitable for work involving agents of unknown or minimal potential

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hazard to laboratory personnel and the environment. The laboratory is not separated from the general
traffic patterns in the building. Work is generally conducted on open bench tops. Special containment
equipment is not required or generally used. Laboratory personnel have specific training in theprocedures conducted in the laboratory and are supervised by a scientist with general training in
microbiology or a related science (see Appendix G-III-A, Footnotes and References of Appendix G).
Appendix G-III-N. Biosafety Level 2 is similar to Level 1 and is suitable for work involving agents ofmoderate potential hazard to personnel and the environment. It differs in that: (1) laboratory personnel
have specific training in handling pathogenic agents and are directed by competent scientists; (2) accessto the laboratory is limited when work is being conducted; and (3) certain procedures in which infectious
aerosols are created are conducted in biological safety cabinets or other physical containment equipment(see Appendix G-III-A, Footnotes and References of Appendix G).
Appendix G-III-O. Office of Research Safety, National Cancer Institute, and the Special Committee of
Safety and Health Experts, Laboratory Safety Monograph: A Supplement to the NIH Guidelines for
Recombinant DNA Research, NIH, Bethesda, Maryland 1978.
Appendix G-III-P. Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production
facilities in which work is conducted with indigenous or exotic agents which may cause serious or
potentially lethal disease as a result of exposure by the inhalation route. Laboratory personnel havespecific training in handling pathogenic and potentially lethal agents and are supervised by competentscientists who are experienced in working with these agents. All procedures involving the manipulation of
infectious material are conducted within biological safety cabinets or other physical containment devices
or by personnel wearing appropriate personal protective clothing and devices. The laboratory has special
engineering and design features. It is recognized, however, that many existing facilities may not have all
the facility safeguards recommended for BL3 (e.g., access zone, sealed penetrations, and directional
airflow, etc.). In these circumstances, acceptable safety may be achieved for routine or repetitive
operations (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, andsusceptibility testing) in laboratories where facility features satisfy BL2 recommendations provided therecommended "Standard Microbiological Practices," "Special Practices," and "Containment Equipment"for BL3 are rigorously followed. The decision to implement this modification of BL3 recommendations
should be made only by the Principal Investigator.
***********************************************************************************************************************
APPENDIX H.

SHIPMENT

Recombinant or synthetic nucleic acid molecules contained in an organism or in a viral genome shall beshipped under the applicable regulations of the U.S. Postal Service (39 Code of Federal Regulations, Part3); the Public Health Service (42 Code of Federal Regulations, Part 72); the U.S. Department ofAgriculture (9 Code of Federal Regulations, Subchapters D and E; 7 CFR, Part 340); and/or the U.S.
Department of Transportation (49 Code of Federal Regulations, Parts 171-179).
Note. A host-vector system may be proposed for certification by the NIH Director in accordance with theprocedures set forth in Appendix I-II, Certification of Host-Vector Systems. In order to ensure protection
for proprietary data, any public notice regarding a host-vector system which is designated by theinstitution as proprietary under Section IV-D, Voluntary Compliance, will be issued only after consultation
with the institution as to the content of the notice (see Section IV-D-3, Certification of Host-Vector
Systems - Voluntary Compliance).
Appendix H-I. Host organisms or viruses will be shipped as etiologic agents, regardless of whether they
contain recombinant or synthetic nucleic acid molecules, if they are regulated as human pathogens by the
Public Health Service (42 Code of Federal Regulations, Part 72) or as animal pathogens or plant pests
under the U.S. Department of Agriculture, Animal and Plant Health Inspection Service (Titles 9 and 7
Code of Federal Regulations, respectively).

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Appendix H-II. Host organisms and viruses will be shipped as etiologic agents if they containrecombinant or synthetic nucleic acid molecules when: (i) the recombinant or synthetic nucleic acidmolecule includes the complete genome of a host organism or virus regulated as a human or animal
pathogen or a plant pest; or (ii) the recombinant or synthetic nucleic acid molecule codes for a toxin orother factor directly involved in eliciting human, animal, or plant disease or inhibiting plant growth, and is
carried on an expression vector or within the host chromosome and/or when the host organism contains a
conjugation proficient plasmid or a generalized transducing phage; or (iii) the recombinant or synthetic
nucleic acid molecule comes from a host organism or virus regulated as a human or animal pathogen or
as a plant pest and has not been adequately characterized to demonstrate that it does not code for a
factor involved in eliciting human, animal, or plant disease.
Appendix H-III. Footnotes and References of Appendix H
For further information on shipping etiologic agents contact: (i) The Centers for Disease Control andPrevention, ATTN: Biohazards Control Office, 1600 Clifton Road, Atlanta, Georgia 30333, (404) 6393883, FTS 236-3883; (ii) The U.S. Department of Transportation, ATTN: Office of Hazardous Materials
Transportation, 400 7th Street, S.W., Washington, DC 20590, (202) 366-4545; or (iii) U.S. Department ofAgriculture, ATTN: Animal and Plant Health Inspection Service (APHIS), Veterinary Services, National
Center for Import-Export, Products Program, 4700 River Road, Unit 40, Riverdale, Maryland 20737.
Phone: (301) 734-8499; Fax: (301) 734-8226.
***********************************************************************************************************************
APPENDIX I.

BIOLOGICAL CONTAINMENT (See Appendix E, Certified Host-Vector Systems)

Appendix I-I.

Levels of Biological Containment

In consideration of biological containment, the vector (plasmid, organelle, or virus) for the recombinant orsynthetic nucleic acid molecule and the host (bacterial, plant, or animal cell) in which the vector is
propagated in the laboratory will be considered together. Any combination of vector and host which is toprovide biological containment shall be chosen or constructed so that the following types of "escape" areminimized: (i) survival of the vector in its host outside the laboratory, and (ii) transmission of the vector
from the propagation host to other non-laboratory hosts. The following levels of biological containment(host-vector systems) for prokaryotes are established. Appendices I-I-A through I-II-B describe levels of
biological containment (host-vector systems) for prokaryotes. Specific criteria will depend on theorganisms to be used.
Appendix I-I-A. Host-Vector 1 Systems
Host-Vector 1 systems provide a moderate level of containment. Specific Host-Vector 1 systems are:Appendix I-I-A-1.

Escherichia coli K-12 Host-Vector 1 Systems (EK1)

The host is always Escherichia coli K-12 or a derivative thereof, and the vectors include non-conjugativeplasmids (e.g., pSC101, Co1E1, or derivatives thereof (see Appendices I-III-A through G, Footnotes andReferences of Appendix I) and variants of bacteriophage, such as lambda (see Appendices I-III-H throughO, Footnotes and References of Appendix I). The Escherichia coli K-12 hosts shall not containconjugation-proficient plasmids, whether autonomous or integrated, or generalized transducing phages.
Appendix I-I-A-2.

Other Host-Vector 1 Systems

At a minimum, hosts and vectors shall be comparable in containment to Escherichia coli K-12 with a nonconjugative plasmid or bacteriophage vector. Appendix I-II, Certification of Host-Vector Systems,
describes the data to be considered and mechanism for approval of Host-Vector 1 systems.

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Appendix I-I-B. Host-Vector 2 Systems (EK2)
Host-Vector 2 Systems provide a high level of biological containment as demonstrated by data fromsuitable tests performed in the laboratory. Escape of the recombinant or synthetic nucleic acid molecule
either via survival of the organisms or via transmission of the recombinant or synthetic nucleic acidmolecule to other organisms should be < 1/108 under specified conditions. Specific Host-Vector 2
systems are:Appendix I-I-B-1. For Escherichia coli K-12 Host-Vector 2 systems (EK2) in which the vector is a
plasmid, no more than 1/108 host cells shall perpetuate a cloned DNA fragment under the specified nonpermissive laboratory conditions designed to represent the natural environment, either by survival of theoriginal host or as a consequence of transmission of the cloned DNA fragment.
Appendix I-I-B-2. For Escherichia coli K-12 Host-Vector 2 systems (EK2) in which the vector is a phage,no more than 1/108 phage particles shall perpetuate a cloned DNA fragment under the specified nonpermissive laboratory conditions designed to represent the natural environment, either as a prophage (inthe inserted or plasmid form) in the laboratory host used for phage propagation, or survival in naturalenvironments and transferring a cloned DNA fragment to other hosts (or their resident prophages).
Appendix I-II.

Certification of Host-Vector Systems

Appendix I-II-A. Responsibility
Host-Vector 1 systems (other than Escherichia coli K-12) and Host-Vector 2 systems may not bedesignated as such until they have been certified by the NIH Director. Requests for certification of hostvector systems may be submitted to the Office of Science Policy, National Institutes of Health, preferably
by e-mail to: NIHGuidelines@od.nih.gov; additional contact information is also available here and on the
OSP website (www.osp.od.nih.gov). Proposed host-vector systems will be reviewed based on theconstruction, properties, and testing of the proposed host-vector system by ad hoc experts. The NIH
Director is responsible for certification of host-vector systems. Minor modifications to existing host-vector
systems (i.e., those that are of minimal or no consequence to the properties relevant to containment) may
be certified by the NIH Director (see Section IV-C-1-b-(2)-(f), Minor Actions). Once a host-vector system
has been certified by the NIH Director, a notice of certification will be sent by NIH OSP to the applicant
and to the Institutional Biosafety Committee Chairs. A list of all currently certified host-vector systems isavailable from the Office of Science Policy, National Institutes of Health, preferably by submitting arequest for this information to: NIHGuidelines@od.nih.gov; additional contact information is also available
here and on the OSP website (www.osp.od.nih.gov). The NIH Director may rescind the certification of ahost-vector system (see Section IV-C-1-b-(2)-(g), Minor Actions). If certification is rescinded, NIH will
instruct investigators to transfer cloned DNA into a different system or use the clones at a higher level ofphysical containment level, unless NIH determines that the already constructed clones incorporate
adequate biological containment. Certification of a host-vector system does not extend to modificationsof either the host or vector component of that system. Such modified systems shall be independently
certified by the NIH Director. If modifications are minor, it may only be necessary for the investigator to
submit data showing that the modifications have either improved or not impaired the major phenotypic
traits on which the containment of the system depends. Substantial modifications to a certified hostvector system requires submission of complete testing data.
Appendix I-II-B. Data to be Submitted for Certification
Appendix I-II-B-1.

Host-Vector 1 Systems Other than Escherichia coli K-12

The following types of data shall be submitted, modified as appropriate for the particular system underconsideration: (i) a description of the organism and vector; the strain's natural habitat and growthrequirements; its physiological properties, particularly those related to its reproduction, survival, and themechanisms by which it exchanges genetic information; the range of organisms with which this organism

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normally exchanges genetic information and the type of information exchanged; and any relevant
information about its pathogenicity or toxicity; (ii) a description of the history of the particular strains andvectors to be used, including data on any mutations which render this organism less able to survive ortransmit genetic information; and (iii) a general description of the range of experiments contemplated withemphasis on the need for developing such an Host-Vector 1 system.
Appendix I-II-B-2.

Host-Vector 2 Systems

Investigators planning to request Host-Vector 2 systems certification may obtain instructions from NIH
OSP concerning data to be submitted (see Appendices I-III-N and O, Footnotes and References ofAppendix I). In general, the following types of data are required: (i) description of construction steps withindication of source, properties, and manner of introduction of genetic traits; (ii) quantitative data on thestability of genetic traits that contribute to the containment of the system; (iii) data on the survival of thehost-vector system under non-permissive laboratory conditions designed to represent the relevant naturalenvironment; (iv) data on transmissibility of the vector and/or a cloned DNA fragment under both
permissive and non-permissive conditions; (v) data on all other properties of the system which affectcontainment and utility, including information on yields of phage or plasmid molecules, ease of DNA
isolation, and ease of transfection or transformation; and (vi) in some cases, the investigator may be
asked to submit data on survival and vector transmissibility from experiments in which the host-vector is
fed to laboratory animals or one or more human subjects. Such in vivo data may be required to confirm
the validity of predicting in vivo survival on the basis of in vitro experiments. Data shall be submitted to
the Office of Science Policy, National Institutes of Health, preferably by e-mail to:
NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website(www.osp.od.nih.gov). Investigators are encouraged to publish their data on the construction, properties,
and testing of proposed Host Vector 2 systems prior to consideration of the system by NIH. Specificinstructions concerning the submission of data for proposed Escherichia coli K-12 Host-Vector 2 system(EK2) involving either plasmids or bacteriophage in Escherichia coli K-12, are available from the Office of
Science Policy, National Institutes of Health, preferably by submitting a request for this information to:
NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website(www.osp.od.nih.gov).
Appendix I-III.

Footnotes and References of Appendix I

Appendix I-III-A. Hersfield, V., H. W. Boyer, C. Yanofsky, M. A. Lovett, and D. R. Helinski, Plasmid
Co1E1 as a Molecular Vehicle for Cloning and Amplification of DNA. Proc. Nat. Acad. Sci., 1974, 71, pp.
3455-3459.
Appendix I-III-B. Wensink, P. C., D. J. Finnegan, J. E. Donelson, and D. S. Hogness, A System for
Mapping DNA Sequences in the Chromosomes of Drosophila Melanogaster. Cell, 1974, 3, pp. 315-335.
Appendix I-III-C. Tanaka, T., and B. Weisblum, Construction of a Colicin El-R Factor Composite Plasmidin Vitro: Means for Amplification of Deoxyribonucleic Acid. J. Bacteriol., 1975, 121, pp. 354-362.
Appendix I-III-D. Armstrong, K. A., V. Hershfield, and D. R. Helinski, Gene Cloning and Containment
Properties of Plasmid Col E1 and Its Derivatives, Science, 1977, 196, pp. 172-174.
Appendix I-III-E. Bolivar, F., R. L. Rodriguez, M. C. Betlack, and H. W. Boyer, Construction andCharacterization of New Cloning Vehicles: I. Ampicillin-Resistant Derivative of PMB9, Gene, 1977, 2, pp.
75-93.
Appendix I-III-F. Cohen, S. N., A. C. W. Chang, H. Boyer, and R. Helling. Construction of Biologically
Functional Bacterial Plasmids in Vitro. Proc. Natl. Acad, Sci., 1973, 70, pp. 3240-3244.
Appendix I-III-G. Bolivar, F., R. L. Rodriguez, R. J. Greene, M. C.Batlack, H. L. Reyneker, H. W. Boyer,
J. H. Cross, and S. Falkow, 1977, Construction and Characterization of New Cloning Vehicles II. A MultiPurpose Cloning System, Gene, 1977, 2, pp. 95-113.

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Appendix I-III-H. Thomas, M., J. R. Cameron, and R. W. Davis (1974). Viable Molecular Hybrids of
Bacteriophage Lambda and Eukaryotic DNA. Proc. Nat. Acad. Sci., 1974, 71, pp. 4579-4583.
Appendix I-III-I. Murray, N. E., and K. Murray, Manipulation of Restriction Targets in Phage Lambda toForm Receptor Chromosomes for DNA Fragments. Nature, 1974, 51, pp. 476-481.
Appendix I-III-J. Ramback, A., and P. Tiollais (1974). Bacteriophage Having EcoRI Endonuclease Sites
Only in the Non-Essential Region of the Genome. Proc. Nat. Acad. Sci., 1974, 71, pp. 3927-3820.
Appendix I-III-K. Blattner, F. R., B. G. Williams, A. E. Bleche, K. Denniston-Thompson, H. E. Faber, L.
A. Furlong, D. J. Gunwald, D. O. Kiefer, D. D. Moore, J. W. Shumm, E. L. Sheldon, and O. Smithies,
Charon Phages: Safer Derivatives of Bacteriophage Lambda for DNA Cloning, Science 1977, 196, pp.
163-169.
Appendix I-III-L. Donoghue, D. J., and P. A. Sharp, An Improved Lambda Vector: Construction of Model
Recombinants Coding for Kanamycin Resistance, Gene, 1977, 1, pp. 209-227.
Appendix I-III-M. Leder, P., D. Tiemeier and L. Enquist (1977), EK2 Derivatives of Bacteriophage
Lambda Useful in the Cloning of DNA from Higher Organisms: The λgt WES System, Science, 1977,
196, pp. 175-177.
Appendix I-III-N. Skalka, A., Current Status of Coliphage AEK2 Vectors, Gene, 1978, 3, pp. 29-35.
Appendix I-III-O. Szybalski, W., A. Skalka, S. Gottesman, A. Campbell, and D. Botstein, Standardized
Laboratory Tests for EK2 Certification, Gene, 1978, 3, pp. 36-38.
***********************************************************************************************************************
APPENDIX J.

BIOTECHNOLOGY RESEARCH SUBCOMMITTEEThe National Science and Technology Council's Committee on Fundamental Science determined that a
subcommittee should be continued to identify and coordinate Federal research efforts, identify researchneeds, stimulating international cooperation, and assess national and international policy issues
concerning biotechnology sciences. The primary emphasis will be on scientific issues to increase theoverall effectiveness and productivity of the Federal investment in biotechnology sciences, especiallyregarding issues which cut across agency boundaries. This subcommittee is called the Biotechnology
Research Subcommittee.
Membership of the Biotechnology Research Subcommittee will include Federal agencies that support
biotechnology research. Agencies represented are: U.S. Department of Agriculture, Department ofCommerce, Department of Defense, Department of Energy, Department of Health and Human Services,
Department of Interior, Department of Justice, Department of State, Department of Veterans Affairs,
Agency for International Development, Environmental Protection Agency, National Aeronautics andSpace Administration, and National Science Foundation. The Biotechnology Research Subcommittee willfunction in an advisory capacity to the Committee on Fundamental Science, the Director of the Office of
Science and Technology Policy, and the Executive Office of the President. The Biotechnology Research
Subcommittee will review the scientific aspects of proposed regulations and guidelines as they aredeveloped.
The primary responsibilities of the Biotechnology Research Subcommittee are to: (i) describe and review
current Federal efforts in biotechnology research; (ii) identify and define the priority areas for future
Federal biotechnology research, including areas needing greater emphasis, describing the role of eachagency in those areas, and delineate where interagency cooperation would enhance progress in thebiotechnology sciences, with an emphasis on integrated research efforts, where appropriate; (iii) assess

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major international efforts in the biotechnology sciences and develop mechanisms for international
collaboration. For example, activities of the U.S.-European Community Task Force on Biotechnology
have been coordinated through the Biotechnology Research Subcommittee; (iv) identify and review
national and international policy issues (such as public education) associated with biotechnology; and (v)provide reviews, analyses, and recommendations to the Chairs of the Committee on Fundamental
Science on scientific issues related to regulations and the applications of biotechnology research andbiotechnology policies and issues.
In 1990, the Biotechnology Research Subcommittee replaced the Biotechnology Sciences Coordinating
Committee. Both the Biotechnology Research Subcommittee and the Biotechnology Sciences
Coordinating Committee previously functioned under the Federal Coordinating Council on Science,
Engineering, and Technology (FCCSET). While regulatory issues became the primary focus of the
Biotechnology Sciences Coordinating Committee, the Biotechnology Research Subcommittee focuses onscientific issues, although it will still provide scientific support for regulatory responsibilities.
***********************************************************************************************************************
APPENDIX K. PHYSICAL CONTAINMENT FOR LARGE SCALE USES OF ORGANISMSCONTAINING RECOMBINANT OR SYNTHETIC NUCLEIC ACID MOLECULES
Appendix K specifies physical containment guidelines for large-scale (greater than 10 liters of culture)research or production involving viable organisms containing recombinant or synthetic nucleic acidmolecules. It shall apply to large-scale research or production activities as specified in Section III-D-6,
Experiments Involving More than 10 Liters of Culture. It is important to note that this appendix addresses
only the biological hazard associated with organisms containing recombinant or synthetic nucleic acidmolecules. Other hazards accompanying the large-scale cultivation of such organisms (e.g., toxic
properties of products; physical, mechanical, and chemical aspects of downstream processing) are notaddressed and shall be considered separately, albeit in conjunction with this appendix.
All provisions shall apply to large-scale research or production activities with the following modifications:(i) Appendix K shall supersede Appendix G, Physical Containment, when quantities in excess of 10 litersof culture are involved in research or production. Appendix K-II applies to Good Large Scale Practice; (ii)the institution shall appoint a Biological Safety Officer if it engages in large-scale research or production
activities involving viable organisms containing recombinant or synthetic nucleic acid molecules. The
duties of the Biological Safety Officer shall include those specified in Section IV-B-3, Biological Safety
Officer; (iii) the institution shall establish and maintain a health surveillance program for personnel
engaged in large-scale research or production activities involving viable organisms containing
recombinant or synthetic nucleic acid molecules which require Biosafety Level (BL) 3 containment at thelaboratory scale. The program shall include: pre assignment and periodic physical and medical
examinations; collection, maintenance, and analysis of serum specimens for monitoring serologicchanges that may result from the employee's work experience; and provisions for the investigation of any
serious, unusual, or extended illnesses of employees to determine possible occupational origin.
Appendix K-I.

Selection of Physical Containment Levels

The selection of the physical containment level required for recombinant or synthetic nucleic acidmolecule research or production involving more than 10 liters of culture is based on the containment
guidelines established in Section III, Experiments Covered by the NIH Guidelines. For purposes of largescale research or production, four physical containment levels are established. The four levels set
containment conditions at those appropriate for the degree of hazard to health or the environment posedby the organism, judged by experience with similar organisms unmodified by recombinant or synthetic
nucleic acid molecule techniques and consistent with Good Large Scale Practice. The four biosafetylevels of large-scale physical containment are referred to as Good Large Scale Practice, BL1-Large
Scale, BL2-Large Scale, and BL3-Large Scale. Good Large Scale Practice is recommended for largescale research or production involving viable, non-pathogenic, and non-toxigenic recombinant or synthetic

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strains derived from host organisms that have an extended history of safe large-scale use. Good Large
Scale Practice is recommended for organisms such as those included in Appendix C, Exemptions under
Section III-F-8, which have built-in environmental limitations that permit optimum growth in the large-scalesetting but limited survival without adverse consequences in the environment. BL1-Large Scale isrecommended for large-scale research or production of viable organisms containing recombinant orsynthetic nucleic acid molecules that require BL1 containment at the laboratory scale and that do not
qualify for Good Large Scale Practice. BL2-Large Scale is recommended for large-scale research or
production of viable organisms containing recombinant or synthetic nucleic acid molecules that require
BL2 containment at the laboratory scale. BL3-Large Scale is recommended for large-scale research or
production of viable organisms containing recombinant or synthetic nucleic acid molecules that require
BL3 containment at the laboratory scale. No provisions are made for large-scale research or productionof viable organisms containing recombinant or synthetic nucleic acid molecules that require BL4
containment at the laboratory scale. If necessary, these requirements will be established by NIH on an
individual basis.
Appendix K-II. Good Large Scale Practice (GLSP)Appendix K-II-A. Institutional codes of practice shall be formulated and implemented to assure adequate
control of health and safety matters.
Appendix K-II-B. Written instructions and training of personnel shall be provided to assure that culturesof viable organisms containing recombinant or synthetic nucleic acid molecules are handled prudently
and that the work place is kept clean and orderly.
Appendix K-II-C. In the interest of good personal hygiene, facilities (e.g., hand washing sink, shower,changing room) and protective clothing (e.g., uniforms, laboratory coats) shall be provided that areappropriate for the risk of exposure to viable organisms containing recombinant or synthetic nucleic acidmolecules. Eating, drinking, smoking, applying cosmetics, and mouth pipetting shall be prohibited in thework area.
Appendix K-II-D. Cultures of viable organisms containing recombinant or synthetic nucleic acidmolecules shall be handled in facilities intended to safeguard health during work with microorganisms that
do not require containment.
Appendix K-II-E. Discharges containing viable recombinant or synthetic organisms shall be handled inaccordance with applicable governmental environmental regulations.
Appendix K-II-F. Addition of materials to a system, sample collection, transfer of culture fluids
within/between systems, and processing of culture fluids shall be conducted in a manner that maintains
employee's exposure to viable organisms containing recombinant or synthetic nucleic acid molecules at alevel that does not adversely affect the health and safety of employees.
Appendix K-II-G. The facility's emergency response plan shall include provisions for handling spills.
Appendix K-III. Biosafety Level 1 (BL1) - Large Scale
Appendix K-III-A. Spills and accidents which result in overt exposures to organisms containing
recombinant or synthetic nucleic acid molecules are immediately reported to the Laboratory Director.
Medical evaluation, surveillance, and treatment are provided as appropriate and written records aremaintained.
Appendix K-III-B. Cultures of viable organisms containing recombinant or synthetic nucleic acidmolecules shall be handled in a closed system (e.g., closed vessel used for the propagation and growthof cultures) or other primary containment equipment (e.g., biological safety cabinet containing a centrifuge
used to process culture fluids) which is designed to reduce the potential for escape of viable organisms.
Volumes less than 10 liters may be handled outside of a closed system or other primary containment

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equipment provided all physical containment requirements specified in Appendix G-II-A, PhysicalContainment Levels--Biosafety Level 1, are met.
Appendix K-III-C. Culture fluids (except as allowed in Appendix K-III-D) shall not be removed from aclosed system or other primary containment equipment unless the viable organisms containing
recombinant or synthetic nucleic acid molecules have been inactivated by a validated inactivation
procedure. A validated inactivation procedure is one which has been demonstrated to be effective using
the organism that will serve as the host for propagating the recombinant or synthetic nucleic acidmolecules. Culture fluids that contain viable organisms or viral vectors intended as final product may beremoved from the primary containment equipment by way of closed systems for sample analysis, further
processing or final fill.
Appendix K-III-D. Sample collection from a closed system, the addition of materials to a closed system,and the transfer of culture fluids from one closed system to another shall be conducted in a manner which
minimizes the release of aerosols or contamination of exposed surfaces.
Appendix K-III-E. Exhaust gases removed from a closed system or other primary containment
equipment shall be treated by filters which have efficiencies equivalent to high efficiency particulate
air/HEPA filters or by other equivalent procedures (e.g., incineration) to minimize the release of viableorganisms containing recombinant or synthetic nucleic acid molecules to the environment.
Appendix K-III-F. A closed system or other primary containment equipment that has contained viableorganisms containing recombinant or synthetic nucleic acid molecules shall not be opened formaintenance or other purposes unless it has been sterilized by a validated sterilization procedure except
when the culture fluids contain viable organisms or vectors intended as final product as described inAppendix K-III-C above. A validated sterilization procedure is one which has been demonstrated to beeffective using the organism that will serve as the host for propagating the recombinant or synthetic
nucleic acid molecules.
Appendix K-III-G. Emergency plans required by Sections IV-B-2-b-(6), Institutional Biosafety Committee,
and IV-B-3-c-(3), Biological Safety Officer, shall include methods and procedures for handling large
losses of culture on an emergency basis.
Appendix K-IV. Biosafety Level 2 (BL2) - Large Scale
Appendix K-IV-A. Spills and accidents which result in overt exposures to organisms containing
recombinant or synthetic nucleic acid molecules are immediately reported to the Biological Safety Officer,
Institutional Biosafety Committee, NIH OSP, and other appropriate authorities (if applicable). Reports to
NIH OSP shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mailto: NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP
website (www.osp.od.nih.gov). Medical evaluation, surveillance, and treatment are provided as
appropriate and written records are maintained.
Appendix K-IV-B. Cultures of viable organisms containing recombinant or synthetic nucleic acidmolecules shall be handled in a closed system (e.g., closed vessel used for the propagation and growthof cultures) or other primary containment equipment (e.g., Class III biological safety cabinet containing acentrifuge used to process culture fluids) which is designed to prevent the escape of viable organisms.
Volumes less than 10 liters may be handled outside of a closed system or other primary containment
equipment provided all physical containment requirements specified in Appendix G-II-B, PhysicalContainment Levels--Biosafety Level 2, are met.
Appendix K-IV-C. Culture fluids (except as allowed in Appendix K-IV-D) shall not be removed from aclosed system or other primary containment equipment unless the viable organisms containing
recombinant or synthetic nucleic acid molecules have been inactivated by a validated inactivation
procedure. A validated inactivation procedure is one which has been demonstrated to be effective using
the organism that will serve as the host for propagating the recombinant or synthetic nucleic acid

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molecules. Culture fluids that contain viable organisms or viral vectors intended as final product may beremoved from the primary containment equipment by way of closed systems for sample analysis, further
processing or final fill.
Appendix K-IV-D. Sample collection from a closed system, the addition of materials to a closed system,and the transfer of cultures fluids from one closed system to another shall be conducted in a manner
which prevents the release of aerosols or contamination of exposed surfaces.
Appendix K-IV-E. Exhaust gases removed from a closed system or other primary containment
equipment shall be treated by filters which have efficiencies equivalent to high efficiency particulate
air/HEPA filters or by other equivalent procedures (e.g., incineration) to prevent the release of viableorganisms containing recombinant or synthetic nucleic acid molecules to the environment.
Appendix K-IV-F. A closed system or other primary containment equipment that has contained viableorganisms containing recombinant or synthetic nucleic acid molecules shall not be opened formaintenance or other purposes unless it has been sterilized by a validated sterilization procedure except
when the culture fluids contain viable organisms or vectors intended as final product as described inAppendix K-IV-C above. A validated sterilization procedure is one which has been demonstrated to beeffective using the organisms that will serve as the host for propagating the recombinant or synthetic
nucleic acid molecules.
Appendix K-IV-G. Rotating seals and other mechanical devices directly associated with a closed systemused for the propagation and growth of viable organisms containing recombinant or synthetic nucleic acidmolecules shall be designed to prevent leakage or shall be fully enclosed in ventilated housings that are
exhausted through filters which have efficiencies equivalent to high efficiency particulate air/HEPA filtersor through other equivalent treatment devices.
Appendix K-IV-H. A closed system used for the propagation and growth of viable organisms containing
recombinant or synthetic nucleic acid molecules and other primary containment equipment used tocontain operations involving viable organisms containing sensing devices that monitor the integrity ofcontainment during operations.
Appendix K-IV-I. A closed system used for the propagation and growth of viable organisms containing
the recombinant or synthetic nucleic acid molecules shall be tested for integrity of the containment
features using the organism that will serve as the host for propagating recombinant or synthetic nucleic
acid molecules. Testing shall be accomplished prior to the introduction of viable organisms containing
recombinant or synthetic nucleic acid molecules and following modification or replacement of essential
containment features. Procedures and methods used in the testing shall be appropriate for theequipment design and for recovery and demonstration of the test organism. Records of tests and results
shall be maintained on file.
Appendix K-IV-J. A closed system used for the propagation and growth of viable organisms containing
recombinant or synthetic nucleic acid molecules shall be permanently identified. This identification shallbe used in all records reflecting testing, operation, and maintenance and in all documentation relating touse of this equipment for research or production activities involving viable organisms containing
recombinant or synthetic nucleic acid molecules.
Appendix K-IV-K. The universal biosafety sign shall be posted on each closed system and primarycontainment equipment when used to contain viable organisms containing recombinant or synthetic
nucleic acid molecules.
Appendix K-IV-L. Emergency plans required by Sections IV-B-2-b-(6), Institutional Biosafety Committee,
and IV-B-3-c-(3), Biological Safety Officer, shall include methods and procedures for handling large
losses of culture on an emergency basis.

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Appendix K-V. Biosafety Level 3 (BL3) - Large Scale
Appendix K-V-A. Spills and accidents which result in overt exposures to organisms containing
recombinant or synthetic nucleic acid molecules are immediately reported to the Biological Safety Officer,
Institutional Biosafety Committee, NIH OSP, and other appropriate authorities (if applicable). Reports to
NIH OSP shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mailto: NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP
website (www.osp.od.nih.gov). Medical evaluation, surveillance, and treatment are provided as
appropriate and written records are maintained.
Appendix K-V-B. Cultures of viable organisms containing recombinant or synthetic nucleic acidmolecules shall be handled in a closed system (e.g., closed vessels used for the propagation and growthof cultures) or other primary containment equipment (e.g., Class III biological safety cabinet containing acentrifuge used to process culture fluids) which is designed to prevent the escape of viable organisms.
Volumes less than 10 liters may be handled outside of a closed system provided all physical containment
requirements specified in Appendix G-II-C, Physical Containment Levels--Biosafety Level 3, are met.
Appendix K-V-C. Culture fluids (except as allowed in Appendix K-V-D) shall not be removed from aclosed system or other primary containment equipment unless the viable organisms containing
recombinant or synthetic nucleic acid molecules have been inactivated by a validated inactivation
procedure. A validated inactivation procedure is one which has been demonstrated to be effective using
the organisms that will serve as the host for propagating the recombinant or synthetic nucleic acidmolecules. Culture fluids that contain viable organisms or viral vectors intended as final product may beremoved from the primary containment equipment by way of closed systems for sample analysis, further
processing or final fill.
Appendix K-V-D. Sample collection from a closed system, the addition of materials to a closed system,and the transfer of culture fluids from one closed system to another shall be conducted in a manner which
prevents the release of aerosols or contamination of exposed surfaces.
Appendix K-V-E. Exhaust gases removed from a closed system or other primary containment
equipment shall be treated by filters which have efficiencies equivalent to high efficiency particulate
air/HEPA filters or by other equivalent procedures (e.g., incineration) to prevent the release of viableorganisms containing recombinant or synthetic nucleic acid molecules to the environment.
Appendix K-V-F. A closed system or other primary containment equipment that has contained viableorganisms containing recombinant or synthetic nucleic acid molecules shall not be opened formaintenance or other purposes unless it has been sterilized by a validated sterilization procedure except
when the culture fluids contain viable organisms or vectors intended as final product as described inAppendix K-V-C above. A validated sterilization procedure is one which has been demonstrated to beeffective using the organisms that will serve as the host for propagating the recombinant or synthetic
nucleic acid molecules.
Appendix K-V-G. A closed system used for the propagation and growth of viable organisms containing
recombinant or synthetic nucleic acid molecules shall be operated so that the space above the culture
level will be maintained at a pressure as low as possible, consistent with equipment design, in order tomaintain the integrity of containment features.
Appendix K-V-H. Rotating seals and other mechanical devices directly associated with a closed systemused to contain viable organisms containing recombinant or synthetic nucleic acid molecules shall be
designed to prevent leakage or shall be fully enclosed in ventilated housings that are exhausted throughfilters which have efficiencies equivalent to high efficiency particulate air/HEPA filters or through otherequivalent treatment devices.
Appendix K-V-I. A closed system used for the propagation and growth of viable organisms containing
recombinant or synthetic nucleic acid molecules and other primary containment equipment used toPage 102 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

contain operations involving viable organisms containing recombinant or synthetic nucleic acid molecules
shall include monitoring or sensing devices that monitor the integrity of containment during operations.
Appendix K-V-J. A closed system used for the propagation and growth of viable organisms containing
recombinant or synthetic nucleic acid molecules shall be tested for integrity of the containment featuresusing the organisms that will serve as the host for propagating the recombinant or synthetic nucleic acidmolecules. Testing shall be accomplished prior to the introduction of viable organisms containing
recombinant or synthetic nucleic acid molecules and following modification or replacement of essential
containment features. Procedures and methods used in the testing shall be appropriate for theequipment design and for recovery and demonstration of the test organism. Records of tests and results
shall be maintained on file.
Appendix K-V-K. A closed system used for the propagation and growth of viable organisms containing
recombinant or synthetic nucleic acid molecules shall be permanently identified. This identification shallbe used in all records reflecting testing, operation, maintenance, and use of this equipment for research
production activities involving viable organisms containing recombinant or synthetic nucleic acidmolecules.
Appendix K-V-L. The universal biosafety sign shall be posted on each closed system and primarycontainment equipment when used to contain viable organisms containing recombinant or synthetic
nucleic acid molecules.
Appendix K-V-M. Emergency plans required by Sections IV-B-2-b-(6), Institutional Biosafety Committee,
and IV-B-3-c-(3), Biological Safety Officer, shall include methods and procedures for handling large
losses of culture on an emergency basis.
Appendix K-V-N. Closed systems and other primary containment equipment used in handling cultures of
viable organisms containing recombinant or synthetic nucleic acid molecules shall be located within acontrolled area which meets the following requirements:
Appendix K-V-N-1. The controlled area shall have a separate entry area. The entry area shall be adouble-doored space such as an air lock, anteroom, or change room that separates the controlled areafrom the balance of the facility.
Appendix K-V-N-2. The surfaces of walls, ceilings, and floors in the controlled area shall be such as topermit ready cleaning and decontamination.
Appendix K-V-N-3. Penetrations into the controlled area shall be sealed to permit liquid or vapor phasespace decontamination.
Appendix K-V-N-4. All utilities and service or process piping and wiring entering the controlled area shallbe protected against contamination.
Appendix K-V-N-5. Hand washing facilities equipped with foot, elbow, or automatically operated valves
shall be located at each major work area and near each primary exit.
Appendix K-V-N-6. A shower facility shall be provided. This facility shall be located in close proximity to
the controlled area.
Appendix K-V-N-7. The controlled area shall be designed to preclude release of culture fluids outside
the controlled area in the event of an accidental spill or release from the closed systems or other primarycontainment equipment.
Appendix K-V-N-8. The controlled area shall have a ventilation system that is capable of controlling air
movement. The movement of air shall be from areas of lower contamination potential to areas of higher
contamination potential. If the ventilation system provides positive pressure supply air, the system shallPage 103 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

operate in a manner that prevents the reversal of the direction of air movement or shall be equipped withan alarm that would be actuated in the event that reversal in the direction of air movement were to occur.
The exhaust air from the controlled area shall not be recirculated to other areas of the facility. The
exhaust air from the controlled area may not be discharged to the outdoors without being high efficiency
particulate air/HEPA filtered, subjected to thermal oxidation, or otherwise treated to prevent the release of
viable organisms.
Appendix K-V-O. The following personnel and operational practices shall be required:
Appendix K-V-O-1. Personnel entry into the controlled area shall be through the entry area specified inAppendix K-V-N-1.
Appendix K-V-O-2. Persons entering the controlled area shall exchange or cover their personal clothingwith work garments such as jump suits, laboratory coats, pants and shirts, head cover, and shoes or shoecovers. On exit from the controlled area the work clothing may be stored in a locker separate from thatused for personal clothing or discarded for laundering. Clothing shall be decontaminated before
laundering.
Appendix K-V-O-3. Entry into the controlled area during periods when work is in progress shall be
restricted to those persons required to meet program or support needs. Prior to entry, all persons shallbe informed of the operating practices, emergency procedures, and the nature of the work conducted.
Appendix K-V-O-4. Persons under 18 years of age shall not be permitted to enter the controlled area.
Appendix K-V-O-5. The universal biosafety sign shall be posted on entry doors to the controlled area
and all internal doors when any work involving the organism is in progress. This includes periods when
decontamination procedures are in progress. The sign posted on the entry doors to the controlled area
shall include a statement of agents in use and personnel authorized to enter the controlled area.
Appendix K-V-O-6. The controlled area shall be kept neat and clean.
Appendix K-V-O-7. Eating, drinking, smoking, and storage of food are prohibited in the controlled area.
Appendix K-V-O-8. Animals and plants shall be excluded from the controlled area.
Appendix K-V-O-9. An effective insect and rodent control program shall be maintained.
Appendix K-V-O-10. Access doors to the controlled area shall be kept closed, except as necessary foraccess, while work is in progress. Service doors leading directly outdoors shall be sealed and locked
while work is in progress.
Appendix K-V-O-11. Persons shall wash their hands when exiting the controlled area.
Appendix K-V-O-12. Persons working in the controlled area shall be trained in emergency procedures.
Appendix K-V-O-13. Equipment and materials required for the management of accidents involving
viable organisms containing recombinant or synthetic nucleic acid molecules shall be available in thecontrolled area.
Appendix K-V-O-14. The controlled area shall be decontaminated in accordance with establishedprocedures following spills or other accidental release of viable organisms containing recombinant orsynthetic nucleic acid molecules.

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Appendix K - Table 1.
Comparison of Good Large Scale Practice (GLSP) and Biosafety Level(BL) - Large Scale (LS) Practice (See Appendix K-VI-A, Footnotes Of Appendix K)
CRITERION[See Appendix K-VI-B, Footnotes of Appendix K]

GLSP

BL1-LS

BL2-LS

BL3-LS

1.

Formulate and implement institutional codes of practice forsafety of personnel and adequate control of hygiene andsafety measures.

K-II-A

G-I

2.

Provide adequate written instructions and training of
personnel to keep work place clean and tidy and to keep
exposure to biological, chemical or physical agents at alevel that does not adversely affect health and safety of
employees.

K-II-B

G-I

3.

Provide changing and hand washing facilities as well asprotective clothing, appropriate to the risk, to be worn
during work.

K-II-C

G-II-A-1-h

G-II-B-2-f

G-II-C-2-i

4.

Prohibit eating, drinking, smoking, mouth pipetting, andapplying cosmetics in the work place.

K-II-C

G-II-A-1-d
G-II-A-1-e

G-II-B-1-d
G-II-B-1-e

G-II-C-1-c
G-II-C-1-d

5.

Internal accident reporting.

K-II-G

K-III-A

K-IV-A

K-V-A

6.

Medical surveillance.

NR

NR

7.

Viable organisms should be handled in a system thatphysically separates the process from the external
environment (closed system or other primarycontainment).

NR

K-III-B

K-IV-B

K-V-B

8.

Culture fluids not removed from a system until organisms
are inactivated.

NR

K-III-C

K-IV-C

K-V-C

9.

Inactivation of waste solutions and materials with respect
to their biohazard potential.

K-II-E

K-III-C

K-IV-C

K-V-C

10.

Control of aerosols by engineering or procedural controls
to prevent or minimize release of organisms during
sampling from a system, addition of materials to a system,transfer of cultivated cells, and removal of material,
products, and effluent from a system.

Minimize
Procedure
K-II-F

Minimize
Engineer
K-III-B
K-III-D

Prevent
Engineer
K-IV-B
K-IV-D

Prevent
Engineer
K-V-B
K-V-D

11.

Treatment of exhaust gases from a closed system tominimize or prevent release of viable organisms.

NR

Minimize
K-III-E

Prevent
K-IV-E

Prevent
K-V-E

12.

Closed system that has contained viable organisms not tobe opened until sterilized by a validated procedure.

NR

K-III-F

K-IV-F

K-V-F

13.

Closed system to be maintained at as a low pressure as
possible to maintain integrity of containment features.

NR

NR

NR

K-V-G

14.

Rotating seals and other penetrations into closed systemdesigned to prevent or minimize leakage.

NR

NR

Prevent
K-IV-G

Prevent
K-V-H

15.

Closed system shall incorporate monitoring or sensing
devices to monitor the integrity of containment.

NR

NR

K-IV-H

K-V-I

16.

Validated integrity testing of closed containment system.

NR

NR

K-IV-I

K-V-J

17.

Closed system to be permanently identified for record
keeping purposes.

NR

NR

K-IV-J

K-V-K

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CRITERION[See Appendix K-VI-B, Footnotes of Appendix K]

GLSP

BL1-LS

BL2-LS

BL3-LS

18.

Universal biosafety sign to be posted on each closed
system.

NR

NR

K-IV-K

K-V-L

19.

Emergency plans required for handling large losses ofcultures.

K-II-G

K-III-G

K-IV-L

K-V-M

20.

Access to the work place.

NR

G-II-A-1-a

G-II-B-1-a

K-V-N

21.

Requirements for controlled access area.

NR

NR

NR

K-V-N&O
NR = not required

Appendix K-VI. Footnotes of Appendix K
Appendix K-VI-A. This table is derived from the text in Appendices G (Physical Containment) and K and
is not to be used in lieu of Appendices G and K.
Appendix K-VI-B. The criteria in this grid address only the biological hazards associated with organisms
containing recombinant or synthetic nucleic acid. Other hazards accompanying the large-scale cultivationof such organisms (e.g., toxic properties of products; physical, mechanical, and chemical aspects ofdownstream processing) are not addressed and shall be considered separately, albeit in conjunction withthis grid.
Appendix K-VII. Definitions to Accompany Containment Grid and Appendix K
Appendix K-VII-A. Accidental Release. An accidental release is the unintentional discharge of a
microbiological agent (i.e., microorganism or virus) or eukaryotic cell due to a failure in the containment
system.
Appendix K-VII-B. Biological Barrier. A biological barrier is an impediment (naturally occurring or
introduced) to the infectivity and/or survival of a microbiological agent or eukaryotic cell once it has beenreleased into the environment.
Appendix K-VII-C. Closed System. A closed system is one in which by its design and proper operation,
prevents release of a microbiological agent or eukaryotic cell contained therein.
Appendix K-VII-D. Containment. Containment is the confinement of a microbiological agent or
eukaryotic cell that is being cultured, stored, manipulated, transported, or destroyed in order to prevent or
limit its contact with people and/or the environment. Methods used to achieve this include: physical andbiological barriers and inactivation using physical or chemical means.
Appendix K-VII-E. De minimis Release. De minimis release is the release of: (i) viable microbiologicalagents or eukaryotic cells that does not result in the establishment of disease in healthy people, plants, oranimals; or (ii) in uncontrolled proliferation of any microbiological agents or eukaryotic cells.
Appendix K-VII-F. Disinfection. Disinfection is a process by which viable microbiological agents or
eukaryotic cells are reduced to a level unlikely to produce disease in healthy people, plants, or animals.
Appendix K-VII-G. Good Large Scale Practice Organism. For an organism to qualify for Good Large
Scale Practice consideration, it must meet the following criteria [Reference: Organization for Economic
Cooperation and Development, Recombinant DNA Safety Considerations, 1987, p. 34-35]: (i) the hostorganism should be non-pathogenic, should not contain adventitious agents and should have an
extended history of safe large-scale use or have built-in environmental limitations that permit optimum
growth in the large-scale setting but limited survival without adverse consequences in the environment;Page 106 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)(ii) the recombinant or synthetic nucleic acid molecule-engineered organism should be non-pathogenic,
should be as safe in the large-scale setting as the host organism, and without adverse consequences inthe environment; and (iii) the vector/insert should be well characterized and free from known harmful
sequences; should be limited in size as much as possible to the DNA required to perform the intended
function; should not increase the stability of the construct in the environment unless that is a requirementof the intended function; should be poorly mobilizable; and should not transfer any resistance markers to
microorganisms unknown to acquire them naturally if such acquisition could compromise the use of adrug to control disease agents in human or veterinary medicine or agriculture.
Appendix K-VII-H. Inactivation. Inactivation is any process that destroys the ability of a specificmicrobiological agent or eukaryotic cell to self-replicate.
Appendix K-VII-I. Incidental Release. An incidental release is the discharge of a microbiological agent
or eukaryotic cell from a containment system that is expected when the system is appropriately designed
and properly operated and maintained.
Appendix K-VII-J. Minimization. Minimization is the design and operation of containment systems inorder that any incidental release is a de minimis release.
Appendix K-VII-K. Pathogen. A pathogen is any microbiological agent or eukaryotic cell containing
sufficient genetic information, which upon expression of such information, is capable of producing diseasein healthy people, plants, or animals.
Appendix K-VII-L. Physical Barrier. A physical barrier is considered any equipment, facilities, or
devices (e.g., fermentors, factories, filters, thermal oxidizers) which are designed to achieve containment.
Appendix K-VII-M. Release. Release is the discharge of a microbiological agent or eukaryotic cell froma containment system. Discharges can be incidental or accidental. Incidental releases are de minimis innature; accidental releases may be de minimis in nature.
***********************************************************************************************************************
APPENDIX L. PHYSICAL AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT ORSYNTHETIC NUCLEIC ACID MOLECULE RESEARCH INVOLVING PLANTS
Appendix L specifies physical and biological containment conditions and practices suitable to thegreenhouse conduct of experiments involving recombinant or synthetic nucleic acid molecule-containing
plants, plant-associated microorganisms, and small animals. All provisions of the NIH Guidelines apply toplant research activities with the following modifications:
Appendix L shall supersede Appendix G (Physical Containment) when the research plants are of a size,
number, or have growth requirements that preclude the use of containment conditions described inAppendix G. The plants covered in Appendix L include but are not limited to mosses, liverworts,
macroscopic algae, and vascular plants including terrestrial crops, forest, and ornamental species.
Plant-associated microorganisms include viroids, virusoids, viruses, bacteria, fungi, protozoans, certain
small algae, and microorganisms that have a benign or beneficial association with plants, such as certain
Rhizobium species and microorganisms known to cause plant diseases. The appendix applies tomicroorganisms which are being modified with the objective of fostering an association with plants.
Plant-associated small animals include those arthropods that: (i) are in obligate association with plants,(ii) are plant pests, (iii) are plant pollinators, or (iv) transmit plant disease agents, as well as other small
animals such as nematodes for which tests of biological properties necessitate the use of plants.
Microorganisms associated with such small animals (e.g., pathogens or symbionts) are included.

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The Institutional Biosafety Committee shall include at least one individual with expertise in plant, plant
pathogen, or plant pest containment principles when experiments utilizing Appendix L require priorapproval by the Institutional Biosafety Committee.
Appendix L-I.

General Plant Biosafety Levels

Appendix L-I-A. The principal purpose of plant containment is to avoid the unintentional transmission ofa recombinant or synthetic nucleic acid molecule-containing plant genome, including nuclear or organelle
hereditary material or release of recombinant or synthetic nucleic acid molecule-derived organisms
associated with plants.
Appendix L-I-B. The containment principles are based on the recognition that the organisms that areused pose no health threat to humans or higher animals (unless deliberately modified for that purpose),and that the containment conditions minimize the possibility of an unanticipated deleterious effect onorganisms and ecosystems outside of the experimental facility, e.g., the inadvertent spread of a serious
pathogen from a greenhouse to a local agricultural crop or the unintentional introduction andestablishment of an organism in a new ecosystem.
Appendix L-I-C. Four biosafety levels, referred to as Biosafety Level (BL) 1 - Plants (P), BL2-P, BL3-P,
and BL4-P, are established in Appendix L-II, Physical Containment Levels. The selection of containment
levels required for research involving recombinant or synthetic nucleic acid molecules in plants or
associated with plants is specified in Appendix L-III, Biological Containment Practices. These biosafetylevels are described in Appendix L-II, Physical Containment Levels. This appendix describes greenhousepractices and special greenhouse facilities for physical containment.
Appendix L-I-D. BL1-P through BL4-P are designed to provide differential levels of biosafety for plants inthe absence or presence of other experimental organisms that contain recombinant or synthetic nucleic
acid molecules. These biosafety levels, in conjunction with biological containment conditions described inAppendix L-III, Biological Containment Practices, provide flexible approaches to ensure the safe conductof research.
Appendix L-I-E. For experiments in which plants are grown at the BL1 through BL4 laboratory settings,
containment practices shall be followed as described in Appendix G, Physical Containment. These
containment practices include the use of plant tissue culture rooms, growth chambers within laboratory
facilities, or experiments performed on open benches. Additional biological containment practices should
be added by the Greenhouse Director or Institutional Biosafety Committee as necessary (see Appendix LIII, Biological Containment Practices), if botanical reproductive structures are produced that have thepotential of being released.
Appendix L-II.

Physical Containment Levels

Appendix L-II-A.

Biosafety Level 1 - Plants (BL1-P)

Appendix L-II-A-1.

Standard Practices (BL1-P)

Appendix L-II-A-1-a.

Greenhouse Access (BL1-P)

Appendix L-II-A-1-a-(1). Access to the greenhouse shall be limited or restricted, at the discretion of theGreenhouse Director, when experiments are in progress.
Appendix L-II-A-1-a-(2). Prior to entering the greenhouse, personnel shall be required to read and follow
instructions on BL1-P greenhouse practices and procedures. All procedures shall be performed inaccordance with accepted greenhouse practices that are appropriate to the experimental organism.
Appendix L-II-A-1-b.

Records (BL1-P)

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Appendix L-II-A-1-b-(1). A record shall be kept of experiments currently in progress in the greenhousefacility.
Appendix L-II-A-1-c.

Decontamination and Inactivation (BL1-P)

Appendix L-II-A-1-c-(1). Experimental organisms shall be rendered biologically inactive by appropriate
methods before disposal outside of the greenhouse facility.
Appendix L-II-A-1-d.

Control of Undesired Species and Motile Macroorganisms (BL1-P)

Appendix L-II-A-1-d-(1). A program shall be implemented to control undesired species (e.g., weed,
rodent, or arthropod pests and pathogens), by methods appropriate to the organisms and in accordancewith applicable state and Federal laws.
Appendix L-II-A-1-d-(2). Arthropods and other motile macroorganisms shall be housed in appropriate
cages. If macroorganisms (e.g., flying arthropods or nematodes) are released within the greenhouse,precautions shall be taken to minimize escape from the greenhouse facility.
Appendix L-II-A-1-e.

Concurrent Experiments Conducted in the Greenhouse (BL1-P)

Appendix L-II-A-1-e-(1). Experiments involving other organisms that require a containment level lower
than BL1-P may be conducted in the greenhouse concurrently with experiments that require BL1-P
containment, provided that all work is conducted in accordance with BL1-P greenhouse practices.
Appendix L-II-A-2.

Facilities (BL1-P)

Appendix L-II-A-2-a.

Definitions (BL1-P)

Appendix L-II-A-2-a-(1). The term "greenhouse" refers to a structure with walls, a roof, and a floor
designed and used principally for growing plants in a controlled and protected environment. The walls
and roof are usually constructed of transparent or translucent material to allow passage of sunlight forplant growth.
Appendix L-II-A-2-a-(2). The term "greenhouse facility" includes the actual greenhouse rooms orcompartments for growing plants, including all immediately contiguous hallways and head-house areas,
and is considered part of the confinement area.
Appendix L-II-A-2-b.

Greenhouse Design (BL1-P)

Appendix L-II-A-2-b-(1). The greenhouse floor may be composed of gravel or other porous material. At
a minimum, impervious (e.g., concrete) walkways are recommended.
Appendix L-II-A-2-b-(2). Windows and other openings in the walls and roof of the greenhouse facility
may be open for ventilation as needed for proper operation and do not require any special barrier tocontain or exclude pollen, microorganisms, or small flying animals (e.g., arthropods and birds); however,screens are recommended.
Appendix L-II-B.

Biosafety Level 2 - Plants (BL2-P)

Appendix L-II-B-1.

Standard Practices (BL2-P)

Appendix L-II-B-1-a.

Greenhouse Access (BL2-P)

Appendix L-II-B-1-a-(1). Access to the greenhouse shall be limited or restricted, at the discretion of theGreenhouse Director, to individuals directly involved with the experiments when they are in progress.

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Appendix L-II-B-1-a-(2). Personnel shall be required to read and follow instructions on BL2-P practices
and procedures. All procedures shall be conducted in accordance with accepted greenhouse practices
that are appropriate to the experimental organisms.
Appendix L-II-B-1-b.

Records (BL2-P)

Appendix L-II-B-1-b-(1). A record shall be kept of experimental plants, microorganisms, or small animals
that are brought into or removed from the greenhouse facility.
Appendix L-II-B-1-b-(2). A record shall be kept of experiments currently in progress in the greenhousefacility.
Appendix L-II-B-1-b-(3). The Principal Investigator shall report any greenhouse accident involving theinadvertent release or spill of microorganisms to the Greenhouse Director, Institutional Biosafety
Committee, NIH OSP and other appropriate authorities immediately (if applicable). Reports to the NIH
OSP shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:
NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website(www.osp.od.nih.gov). Documentation of any such accident shall be prepared and maintained.
Appendix L-II-B-1-c.

Decontamination and Inactivation (BL2-P)

Appendix L-II-B-1-c-(1). Experimental organisms shall be rendered biologically inactive by appropriate
methods before disposal outside of the greenhouse facility.
Appendix L-II-B-1-c-(2). Decontamination of run-off water is not necessarily required. If part of thegreenhouse is composed of gravel or similar material, appropriate treatments should be made periodicallyto eliminate, or render inactive, any organisms potentially entrapped by the gravel.
Appendix L-II-B-1-d.

Control of Undesired Species and Motile Macroorganisms (BL2-P)

Appendix L-II-B-1-d-(1). A program shall be implemented to control undesired species (e.g., weed,
rodent, or arthropod pests and pathogens) by methods appropriate to the organisms and in accordancewith applicable state and Federal laws.
Appendix L-II-B-1-d-(2). Arthropods and other motile macroorganisms shall be housed in appropriate
cages. If macroorganisms (e.g., flying arthropods or nematodes) are released within the greenhouse,precautions shall be taken to minimize escape from the greenhouse facility.
Appendix L-II-B-1-e.

Concurrent Experiments Conducted in the Greenhouse (BL2-P)

Appendix L-II-B-1-e-(1). Experiments involving other organisms that require a containment level lower
than BL2-P may be conducted in the greenhouse concurrently with experiments that require BL2-P
containment provided that all work is conducted in accordance with BL2-P greenhouse practices.
Appendix L-II-B-1-f.

Signs (BL2-P)

Appendix L-II-B-1-f-(1). A sign shall be posted indicating that a restricted experiment is in progress. The
sign shall indicate the following: (i) the name of the responsible individual, (ii) the plants in use, and (iii)
any special requirements for using the area.
Appendix L-II-B-1-f-(2). If organisms are used that have a recognized potential for causing serious
detrimental impacts on managed or natural ecosystems, their presence shall be indicated on a signposted on the greenhouse access doors.
Appendix L-II-B-1-f-(3). If there is a risk to human health, a sign shall be posted incorporating theuniversal biosafety symbol.

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Appendix L-II-B-1-g.

(April 2024)

Transfer of Materials (BL2-P)

Appendix L-II-B-1-g-(1). Materials containing experimental microorganisms, which are brought into or
removed from the greenhouse facility in a viable or intact state, shall be transferred in a closed
non-breakable container.
Appendix L-II-B-1-h.

Greenhouse Practices Manual (BL2-P)

Appendix L-II-B-1-h-(1). A greenhouse practices manual shall be prepared or adopted. This manual
shall: (i) advise personnel of the potential consequences if such practices are not followed, and (ii)outline contingency plans to be implemented in the event of the unintentional release of organisms.
Appendix L-II-B-2.

Facilities (BL2-P)

Appendix L-II-B-2-a.

Definitions (BL2-P)

Appendix L-II-B-2-a-(1). The term "greenhouse" refers to a structure with walls, a roof, and a floor
designed and used principally for growing plants in a controlled and protected environment. The walls
and roof are usually constructed of transparent or translucent material to allow passage of sunlight forplant growth.
Appendix L-II-B-2-a-(2). The term "greenhouse facility" includes the actual greenhouse rooms orcompartments for growing plants, including all immediately contiguous hallways and head-house areasand is considered part of the confinement area.
Appendix L-II-B-2-b.

Greenhouse Design (BL2-P)

Appendix L-II-B-2-b-(1). A greenhouse floor composed of an impervious material. Concrete isrecommended, but gravel or other porous material under benches is acceptable unless propagules ofexperimental organisms are readily disseminated through soil. Soil beds are acceptable unlesspropagules of experimental organisms are readily disseminated through soil.
Appendix L-II-B-2-b-(2). Windows and other openings in the walls and roof of the greenhouse facility
may be open for ventilation as needed for proper operation and do not require any special barrier to
exclude pollen or microorganisms; however, screens are required to exclude small flying animals (e.g.,arthropods and birds).
Appendix L-II-B-2-c.

Autoclaves (BL2-P)

Appendix L-II-B-2-c-(1). An autoclave shall be available for the treatment of contaminated greenhousematerials.
Appendix L-II-B-2-d.

Supply and Exhaust Air Ventilation Systems (BL2-P)

Appendix L-II-B-2-d-(1). If intake fans are used, measures shall be taken to minimize the ingress ofarthropods. Louvers or fans shall be constructed such that they can only be opened when the fan is inoperation.
Appendix L-II-B-2-e.

Other (BL2-P)

Appendix L-II-B-2-e-(1). BL2-P greenhouse containment requirements may be satisfied by using a
growth chamber or growth room within a building provided that the external physical structure limits
access and escape of microorganisms and macroorganisms in a manner that satisfies the intent of theforegoing clauses.

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Appendix L-II-C.

Biosafety Level 3 - Plants (BL3-P)

Appendix L-II-C-1.

Standard Practices (BL3-P)

Appendix L-II-C-1-a.

Greenhouse Access (BL3-P)(April 2024)

Appendix L-II-C-1-a-(1). Authorized entry into the greenhouse shall be restricted to individuals who are
required for program or support purposes. The Greenhouse Director shall be responsible for assessing
each circumstance and determining those individuals who are authorized to enter the greenhouse facility.
Appendix L-II-C-1-a-(2). Prior to entering the greenhouse, personnel shall be required to read and follow
instructions on BL3-P practices and procedures. All procedures shall be conducted in accordance withaccepted greenhouse practices that are appropriate to the experimental organisms.
Appendix L-II-C-1-b.

Records (BL3-P)

Appendix L-II-C-1-b-(1). A record shall be kept of experimental plants, microorganisms, or small animals
that are brought into or removed from the greenhouse facility.
Appendix L-II-C-1-b-(2). A record shall be kept of experiments currently in progress in the greenhousefacility.
Appendix L-II-C-1-b-(3). The Principal Investigator shall report any greenhouse accident involving theinadvertent release or spill of microorganisms to the Biological Safety Officer, Greenhouse Director,
Institutional Biosafety Committee, NIH OSP, and other appropriate authorities immediately (if applicable).
Reports to the NIH OSP shall be sent to the Office of Science Policy, National Institutes of Health,preferably by e-mail to: NIHGuidelines@od.nih.gov; additional contact information is also available here
and on the OSP website (www.osp.od.nih.gov). Documentation of any such accident shall be preparedand maintained.
Appendix L-II-C-1-c.

Decontamination and Inactivation (BL3-P)

Appendix L-II-C-1-c-(1). All experimental materials shall be sterilized in an autoclave or renderedbiologically inactive by appropriate methods before disposal, except those that are to remain in a viable orintact state for experimental purposes; including water that comes in contact with experimental
microorganisms or with material exposed to such microorganisms, and contaminated equipment andsupplies.
Appendix L-II-C-1-d.

Control of Undesired Species and Motile Macroorganisms (BL3-P)

Appendix L-II-C-1-d-(1). A program shall be implemented to control undesired species (e.g., weed,
rodent, or arthropod pests and pathogens) by methods appropriate to the organisms and in accordancewith applicable state and Federal laws.
Appendix L-II-C-1-d-(2). Arthropods and other motile macroorganisms shall be housed in appropriate
cages. When appropriate to the organism, experiments shall be conducted within cages designed tocontain the motile organisms.
Appendix L-II-C-1-e.

Concurrent Experiments Conducted in the Greenhouse (BL3-P)

Appendix L-II-C-1-e-(1). Experiments involving organisms that require a containment level lower than
BL3-P may be conducted in the greenhouse concurrently with experiments that require BL3-P
containment provided that all work is conducted in accordance with BL3-P greenhouse practices.
Appendix L-II-C-1-f.

Signs (BL3-P)

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Appendix L-II-C-1-f-(1). A sign shall be posted indicating that a restricted experiment is in progress. The
sign shall indicate the following: (i) the name of the responsible individual, (ii) the plants in use, and (iii)
any special requirements for using the area.
Appendix L-II-C-1-f-(2). If organisms are used that have a recognized potential for causing serious
detrimental impacts on managed or natural ecosystems, their presence should be indicated on a signposted on the greenhouse access doors.
Appendix L-II-C-1-f-(3). If there is a risk to human health, a sign shall be posted incorporating theuniversal biosafety symbol.
Appendix L-II-C-1-g.

Transfer of Materials (BL3-P)

Appendix L-II-C-1-g-(1). Experimental materials that are brought into or removed from the greenhousefacility in a viable or intact state shall be transferred to a non-breakable sealed secondary container. At
the time of transfer, if the same plant species, host, or vector are present within the effective
dissemination distance of propagules of the experimental organism, the surface of the secondary
container shall be decontaminated. Decontamination may be accomplished by passage through achemical disinfectant or fumigation chamber or by an alternative procedure that has demonstrated
effective inactivation of the experimental organism.
Appendix L-II-C-1-h.

Greenhouse Practices Manual (BL3-P)

Appendix L-II-C-1-h-(1). A greenhouse practices manual shall be prepared or adopted. This manual
shall: (i) advise personnel of the potential consequences if such practices are not followed, and (ii)outline contingency plans to be implemented in the event of the unintentional release of organisms withrecognized potential for serious detrimental impact.
Appendix L-II-C-1-i.

Protective Clothing (BL3-P)

Appendix L-II-C-1-i-(1). Disposable clothing (e.g., solid front or wrap-around gowns, scrub suits, or otherappropriate clothing) shall be worn in the greenhouse if deemed necessary by the Greenhouse Director
because of potential dissemination of the experimental microorganisms.
Appendix L-II-C-1-i-(2). Protective clothing shall be removed before exiting the greenhouse anddecontaminated prior to laundering or disposal.
Appendix L-II-C-1-j.

Other (BL3-P)

Appendix L-II-C-1-j-(1). Personnel are required to thoroughly wash their hands upon exiting thegreenhouse.
Appendix L-II-C-1-j-(2). All procedures shall be performed carefully to minimize the creation of aerosols
and excessive splashing of potting material/soil during watering, transplanting, and all experimental
manipulations.
Appendix L-II-C-2.

Facilities (BL3-P)

Appendix L-II-C-2-a.

Definitions (BL3-P)

Appendix L-II-C-2-a-(1). The term "greenhouse" refers to a structure with walls, roof, and floor designed
and used principally for growing plants in a controlled and protected environment. The walls and roof areusually constructed of transparent or translucent material to allow passage of sunlight for plant growth.
Appendix L-II-C-2-a-(2). The term "greenhouse facility" includes the actual greenhouse rooms orcompartments for growing plants, including all immediately contiguous hallways and head-house areas,

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and is considered part of the confinement area. The need to maintain negative pressure should beconsidered when constructing or renovating the greenhouse.
Appendix L-II-C-2-b.

Greenhouse Design (BL3-P)

Appendix L-II-C-2-b-(1). The greenhouse floor shall be composed of concrete or other impervious
material with provision for collection and decontamination of liquid run-off.
Appendix L-II-C-2-b-(2). Windows shall be closed and sealed. All glazing shall be resistant to breakage(e.g., double-pane tempered glass or equivalent).
Appendix L-II-C-2-b-(3). The greenhouse shall be a closed self-contained structure with a continuous
covering that is separated from areas that are open to unrestricted traffic flow. The minimum requirement
for greenhouse entry shall be passage through two sets of self-closing locking doors.
Appendix L-II-C-2-b-(4). The greenhouse facility shall be surrounded by a security fence or protected byequivalent security measures.
Appendix L-II-C-2-b-(5). Internal walls, ceilings, and floors shall be resistant to penetration by liquids
and chemicals to facilitate cleaning and decontamination of the area. All penetrations into thesestructures and surfaces (e.g., plumbing and utilities) shall be sealed.
Appendix L-II-C-2-b-(6). Bench tops and other work surfaces should have seamless surfaces that areimpervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
Appendix L-II-C-2-b-(7). The greenhouse contains a foot, elbow, or automatically operated sink, which islocated near the exit door for hand washing.
Appendix L-II-C-2-c.

Autoclaves (BL3-P)

Appendix L-II-C-2-c-(1). An autoclave shall be available for decontaminating materials within thegreenhouse facility. A double-door autoclave is recommended (not required) for the decontamination ofmaterials passing out of the greenhouse facility.
Appendix L-II-C-2-d.

Supply and Exhaust Air Ventilation Systems (BL3-P)

Appendix L-II-C-2-d-(1). An individual supply and exhaust air ventilation system shall be provided. The
system maintains pressure differentials and directional airflow, as required, to assure inward (or zero)
airflow from areas outside of the greenhouse.
Appendix L-II-C-2-d-(2). The exhaust air from the greenhouse facility shall be filtered through highefficiency particulate air-HEPA filters and discharged to the outside. The filter chambers shall be
designed to allow in situ decontamination before filters are removed and to facilitate certification testing
after they are replaced. Air filters shall be 80-85% average efficiency by the American Society of Heating,
Refrigerating, and Air Conditioning Engineers (ASHRAE) Standard 52-68 test method using atmosphere
dust. Air supply fans shall be equipped with a back-flow damper that closes when the air supply fan is off.
Alternatively, a HEPA filter may be used on the air supply system instead of the filters and damper. The
supply and exhaust airflow shall be interlocked to assure inward (or zero) airflow at all times.
Appendix L-II-C-2-e.

Other (BL3-P)

Appendix L-II-C-2-e-(1). BL3-P greenhouse containment requirements may be satisfied using a growthchamber or growth room within a building provided that the location, access, airflow patterns, andprovisions for decontamination of experimental materials and supplies meet the intent of the foregoingclauses.

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Appendix L-II-C-2-e-(2). Vacuum lines shall be protected with high efficiency particulate air/HEPA orequivalent filters and liquid disinfectant traps.
Appendix L-II-D.

Biosafety Level 4 - Plants (BL4-P)

Appendix L-II-D-1.

Standard Practices (BL4-P)

Appendix L-II-D-1-a.

Greenhouse Access (BL4-P)

Appendix L-II-D-1-a-(1). Authorized entry into the greenhouse shall be restricted to individuals who are
required for program or support purposes. The Greenhouse Director shall be responsible for assessing
each circumstance and determining those individuals who are authorized to enter the greenhouse facility
or work in the greenhouse during experiments.
Appendix L-II-D-1-a-(2). Access shall be managed by the Greenhouse Director, Biological Safety
Officer, or other individual responsible for physical security of the greenhouse facility; and access limitedby means of secure, locked doors.
Appendix L-II-D-1-a-(3). Prior to entering, individuals shall be advised of the potential environmental
hazards and instructed on appropriate safeguards for ensuring environmental safety. Individuals
authorized to enter the greenhouse facility shall comply with the instructions and all other applicableentry/exit procedures.
Appendix L-II-D-1-a-(4). Personnel shall enter and exit the greenhouse facility only through the clothingchange and shower rooms and shall shower each time they exit the greenhouse facility. Personnel shalluse the airlocks to enter or exit the laboratory only in an emergency. In the event of an emergency, every
reasonable effort should be made to prevent the possible transport of viable propagules fromcontainment.
Appendix L-II-D-1-a-(5). Prior to entering the greenhouse, personnel shall be required to read and follow
instructions on BL4-P practices and procedures.
Appendix L-II-D-1-b.

Records (BL4-P)

Appendix L-II-D-1-b-(1). A record shall be kept of all experimental materials brought into or removed
from the greenhouse.
Appendix L-II-D-1-b-(2). A record shall be kept of experiments currently in progress in the greenhousefacility.
Appendix L-II-D-1-b-(3). A record shall be kept of all personnel entering and exiting the greenhousefacility, including the date and time of each entry.
Appendix L-II-D-1-b-(4). The Principal Investigator shall report any greenhouse accident involving theinadvertent release or spill of microorganisms to the Biological Safety Officer, Greenhouse Director,
Institutional Biosafety Committee, NIH OSP, and other appropriate authorities immediately (if applicable).
Reports to the NIH OSP shall be sent to the Office of Science Policy, National Institutes of Health,preferably by e-mail to: NIHGuidelines@od.nih.gov; additional contact information is also available here
and on the OSP website (www.osp.od.nih.gov). Documentation of any such accident shall be preparedand maintained.
Appendix L-II-D-1-c.

Decontamination and Inactivation (BL4-P)

Appendix L-II-D-1-c-(1). All materials, except for those that are to remain in a viable or intact state forexperimental purposes, shall be autoclaved prior to removal from the maximum containment greenhouse.
Equipment or material that could be damaged by high temperatures or steam shall be decontaminated by

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alternative methods (e.g., gas or vapor sterilization) in an airlock or chamber designed for this purpose.
Appendix L-II-D-1-c-(2). Water that comes in contact with experimental microorganisms or with material
exposed to such microorganisms (e.g., run-off from watering plants) shall be collected anddecontaminated before disposal.
Appendix L-II-D-1-c-(3). Standard microbiological procedures shall be followed for decontamination ofequipment and materials. Spray or liquid waste or rinse water from containers used to apply theexperimental microorganisms shall be decontaminated before disposal.
Appendix L-II-D-1-d.

Control of Undesired Species and Motile Macroorganisms (BL4-P)

Appendix L-II-D-1-d-(1). A chemical control program shall be implemented to eliminate undesired pests
and pathogens in accordance with applicable state and Federal laws.
Appendix L-II-D-1-d-(2). Arthropods and other motile macroorganisms used in conjunction withexperiments requiring BL4-P level physical containment shall be housed in appropriate cages. Whenappropriate to the organism, experiments shall be conducted within cages designed to contain the motile
organisms.
Appendix L-II-D-1-e.

Concurrent Experiments Conducted in the Greenhouse (BL4-P)

Appendix L-II-D-1-e-(1). Experiments involving organisms that require a containment level lower than
BL4-P may be conducted in the greenhouse concurrently with experiments that require BL4-P
containment provided that all work is conducted in accordance with BL4-P greenhouse practices. Whenthe experimental microorganisms in use require a containment level lower than BL4-P, greenhousepractices reflect the level of containment required by the highest containment level microorganisms being
tested.
Appendix L-II-D-1-f.

Signs (BL4-P)

Appendix L-II-D-1-f-(1). A sign shall be posted indicating that a restricted experiment is in progress. The
sign shall indicate the following: (i) the name of the responsible individual, (ii) the plants in use, and (iii)
any special requirements for using the area.
Appendix L-II-D-1-f-(2). If organisms are used that have a recognized potential for causing serious
detrimental impacts on managed or natural ecosystems, their presence shall be indicated by a signposted on the greenhouse access doors.
Appendix L-II-D-1-f-(3). If there is a risk to human health, a sign shall be posted incorporating theuniversal biosafety symbol.
Appendix L-II-D-1-g.

Transfer of Materials (BL4-P)

Appendix L-II-D-1-g-(1). Experimental materials that are brought into or removed from the greenhousein a viable or intact state shall be transferred to a non-breakable, sealed, primary container then enclosed
in a non-breakable, sealed secondary container. These containers shall be removed from thegreenhouse facility through a chemical disinfectant, fumigation chamber, or an airlock designed for thispurpose.
Appendix L-II-D-1-g-(2). Supplies and materials shall be brought into the greenhouse facility through adouble-door autoclave, fumigation chamber, or airlock that is appropriately decontaminated between eachuse. After securing the outer doors, personnel within the greenhouse facility shall retrieve the materials
by opening the interior door of the autoclave, fumigation chamber, or airlock. These doors shall be
secured after the materials are brought into the greenhouse facility.

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Appendix L-II-D-1-h.

(April 2024)

Greenhouse Practices Manual (BL4-P)

Appendix L-II-D-1-h-(1). A greenhouse practices manual shall be prepared or adopted. This manual
shall include contingency plans to be implemented in the event of the unintentional release ofexperimental organisms.
Appendix L-II-D-1-i.

Protective Clothing (BL4-P)

Appendix L-II-D-1-i-(1). Street clothing shall be removed in the outer clothing change room. Complete
laboratory clothing (may be disposable) including undergarments, pants, and shirts, jump suits, shoes,
and hats shall be provided and worn by all personnel entering the greenhouse facility.
Appendix L-II-D-1-i-(2). Personnel shall remove laboratory clothing when exiting the greenhouse facility
and before entering the shower area. This clothing shall be stored in a locker or hamper in the innerchange room.
Appendix L-II-D-1-i-(3). All laboratory clothing shall be autoclaved before laundering.
Appendix L-II-D-2.

Facilities (BL4-P)

Appendix L-II-D-2-a.

Greenhouse Design (BL4-P)

Appendix L-II-D-2-a-(1). The maximum containment greenhouse facility shall consist of a separatebuilding or a clearly demarcated and isolated area within a building. The need to maintain negative
pressure should be considered when constructing or renovating the greenhouse facility.
Appendix L-II-D-2-a-(2). Outer and inner change rooms, separated by a shower, shall be provided for
personnel entering and exiting the greenhouse facility.
Appendix L-II-D-2-a-(3). Windows shall be closed and sealed. All glazing shall be resistant to breakage(e.g., double-pane tempered glass or equivalent).
Appendix L-II-D-2-a-(4). Access doors to the greenhouse shall be self-closing and locking.
Appendix L-II-D-2-a-(5). The greenhouse facility shall be surrounded by a security fence or protected byequivalent security measures.
Appendix L-II-D-2-a-(6). The walls, floors, and ceilings of the greenhouse shall be constructed to form asealed internal shell that facilitates fumigation and is animal and arthropod-proof. These internal surfaces
shall be resistant to penetration and degradation by liquids and chemicals to facilitate cleaning anddecontamination of the area. All penetrations into these structures and surfaces (e.g., plumbing andutilities) shall be sealed.
Appendix L-II-D-2-a-(7). Bench tops and other work surfaces shall have seamless surfaces impervious
to water and resistant to acids, alkalis, organic solvents, and moderate heat.
Appendix L-II-D-2-a-(8). A double-door autoclave, fumigation chamber, or ventilated airlock shall be
provided for passage of all materials, supplies, or equipment that are not brought into the greenhousefacility through the change room.
Appendix L-II-D-2-b.

Autoclaves (BL4-P)

Appendix L-II-D-2-b-(1). A double-door autoclave shall be provided for the decontamination of materials
removed from the greenhouse facility. The autoclave door, which opens to the area external to thegreenhouse facility, shall be sealed to the outer wall and automatically controlled so that it can only be
opened upon completion of the sterilization cycle.

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Appendix L-II-D-2-c.

(April 2024)

Supply and Exhaust Air Ventilation Systems (BL4-P)

Appendix L-II-D-2-c-(1). An individual supply and exhaust air ventilation system shall be provided. The
system shall maintain pressure differentials and directional airflow as required to assure inward (or zero)
airflow from areas outside of the greenhouse. Differential pressure transducers shall be used to sensepressure levels. If a system malfunctions, the transducers shall sound an alarm. A backup source ofpower should be considered. The supply and exhaust airflow shall be interlocked to assure inward (orzero) airflow at all times. The integrity of the greenhouse shall have an air leak rate (decay rate) not toexceed 7 percent per minute (logarithm of pressure against time) over a 20-minute period at 2 inches ofwater gauge pressure. Nominally, this is 0.05 inches of water gauge pressure loss in 1 minute at 2
inches water gauge pressure.
Appendix L-II-D-2-c-(2). Exhaust air from the greenhouse facility shall be filtered through high efficiency
particulate air/HEPA filters and discharged to the outside and dispersed away from occupied buildings
and air intakes. Filter chambers shall be designed to allow in situ decontamination before filters are
removed and to facilitate certification testing after they are replaced. HEPA filters shall be provided totreat air supplied to the greenhouse facility. HEPA filters shall be certified annually.
Appendix L-II-D-2-d.

Other (BL4-P)

Appendix L-II-D-2-d-(1). Sewer vents and other ventilation lines contain high efficiency particulate
air/HEPA filters. HEPA filters shall be certified annually.
Appendix L-II-D-2-d-(2). A pass-through dunk tank, fumigation chamber, or an equivalent method of
decontamination shall be provided to ensure decontamination of materials and equipment that cannot be
decontaminated in the autoclave.
Appendix L-II-D-2-d-(3). Liquid effluent from sinks, floors, and autoclave chambers shall bedecontaminated by heat or chemical treatment before being released from the maximum containment
greenhouse facility. Liquid wastes from shower rooms and toilets may be decontaminated by heat or
chemical treatment. Autoclave and chemical decontamination of liquid wastes shall be evaluated byappropriate standard procedures for autoclaved wastes. Decontamination shall be evaluatedmechanically and biologically using a recording thermometer and an indicator microorganism with adefined heat susceptibility pattern. If liquid wastes are decontaminated with chemical disinfectants, thechemicals used must have demonstrated efficacy against the target or indicator microorganisms.
Appendix L-II-D-2-d-(4). If there is a central vacuum system, it shall not serve areas outside of thegreenhouse facility. In-line high efficiency particulate air/HEPA filters shall be placed as near aspracticable to each use point or vacuum service cock. Other liquid and gas services to the greenhousefacility shall be protected by devices that prevent back-flow. HEPA filters shall be certified annually.
Appendix L-III. Biological Containment Practices
Appropriate selection of the following biological containment practices may be used to meet thecontainment requirements for a given organism. The present list is not exhaustive; there may be otherways of preventing effective dissemination that could possibly lead to the establishment of the organism
or its genetic material in the environment resulting in deleterious consequences to managed or natural
ecosystems.
Appendix L-III-A.

Biological Containment Practices (Plants)

Appendix L-III-A-1. Effective dissemination of plants by pollen or seed can be prevented by one or moreof the following procedures: (i) cover the reproductive structures to prevent pollen dissemination at
flowering and seed dissemination at maturity; (ii) remove reproductive structures by employing malesterile strains, or harvest the plant material prior to the reproductive stage; (iii) ensure that experimental

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plants flower at a time of year when cross-fertile plants are not flowering within the normal pollen
dispersal range of the experimental plant; or (iv) ensure that cross-fertile plants are not growing within the
known pollen dispersal range of the experimental plant.
Appendix L-III-B.

Biological Containment Practices (Microorganisms)

Appendix L-III-B-1. Effective dissemination of microorganisms beyond the confines of the greenhousecan be prevented by one or more of the following procedures: (i) confine all operations to injections of
microorganisms or other biological procedures (including genetic manipulation) that limit replication or
reproduction of viruses and microorganisms or sequences derived from microorganisms, and confine
these injections to internal plant parts or adherent plant surfaces; (ii) ensure that organisms, which canserve as hosts or promote the transmission of the virus or microorganism, are not present within thefarthest distance that the airborne virus or microorganism may be expected to be effectively
disseminated; (iii) conduct experiments at a time of year when plants that can serve as hosts are either
not growing or are not susceptible to productive infection; (iv) use viruses and other microorganisms ortheir genomes that have known arthropod or animal vectors, in the absence of such vectors; (v) use
microorganisms that have an obligate association with the plant; or (vi) use microorganisms that are
genetically disabled to minimize survival outside of the research facility and whose natural mode oftransmission requires injury of the target organism, or assures that inadvertent release is unlikely toinitiate productive infection of organisms outside of the experimental facility.
Appendix L-III-C.

Biological Containment Practices (Macroorganisms)

Appendix L-III-C-1. Effective dissemination of arthropods and other small animals can be prevented byusing one or more of the following procedures: (i) use non-flying, flight-impaired, or sterile arthropods; (ii)use non-motile or sterile strains of small animals; (iii) conduct experiments at a time of year that precludes
the survival of escaping organisms; (iv) use animals that have an obligate association with a plant that is
not present within the dispersal range of the organism; or (v) prevent the escape of organisms present in
run-off water by chemical treatment or evaporation of run-off water. Containment for arthropods is
described in the Arthropod Containment Guidelines and Addendum 1 Containment Practices for
Arthropods Modified with Engineered Transgenes Capable of Gene Drive (see Appendix G-III-B).
***********************************************************************************************************************
APPENDIX M. PHYSICAL AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT ORSYNTHETIC NUCLEIC ACID MOLECULE RESEARCH INVOLVING ANIMALSAppendix M specifies containment and confinement practices for research involving whole animals, boththose in which the animal's genome has been altered by stable introduction of recombinant or synthetic
nucleic acid molecules, or DNA derived therefrom, into the germ-line (transgenic animals) andexperiments involving viable recombinant or synthetic nucleic acid molecule-modified microorganisms
tested on whole animals. The appendix applies to animal research activities with the following
modifications:
Appendix M shall supersede Appendix G (Physical Containment) when research animals are of a size or
have growth requirements that preclude the use of containment for laboratory animals. Some animals
may require other types of containment (see Appendix M-III-D, Footnotes and References for Appendix
M). The animals covered in Appendix M are those species normally categorized as animals including but
not limited to cattle, swine, sheep, goats, horses, and poultry.
The Institutional Biosafety Committee shall include at least one scientist with expertise in animal
containment principles when experiments utilizing Appendix M require Institutional Biosafety Committee
prior approval.
The institution shall establish and maintain a health surveillance program for personnel engaged in animal

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research involving viable recombinant or synthetic nucleic acid molecule-containing microorganisms that
require Biosafety Level (BL) 3 or greater containment in the laboratory.
Appendix M-I.

General Considerations

Appendix M-I-A.Containment Levels
The containment levels required for research involving recombinant or synthetic nucleic acid molecules
associated with or in animals is based on classification of experiments in Section III, Experiments
Covered by the NIH Guidelines. For the purpose of animal research, four levels of containment are
established. These are referred to as BL1-Animals (N), BL2-N, BL3-N, and BL4-N and are described in
the following appendices of Appendix M. The descriptions include: (i) standard practices for physical andbiological containment, and (ii) animal facilities.
Appendix M-I-B.Disposal of Animals (BL1-N through BL4-N)
Appendix M-I-B-1. When an animal covered by Appendix M containing recombinant or synthetic nucleic
acid molecules or a recombinant or synthetic nucleic acid molecule-derived organism is euthanized ordies, the carcass shall be disposed of to avoid its use as food for human beings or animals unless fooduse is specifically authorized by an appropriate Federal agency.
Appendix M-I-B-2. A permanent record shall be maintained of the experimental use and disposal ofeach animal or group of animals.
Appendix M-II. Physical and Biological Containment Levels
Appendix M-II-A.

Biosafety Level 1 - Animals (BL1-N)

Appendix M-II-A-1.

Standard Practices (BL1-N)

Appendix M-II-A-1-a.

Animal Facility Access (BL1-N)

Appendix M-II-A-1-a-(1). The containment area shall be locked.
Appendix M-II-A-1-a-(2). Access to the containment area shall be limited or restricted whenexperimental animals are being held.
Appendix M-II-A-1-a-(3). The containment area shall be patrolled or monitored at frequent intervals.
Appendix M-II-A-1-b.

Other (BL1-N)

Appendix M-II-A-1-b-(1). All genetically engineered neonates shall be permanently marked within 72
hours after birth, if their size permits. If their size does not permit marking, their containers should bemarked. In addition, transgenic animals should contain distinct and biochemically assayable DNA
sequences that allow identification of transgenic animals from among non-transgenic animals.
Appendix M-II-A-1-b-(2) A double barrier shall be provided to separate male and female animals unlessreproductive studies are part of the experiment or other measures are taken to avoid reproductive
transmission. Reproductive incapacitation may be used.
Appendix M-II-A-1-b-(3). The containment area shall be in accordance with state and Federal laws andanimal care requirements.
Appendix M-II-A-2.

Animal Facilities (BL1-N)

Appendix M-II-A-2-a. Animals shall be confined to securely fenced areas or be in enclosed structures

Page 120 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)(animal rooms) to minimize the possibility of theft or unintentional release.
Appendix M-II-B.
Biosafety Level 2 - Animals (BL2-N) (See Appendix M-III-A, Footnotes andReferences for Appendix M)
Appendix M-II-B-1.

Standard Practices (BL2-N)

Appendix M-II-B-1-a.

Animal Facility Access (BL2-N)

Appendix M-II-B-1-a-(1). The containment area shall be locked.
Appendix M-II-B-1-a-(2). The containment area shall be patrolled or monitored at frequent intervals.
Appendix M-II-B-1-a-(3). The containment building shall be controlled and have a locking access.
Appendix M-II-B-1-a-(4). The Animal Facility Director shall establish policies and procedures whereby
only persons who have been advised of the potential hazard and who meet any specific entry
requirements (e.g., vaccination) may enter the laboratory or animal rooms.
Appendix M-II-B-1-a-(5). Animals of the same or different species, which are not involved in the work
being performed, shall not be permitted in the animal area.
Appendix M-II-B-1-b.

Decontamination and Inactivation (BL2-N)

Appendix M-II-B-1-b-(1). Contaminated materials that are decontaminated at a site away from thelaboratory shall be placed in a closed durable leak-proof container prior to removal from the laboratory.
Appendix M-II-B-1-b-(2). Needles and syringes shall be promptly placed in a puncture-resistantcontainer and decontaminated, preferably by autoclaving, before discard or reuse.
Appendix M-II-B-1-c.

Signs (BL2-N)

Appendix M-II-B-1-c-(1). When the animal research requires special provisions for entry (e.g.,
vaccination), a warning sign incorporating the universal biosafety symbol shall be posted on all accessdoors to the animal work area. The sign shall indicate: (i) the agent, (ii) the animal species, (iii) the nameand telephone number of the Animal Facility Director or other responsible individual, and (iv) any special
requirements for entering the laboratory.
Appendix M-II-B-1-d.

Protective Clothing (BL2-N)

Appendix M-II-B-1-d-(1). Laboratory coats, gowns, smocks, or uniforms shall be worn while in the
animal area or attached laboratory. Before entering non-laboratory areas (e.g., cafeteria, library,administrative offices), protective clothing shall be removed and kept in the work entrance area.
Appendix M-II-B-1-d-(2). Special care shall be taken to avoid skin contamination with microorganisms
containing recombinant or synthetic nucleic acid molecules. Impervious and/or protective gloves shall beworn when handling experimental animals and when skin contact with an infectious agent is unavoidable.
Appendix M-II-B-1-e.

Records (BL2-N)

Appendix M-II-B-1-e-(1). Any incident involving spills and accidents that result in environmental releaseor exposures of animals or laboratory workers to organisms containing recombinant or synthetic nucleic
acid molecules shall be reported immediately to the Animal Facility Director, Institutional Biosafety
Committee, NIH OSP, and other appropriate authorities (if applicable). Reports to the NIH OSP shall besent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:
NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website

Page 121 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)(www.osp.od.nih.gov). Medical evaluation, surveillance, and treatment shall be provided as appropriate
and written records maintained. If necessary, the area shall be appropriately decontaminated.
Appendix M-II-B-1-e-(2). When appropriate and giving consideration to the agent handled, baseline
serum samples shall be collected and stored for animal care and other at-risk personnel. Additional
serum specimens may be collected periodically depending on the agent handled and the function of theanimal facility.
Appendix M-II-B-1-f.

Transfer of Materials (BL2-N)

Appendix M-II-B-1-f-(1). Biological materials removed from the animal containment area in a viable orintact state shall be transferred to a non-breakable sealed primary container and then enclosed in a nonbreakable sealed secondary container. All containers, primary and secondary, shall be disinfected before
removal from the animal facility. Advance approval for transfer of material shall be obtained from theAnimal Facility Director. Packages containing viable agents may only be opened in a facility having anequivalent or higher level of physical containment unless the agent is biologically inactivated or incapableof reproduction.
Appendix M-II-B-1-g.

Other (BL2-N)

Appendix M-II-B-1-g-(1). All genetically engineered neonates shall be permanently marked within 72
hours after birth, if their size permits. If their size does not permit marking, their containers should bemarked. In addition, transgenic animals should contain distinct and biochemically assayable DNA
sequences that allow identification of transgenic animals from among non-transgenic animals.
Appendix M-II-B-1-g-(2). Needles and syringes shall be used only for parenteral injection and aspirationof fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable
syringe-needle units (i.e., needle is integral to the syringe) shall be used for the injection or aspiration of
fluids containing organisms that contain recombinant or synthetic nucleic acid molecules. Extreme
caution shall be used when handling needles and syringes to avoid autoinoculation and the generation ofaerosols during use and disposal. Following use, needles shall not be bent, sheared, replaced in theneedle sheath or guard, or removed from the syringe. Needles and syringes shall be promptly placed in apuncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse.
Appendix M-II-B-1-g-(3). Appropriate steps should be taken to prevent horizontal transmission or
exposure of laboratory personnel. If the agent used as a vector is known to be transmitted by a particularroute (e.g., arthropods), special attention should be given to preventing spread by that route. In theabsence of specific knowledge of a particular route of transmission, all potential means of horizontaltransmission (e.g., arthropods, contaminated bedding, or animal waste, etc.) should be prevented.
Appendix M-II-B-1-g-(4). Eating, drinking, smoking, and applying cosmetics shall not be permitted in thework area.
Appendix M-II-B-1-g-(5). Individuals who handle materials and animals containing recombinant orsynthetic nucleic acid molecules shall be required to wash their hands before exiting the containmentarea.
Appendix M-II-B-1-g-(6). A double barrier shall be provided to separate male and female animals unlessreproductive studies are part of the experiment or other measures are taken to avoid reproductive
transmission. Reproductive incapacitation may be used.
Appendix M-II-B-1-g-(7). The containment area shall be in accordance with state and Federal laws andanimal care requirements.
Appendix M-II-B-1-g-(8). A biosafety manual shall be prepared or adopted. Personnel shall be advisedof special hazards and required to read and follow instructions on practices and procedures.

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Appendix M-II-B-2.

(April 2024)

Animal Facilities (BL2-N)

Appendix M-II-B-2-a. Animals shall be contained within an enclosed structure (animal room orequivalent) to minimize the possibility of theft or unintentional release and to avoid arthropod access. The
special provision to avoid the entry or escape of arthropods from the animal areas may be waived if theagent in use is not known to be transmitted by arthropods.
Appendix M-II-B-2-b. Surfaces shall be impervious to water and resistant to acids, alkalis, organic
solvents, and moderate heat.
Appendix M-II-B-2-c. The animal containment area shall be designed so that it can be easily cleaned.
Appendix M-II-B-2-d. Windows that open shall be fitted with fly screens.
Appendix M-II-B-2-e. An autoclave shall be available for decontamination of laboratory wastes.
Appendix M-II-B-2-f. If arthropods are used in the experiment or the agent under study can betransmitted by an arthropod, interior work areas shall be appropriately screened (52 mesh). All perimeter
joints and openings shall be sealed and additional arthropod control mechanisms used to minimize
arthropod entry and propagation, including appropriate screening of access doors or the equivalent.
Appendix M-II-C.
Biosafety Level 3 - Animals (BL3-N) (See Appendix M-III-B, Footnotes andReferences for Appendix M)
Appendix M-II-C-1.

Standard Practices (BL3-N)

Appendix M-II-C-1-a.

Animal Facility Access (BL3-N)

Appendix M-II-C-1-a-(1). The containment area shall be locked.
Appendix M-II-C-1-a-(2). The containment area shall be patrolled or monitored at frequent intervals.
Appendix M-II-C-1-a-(3). The containment building shall be controlled and have a locking access.
Appendix M-II-C-1-a-(4). The Animal Facility Director shall establish policies and procedures whereby
only persons who have been advised of the potential hazard and who meet any specific entry
requirements (e.g., vaccination) shall enter the laboratory or animal rooms.
Appendix M-II-C-1-a-(5). Animal room doors, gates, or other closures shall be kept closed whenexperiments are in progress.
Appendix M-II-C-1-b.

Decontamination and Inactivation (BL3-N)

Appendix M-II-C-1-b-(1). The work surfaces of containment equipment shall be decontaminated whenwork with organisms containing recombinant or synthetic nucleic acid molecules is finished. Where
feasible, plastic-backed paper toweling shall be used on nonporous work surfaces to facilitate clean-up.
Appendix M-II-C-1-b-(2). All animals shall be euthanized at the end of their experimental usefulness andthe carcasses decontaminated before disposal in an approved manner.
Appendix M-II-C-1-b-(3). Needles and syringes shall be promptly placed in a puncture-resistantcontainer and decontaminated, preferably by autoclaving, before discard or reuse.
Appendix M-II-C-1-b-(4). Special safety testing, decontamination procedures, and Institutional Biosafety
Committee approval shall be required to transfer agents or tissue/organ specimens from a BL3-N animal

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facility to a facility with a lower containment classification.
Appendix M-II-C-1-b-(5). Liquid effluent from containment equipment, sinks, biological safety cabinets,
animal rooms, primary barriers, floor drains, and sterilizers shall be decontaminated by heat treatment
before being released into the sanitary system. The procedure used for heat decontamination of liquid
wastes shall be monitored with a recording thermometer. The effectiveness of the heat decontamination
process system shall be revalidated at minimum on a yearly basis with an indicator organism. Morefrequent validation, based on the amount of use or other safety factors, shall be left to the discretion of
the IBC.
Appendix M-II-C-1-c.

Signs (BL3-N)

Appendix M-II-C-1-c-(1). When the animal research requires special provisions for entry (e.g.,
vaccination), a warning sign incorporating the universal biosafety symbol shall be posted on all accessdoors to the animal work area. The sign shall indicate: (i) the agent, (ii) the animal species, (iii) the nameand telephone number of the Animal Facility Director or other responsible individual, and (iv) any special
requirements for entering the laboratory.
Appendix M-II-C-1-d.

Protective Clothing (BL3-N)

Appendix M-II-C-1-d-(1). Full protective clothing that protects the individual (e.g., scrub suits, coveralls,
uniforms) shall be worn in the animal area. Clothing shall not be worn outside the animal containmentarea and shall be decontaminated before laundering or disposal. Personnel shall be required to shower
before exiting the BL3-N area and wearing of personal clothing.
Appendix M-II-C-1-d-(2). Special care shall be taken to avoid skin contamination with microorganisms
containing recombinant or synthetic nucleic acid molecules. Impervious and/or protective gloves shall beworn when handling experimental animals and when skin contact with an infectious agent is unavoidable.
Appendix M-II-C-1-d-(3). Appropriate respiratory protection shall be worn in rooms containing
experimental animals.
Appendix M-II-C-1-e.

Records (BL3-N)

Appendix M-II-C-1-e-(1). Documents regarding experimental animal use and disposal shall bemaintained in a permanent record book.
Appendix M-II-C-1-e-(2). Any incident involving spills and accidents that result in environmental releaseor exposure of animals or laboratory workers to organisms containing recombinant or synthetic nucleic
acid molecules shall be reported immediately to the Biological Safety Office, Animal Facility Director,
Institutional Biosafety Committee, NIH OSP, and other appropriate authorities (if applicable). Reports to
the NIH OSP shall be sent to the Office of Science Policy, National Institutes of Health, preferably by email to: NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP
website (www.osp.od.nih.gov). Medical evaluation, surveillance, and treatment shall be provided as
appropriate and written records maintained. If necessary, the area shall be appropriately
decontaminated.
Appendix M-II-C-1-e-(3). When appropriate and giving consideration to the agent handled, baseline
serum samples shall be collected and stored for animal care and other at-risk personnel. Additional
serum specimens may be collected periodically depending on the agent handled or the function of the
facility.
Appendix M-II-C-1-f.

Transfer of Materials (BL3-N)

Appendix M-II-C-1-f-(1). Biological materials removed from the animal containment laboratory in a viable
or intact state shall be transferred to a non-breakable sealed primary container and then enclosed in a

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non-breakable sealed secondary container. All containers, primary and secondary, shall be disinfected
before removal from the animal facility. Advance approval for transfer of material shall be obtained fromthe Animal Facility Director. Packages containing viable agents may be opened only in a facility having
an equivalent or higher level of physical containment unless the agent is biologically inactivated orincapable of reproduction.
Appendix M-II-C-1-f-(2). Special safety testing, decontamination procedures, and Institutional Biosafety
Committee approval shall be required to transfer agents or tissue/organ specimens from a BL3-N animal
facility to a facility with a lower containment classification.
Appendix M-II-C-1-g.

Other (BL3-N)

Appendix M-II-C-1-g-(1). All genetically engineered neonates shall be permanently marked within 72
hours after birth, if their size permits. If their size does not permit marking, their containers should bemarked. In addition, transgenic animals should contain distinct and biochemically assayable DNA
sequences that allow identification of transgenic animals from among non-transgenic animals.
Appendix M-II-C-1-g-(2). Appropriate steps should be taken to prevent horizontal transmission or
exposure of laboratory personnel. If the agent used as the vector is known to be transmitted by aparticular route (e.g., arthropods), special attention should be given to preventing spread by that route. In
the absence of specific knowledge of a particular route of transmission, all potential means of horizontaltransmission (e.g., arthropods, contaminated bedding, or animal waste) should be prevented.
Appendix M-II-C-1-g-(3). Eating, drinking, smoking, and applying cosmetics shall not be permitted in thework area.
Appendix M-II-C-1-g-(4). Individuals who handle materials and animals containing recombinant orsynthetic nucleic acid molecules shall be required to wash their hands before exiting the containmentarea.
Appendix M-II-C-1-g-(5). Experiments involving other organisms that require containment levels lower
than BL3-N may be conducted in the same area concurrently with experiments requiring BL3-N
containment provided that they are conducted in accordance with BL3-N practices.
Appendix M-II-C-1-g-(6). Animal holding areas shall be cleaned at least once a day and decontaminatedimmediately following any spill of viable materials.
Appendix M-II-C-1-g-(7). All procedures shall be performed carefully to minimize the creation ofaerosols.
Appendix M-II-C-1-g-(8). A double barrier shall be provided to separate male and female animals unlessreproductive studies are part of the experiment or other measures are taken to avoid reproductive
transmission. Reproductive incapacitation may be used.
Appendix M-II-C-1-g-(9). The containment area shall be in accordance with state and Federal laws andanimal care requirements.
Appendix M-II-C-1-g-(10). All animals shall be euthanized at the end of their experimental usefulness
and the carcasses decontaminated before disposal in an approved manner.
Appendix M-II-C-1-g-(11). Personnel shall be required to shower before exiting the BL3-N area andwearing personal clothing.
Appendix M-II-C-1-g-(12). Animals of the same or different species, which are not involved in the work
being performed, shall not be permitted in the animal area.

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Appendix M-II-C-1-g-(13). Needles and syringes shall be used only for parenteral injection andaspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes ordisposable syringe-needle units (i.e., needle is integral to the syringe) shall be used for the injection or
aspiration of fluids containing organisms that contain recombinant or synthetic nucleic acid molecules.
Extreme caution shall be used when handling needles and syringes to avoid autoinoculation and thegeneration of aerosols during use and disposal. Following use, needles shall not be bent, sheared,
replaced in the needle sheath or guard or removed from the syringe. The needles and syringes shall bepromptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before
discard or reuse.
Appendix M-II-C-1-g-(14). A biosafety manual shall be prepared or adopted. Personnel shall be advisedof special hazards and required to read and follow instructions on practices and procedures.
Appendix M-II-C-2.

Animal Facilities (BL3-N)

Appendix M-II-C-2-a. Animals shall be contained within an enclosed structure (animal room orequivalent) to minimize the possibility of theft or unintentional release and avoid arthropod access. The
special provision to avoid the entry or escape of arthropods from the animal areas may be waived if theagent in use is not known to be transmitted by arthropods.
Appendix M-II-C-2-b. The interior walls, floors, and ceilings shall be impervious to water and resistant toacids, alkalis, organic solvents, and moderate heat, to facilitate cleaning. Penetrations in these structuresand surfaces (e.g., plumbing and utilities) shall be sealed.
Appendix M-II-C-2-c. Windows in the animal facility shall be closed, sealed, and breakage resistant(e.g., double-pane tempered glass or equivalent). The need to maintain negative pressure should beconsidered when constructing or renovating the animal facility.
Appendix M-II-C-2-d. An autoclave, incinerator, or other effective means to decontaminate animals andwaste shall be available, preferably within the containment area. If feasible, a double-door autoclave ispreferred and should be positioned to allow removal of material from the containment area.
Appendix M-II-C-2-e. If arthropods are used in the experiment or the agent under study can betransmitted by an arthropod, the interior work area shall be appropriately screened (52 mesh). All
perimeter joints and openings shall be sealed, and additional arthropod control mechanisms used tominimize arthropod entry and propagation, including appropriate screening, or the equivalent of accessdoors.
Appendix M-II-C-2-f. Access doors to the containment area shall be self-closing.
Appendix M-II-C-2-g. The animal area shall be separated from all other areas. Passage through two
sets of doors shall be the basic requirement for entry into the animal area from access corridors or othercontiguous areas. The animal containment area shall be physically separated from access corridors andother laboratories or areas by a double-door clothes change room, equipped with integral showers andairlock.
Appendix M-II-C-2-h. Liquid effluent from containment equipment, sinks, biological safety cabinets,
animal rooms, primary barriers, floor drains, and sterilizers shall be decontaminated by heat treatment
before being released into the sanitary system. The procedure used for heat decontamination of liquid
wastes shall be monitored with a recording thermometer. The effectiveness of the heat decontamination
process system shall be revalidated at minimum on a yearly basis with an indicator organism. Morefrequent validation, based on the amount of use or other safety factors, shall be left to the discretion of
the IBC.
Appendix M-II-C-2-i. An exhaust air ventilation system shall be provided. This system shall create
directional airflow that draws air into the animal room through the entry area. The building exhaust, or thePage 126 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

exhaust from primary containment units, may be used for this purpose if the exhaust air is discharged tothe outside and shall be dispersed away from occupied areas and air intakes. Personnel shall verify thatthe direction of the airflow (into the animal room) is proper.
Appendix M-II-C-2-j. If the agent is transmitted by aerosol, then the exhaust air shall pass through a highefficiency particulate air/HEPA filter.
Appendix M-II-C-2-k. Vacuum lines shall be protected with high efficiency particulate air/HEPA filters
and liquid disinfectant traps.
Appendix M-II-C-2-l. In lieu of open housing in the special animal room, animals held in a BL3-N area
may be housed in partial-containment caging systems (e.g., Horsfall units or gnotobiotic systems, or otherspecial containment primary barriers). Prudent judgment must be exercised to implement this ventilation
system (e.g., animal species) and its discharge location.
Appendix M-II-C-2-m. Each animal area shall contain a foot, elbow, or automatically operated sink forhand washing. The sink shall be located near the exit door.
Appendix M-II-C-2-n. Restraining devices for animals may be required to avoid damage to the integrityof the animal containment facility.
Appendix M-II-D.
Biosafety Level 4 - Animals (BL4-N) (See Appendix M-III-C, Footnotes andReferences for Appendix M)
Appendix M-II-D-1.

Standard Practices (BL4-N)

Appendix M-II-D-1-a.

Animal Facility Access (BL4-N)

Appendix M-II-D-1-a-(1). Individuals under 16 years of age shall not be permitted to enter the animalarea.
Appendix M-II-D-1-a-(2). The containment area shall be locked.
Appendix M-II-D-1-a-(3). The containment area shall be patrolled or monitored at frequent intervals.
Appendix M-II-D-1-a-(4). The containment building shall be controlled and have a locking access.
Appendix M-II-D-1-a-(5). The Animal Facility Director shall establish policies and procedures whereby
only persons who have been advised of the potential hazard and who meet any specific entry
requirements (e.g., vaccination) may enter the laboratory or animal room.
Appendix M-II-D-1-a-(6). Individuals shall enter and exit the animal facility only through the clothingchange and shower rooms.
Appendix M-II-D-1-a-(7). Personnel shall use the airlocks to enter or exit the laboratory only in an
emergency.
Appendix M-II-D-1-a-(8). Animal room doors, gates, and other closures shall be kept closed whenexperiments are in progress.
Appendix M-II-D-1-b.

Decontamination and Inactivation (BL4-N)

Appendix M-II-D-1-b-(1). All contaminated liquid or solid wastes shall be decontaminated before
disposal.
Appendix M-II-D-1-b-(2). The work surfaces and containment equipment shall be decontaminated whenPage 127 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

work with organisms containing recombinant or synthetic nucleic acid molecules is finished. Where
feasible, plastic-backed paper toweling shall be used on nonporous work surfaces to facilitate clean-up.
Appendix M-II-D-1-b-(3). All wastes from animal rooms and laboratories shall be appropriately
decontaminated before disposal in an approved manner.
Appendix M-II-D-1-b-(4). No materials, except for biological materials that are to remain in a viable orintact state, shall be removed from the maximum containment laboratory unless they have been
autoclaved or decontaminated. Equipment or material that might be damaged by high temperatures or
steam shall be decontaminated by gaseous or vapor methods in an airlock or chamber designed for thispurpose.
Appendix M-II-D-1-b-(5). When ventilated suits are required, the animal personnel shower entrance/exitarea shall be equipped with a chemical disinfectant shower to decontaminate the surface of the suit
before exiting the area. A neutralization or water dilution device shall be integral with the chemical
disinfectant discharge piping before entering the heat sterilization system. Entry to this area shall bethrough an airlock fitted with airtight doors.
Appendix M-II-D-1-b-(6). Needles and syringes shall be promptly placed in a puncture-resistantcontainer and decontaminated, preferably by autoclaving, before discard or reuse.
Appendix M-II-D-1-b-(7). Supplies and materials needed in the animal facility shall be brought in by wayof the double-door autoclave, fumigation chamber, or airlock that shall be appropriately decontaminatedbetween each use.
Appendix M-II-D-1-b-(8). An autoclave, incinerator, or other effective means to decontaminate animals
and wastes shall be available, preferably within the containment area. If feasible, a double-door
autoclave is preferred and should be positioned to allow removal of material from the containment area.
Appendix M-II-D-1-b-(9). Liquid effluent from containment equipment, sinks, biological safety cabinets,
animal rooms, primary barriers, floor drains, and sterilizers shall be decontaminated by heat treatment
before being released into the sanitary system. If required by design, regulation, local ordinance orpolicy, liquid wastes from shower rooms and toilets shall be decontaminated with chemical disinfectantsor heat by methods demonstrated to be effective. The procedure used for heat decontamination of liquid
wastes shall be monitored with a recording thermometer. The effectiveness of the heat decontamination
process system shall be revalidated at minimum on a yearly basis with an indicator organism. Morefrequent validation, based on the amount of use or other safety factors, shall be left to the discretion of
the IBC. If required by design, regulation, local ordinance or policy, liquid wastes from the shower shallbe chemically decontaminated using an Environmental Protection Agency-approved germicide. The
efficacy of the chemical treatment process shall be validated with an indicator organism. Chemical
disinfectants shall be neutralized or diluted before release into general effluent waste systems.
Appendix M-II-D-1-c.

Signs (BL4-N)

Appendix M-II-D-1-c-(1). When the animal research requires special provisions for entry (e.g.,
vaccination), a warning sign incorporating the universal biosafety symbol shall be posted on all accessdoors to the animal work area. The sign shall indicate: (i) the agent, (ii) the animal species, (iii) the nameand telephone number of the Animal Facility Director, or other responsible individual, and (iv) any special
requirements for entering the laboratory.
Appendix M-II-D-1-d.

Protective Clothing (BL4-N)

Appendix M-II-D-1-d-(1). Individuals shall enter and exit the animal facility only through the clothingchange and shower rooms. Street clothing shall be removed and kept in the outer clothing change room.
Complete laboratory clothing (may be disposable), including undergarments, pants, shirts, jump suits, andshoes shall be provided for all personnel entering the animal facility. When exiting the BL4-N area andPage 128 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

before proceeding into the shower area, personnel shall remove their laboratory clothing in the innerchange room. All laboratory clothing shall be autoclaved before laundering. Personnel shall shower eachtime they exit the animal facility.
Appendix M-II-D-1-d-(2). A ventilated head-hood or a one-piece positive pressure suit, which isventilated by a life-support system, shall be worn by all personnel entering rooms that containexperimental animals when appropriate. When ventilated suits are required, the animal personnel
shower entrance/exit area shall be equipped with a chemical disinfectant shower to decontaminate thesurface of the suit before exiting the area. A neutralization or water dilution device shall be integral withthe chemical disinfectant discharge piping before entering the heat sterilization system. Entry to this area
shall be through an airlock fitted with airtight doors.
Appendix M-II-D-1-d-(3). Appropriate respiratory protection shall be worn in rooms containing
experimental animals.
Appendix M-II-D-1-e.

Records (BL4-N)

Appendix M-II-D-1-e-(1). Documents regarding experimental animal use and disposal shall bemaintained in a permanent record book.
Appendix M-II-D-1-e-(2). A system shall be established for: (i) reporting laboratory accidents andexposures that are a result of overt exposures to organisms containing recombinant or synthetic nucleic
acid molecules, (ii) employee absenteeism, and (iii) medical surveillance of potential laboratoryassociated illnesses. Permanent records shall be prepared and maintained. Any incident involving spills
and accidents that results in environmental release or exposures of animals or laboratory workers toorganisms containing recombinant or synthetic nucleic acid molecules shall be reported immediately to
the Biological Safety Officer, Animal Facility Director, Institutional Biosafety Committee, NIH OSP, andother appropriate authorities (if applicable). Reports to the NIH OSP shall be sent to the Office of Science
Policy, National Institutes of Health, preferably by e-mail to: NIHGuidelines@od.nih.gov; additional
contact information is also available here and on the OSP website (www.osp.od.nih.gov). Medical
evaluation, surveillance, and treatment shall be provided as appropriate and written records maintained.
If necessary, the area shall be appropriately decontaminated.
Appendix M-II-D-1-e-(3). When appropriate and giving consideration to the agents handled, baseline
serum samples shall be collected and stored for animal care and other at-risk personnel. Additional
serum specimens may be collected periodically depending on the agents handled or the function of the
facility.
Appendix M-II-D-1-e-(4). A permanent record book indicating the date and time of each entry and exit
shall be signed by all personnel.
Appendix M-II-D-1-f.

Transfer of Materials (BL4-N)

Appendix M-II-D-1-f-(1). No materials, except for biological materials that are to remain in a viable orintact state, shall be removed from the maximum containment laboratory unless they have been
autoclaved or decontaminated. Equipment or material that might be damaged by high temperatures or
steam shall be decontaminated by gaseous or vapor methods in an airlock or chamber designed for thispurpose.
Appendix M-II-D-1-f-(2). Biological materials removed from the animal maximum containment laboratory
in a viable or intact state shall be transferred to a non-breakable sealed primary container and thenenclosed in a non-breakable sealed secondary container that shall be removed from the animal facility
through a disinfectant dunk tank, fumigation chamber, or an airlock designed for this purpose. Advanceapproval for transfer of material shall be obtained from the Animal Facility Director. Such packages
containing viable agents can only be opened in another BL4-N animal facility if the agent is biologicallyinactivated or incapable of reproduction. Special safety testing, decontamination procedures, andPage 129 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

Institutional Biosafety Committee approval shall be required to transfer agents or tissue/organ specimensfrom a BL4-N animal facility to one with a lower containment classification.
Appendix M-II-D-1-f-(3). Supplies and materials needed in the animal facility shall be brought in by wayof the double-door autoclave, fumigation chamber, or airlock that shall be appropriately decontaminatedbetween each use. After securing the outer doors, personnel within the animal facility retrieve thematerials by opening the interior doors of the autoclave, fumigation chamber, or airlock. These doors
shall be secured after materials are brought into the animal facility.
Appendix M-II-D-1-g.

Other (BL4-N)

Appendix M-II-D-1-g-(1). All genetically engineered neonates shall be permanently marked within 72
hours after birth, if their size permits. If their size does not permit marking, their containers should bemarked. In addition, transgenic animals should contain distinct and biochemically assayable DNA
sequences that allow identification of transgenic animals from among non-transgenic animals.
Appendix M-II-D-1-g-(2). Eating, drinking, smoking, and applying cosmetics shall not be permitted in thework area.
Appendix M-II-D-1-g-(3). Individuals who handle materials and animals containing recombinant orsynthetic nucleic acid molecules shall be required to wash their hands before exiting the containmentarea.
Appendix M-II-D-1-g-(4). Experiments involving other organisms that require containment levels lower
than BL4-N may be conducted in the same area concurrently with experiments requiring BL4-N
containment provided that they are conducted in accordance with BL4-N practices.
Appendix M-II-D-1-g-(5). Animal holding areas shall be cleaned at least once a day and decontaminatedimmediately following any spill of viable materials.
Appendix M-II-D-1-g-(6). All procedures shall be performed carefully to minimize the creation ofaerosols.
Appendix M-II-D-1-g-(7). A double barrier shall be provided to separate male and female animals.
Animal isolation barriers shall be sturdy and accessible for cleaning. Reproductive incapacitation may be
used.
Appendix M-II-D-1-g-(8). The containment area shall be in accordance with state and Federal laws andanimal care requirements.
Appendix M-II-D-1-g-(9). The life support system for the ventilated suit or head hood is equipped withalarms and emergency back-up air tanks. The exhaust air from the suit area shall be filtered by two setsof high efficiency particulate air/HEPA filters installed in series or incinerated. A duplicate filtration unit,
exhaust fan, and an automatically starting emergency power source shall be provided. The air pressure
within the suit shall be greater than that of any adjacent area. Emergency lighting and communication
systems shall be provided. A double-door autoclave shall be provided for decontamination of wastematerials to be removed from the suit area.
Appendix M-II-D-1-g-(10). Needles and syringes shall be used only for parenteral injection andaspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes ordisposable syringe-needle units (i.e., needle is integral to the syringe) shall be used for the injection or
aspiration of fluids containing organisms that contain recombinant or synthetic nucleic acid molecules.
Extreme caution shall be used when handling needles and syringes to avoid autoinoculation and thegeneration of aerosols during use and disposal. Following use, needles shall not be bent, sheared,
replaced in the needle sheath or guard, or removed from the syringe. The needles and syringes shall bepromptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before

Page 130 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)

discard or reuse.
Appendix M-II-D-1-g-(11). An essential adjunct to the reporting-surveillance system is the availability ofa facility for quarantine, isolation, and medical care of personnel with potential or known laboratoryassociated illnesses.
Appendix M-II-D-1-g-(12). A biosafety manual shall be prepared or adopted. Personnel shall be advisedof special hazards and required to read and follow instructions on practices and procedures.
Appendix M-II-D-1-g-(13). Vacuum lines shall be protected with high efficiency particulate air/HEPA
filters and liquid disinfectant traps.
Appendix M-II-D-2.

Animal Facilities (BL4-N)

Appendix M-II-D-2-a. Animals shall be contained within an enclosed structure (animal room orequivalent) to minimize the possibility of theft or unintentional release and avoid arthropod access.
Appendix M-II-D-2-b. The interior walls, floors, and ceilings shall be impervious to water and resistant toacids, alkalis, organic solvents, and moderate heat, to facilitate cleaning. Penetrations in these structuresand surfaces (e.g., plumbing and utilities) shall be sealed.
Appendix M-II-D-2-c. Windows in the animal facility shall be closed, sealed, and breakage resistant(e.g., double-pane tempered glass or equivalent).
Appendix M-II-D-2-d. An autoclave, incinerator, or other effective means to decontaminate animals andwastes shall be available, preferably within the containment area. If feasible, a double-door autoclave ispreferred and should be positioned to allow removal of material from the containment area.
Appendix M-II-D-2-e. Access doors to the containment area shall be self-closing.
Appendix M-II-D-2-f. All perimeter joints and openings shall be sealed to form an arthropod-proof
structure.
Appendix M-II-D-2-g. The BL4-N laboratory provides a double barrier to prevent the release ofrecombinant or synthetic nucleic acid molecule containing microorganisms into the environment. Designof the animal facility shall be such that if the barrier of the inner facility is breached, the outer barrier will
prevent release into the environment. The animal area shall be separated from all other areas. Passage
through two sets of doors shall be the basic requirement for entry into the animal area from accesscorridors or other contiguous areas. Physical separation of the animal containment area from accesscorridors or other laboratories or activities shall be provided by a double-door clothes change room
equipped with integral showers and airlock.
Appendix M-II-D-2-h. A necropsy room shall be provided within the BL4-N containment area.
Appendix M-II-D-2-i. Liquid effluent from containment equipment, sinks, biological safety cabinets,
animal rooms, primary barriers, floor drains, and sterilizers shall be decontaminated by heat treatment
before being released into the sanitary system. If required by design, regulation, local ordinance orpolicy, liquid wastes from shower rooms and toilets shall be decontaminated with chemical disinfectantsor heat by methods demonstrated to be effective. The procedure used for heat decontamination of liquid
wastes shall be monitored with a recording thermometer. The effectiveness of the heat decontamination
process system shall be revalidated at minimum on a yearly basis with an indicator organism. Morefrequent validation, based on the amount of use or other safety factors, shall be left to the discretion of
the IBC. If required by design, regulation, local ordinance or policy, liquid wastes from the shower shallbe chemically decontaminated using an Environmental Protection Agency-approved germicide. The
efficacy of the chemical treatment process shall be validated with an indicator organism. Chemical
disinfectants shall be neutralized or diluted before release into general effluent waste systems.

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Appendix M-II-D-2-j. A ducted exhaust air ventilation system shall be provided that creates directional
airflow that draws air into the laboratory through the entry area. The exhaust air, which is not recirculated
to any other area of the building, shall be discharged to the outside and dispersed away from theoccupied areas and air intakes. Personnel shall verify that the direction of the airflow (into the animalroom) is proper.
Appendix M-II-D-2-k. Exhaust air from BL4-N containment area shall be double high efficiency
particulate air/HEPA filtered or treated by passing through a certified HEPA filter and an air incinerator
before release to the atmosphere. Double HEPA filters shall be required for the supply air system in a
BL4-N containment area.
Appendix M-II-D-2-l. All high efficiency particulate air/HEPA filters' frames and housings shall becertified to have no detectable smoke [dioctyl phthalate] leaks when the exit face (direction of flow) of thefilter is scanned above 0.01 percent when measured by a linear or logarithmic photometer. The
instrument must demonstrate a threshold sensitivity of at least 1x10-3 micrograms per liter for 0.3
micrometer diameter dioctyl phthalate particles and a challenge concentration of 80-120 micrograms perliter. The air sampling rate should be at least 1 cfm (28.3 liters per minute).
Appendix M-II-D-2-m. If an air incinerator is used in lieu of the second high efficiency particulate
air/HEPA filter, it shall be biologically challenged to prove all viable test agents are sterilized. The
biological challenge must be minimally 1x108 organisms per cubic foot of airflow through the incinerator.
It is universally accepted if bacterial spores are used to challenge and verify that the equipment iscapable of killing spores, then assurance is provided that all other known agents are inactivated by theparameters established to operate the equipment. Test spores meeting this criterion are Bacillus subtilis
var. niger or Bacillus stearothermophilus. The operating temperature of the incinerator shall be
continuously monitored and recorded during use.
Appendix M-II-D-2-n. All equipment and floor drains shall be equipped with deep traps (minimally 5
inches). Floor drains shall be fitted with isolation plugs or fitted with automatic water fill devices.
Appendix M-II-D-2-o. Each animal area shall contain a foot, elbow, or automatically operated sink forhand washing. The sink shall be located near the exit door.
Appendix M-II-D-2-p. Restraining devices for animals may be required to avoid damage to the integrityof the containment animal facility.
Appendix M-II-D-2-q. The supply water distribution system shall be fitted with a back-flow preventer or
break tank.
Appendix M-II-D-2-r. All utilities, liquid and gas services, shall be protected with devices that avoid backflow.
Appendix M-II-D-2-s. Sewer and other atmospheric ventilation lines shall be equipped minimally with asingle high efficiency particulate/HEPA filter. Condensate drains from these type housings shall beappropriately connected to a contaminated or sanitary drain system. The drain position in the housing
dictates the appropriate system to be used.
Appendix M-III. Footnotes and References for Appendix M
Appendix M-III-A. If a recombinant or synthetic nucleic acid molecule is derived from a Class 2 organism
requiring BL2 containment, personnel shall be required to have specific training in handling pathogenic
agents and directed by knowledgeable scientists.
Appendix M-III-B. Personnel who handle pathogenic and potentially lethal agents shall be required tohave specific training and be supervised by knowledgeable scientists who are experienced in working

Page 132 - NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(April 2024)with these agents. BL3-N containment also minimizes escape of recombinant or synthetic nucleic acidmolecule-containing organisms from exhaust air or waste material from the containment area.
Appendix M-III-C. Risk Group 4 and restricted microorganisms (see Appendix B, Classification of
Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References ofSections I through IV) pose a high level of individual risk for acquiring life-threatening diseases topersonnel and/or animals. To import animal or plant pathogens, special approval must be obtained from
U.S. Department of Agriculture, Animal and Plant Health Inspection Service (APHIS), Veterinary Services,
National Center for Import-Export, Products Program, 4700 River Road, Unit 40, Riverdale, MD 20737.
Phone: (301) 734-8499; Fax: (301) 734-8226.
Laboratory staff shall be required to have specific and thorough training in handling extremely hazardous
infectious agents, primary and secondary containment, standard and special practices, and laboratory
design characteristics. The laboratory staff shall be supervised by knowledgeable scientists who aretrained and experienced in working with these agents and in the special containment facilities.
Within work areas of the animal facility, all activities shall be confined to the specially equipped animalrooms or support areas. The maximum animal containment area and support areas shall have special
engineering and design features to prevent the dissemination of microorganisms into the environment via
exhaust air or waste disposal.
Appendix M-III-D. Research with animals, which may not appropriately be conducted under conditions
described in Appendix M, may be conducted safely by applying practices routinely used for controlled
culture of these biota. In aquatic systems, for example, BL1 equivalent conditions could be met byutilizing growth tanks that provide adequate physical means to avoid the escape of the aquatic species,
its gametes, and introduced exogenous genetic material. A mechanism shall be provided to ensure thatneither the organisms nor their gametes can escape into the supply or discharge system of the rearing
container (e.g., tank, aquarium, etc.) Acceptable barriers include appropriate filtration, irradiation, heat
treatment, chemical treatment, etc. Moreover, the top of the rearing container shall be covered to avoid
escape of the organism and its gametes. In the event of tank rupture, leakage, or overflow, theconstruction of the room containing these tanks should prevent the organisms and gametes from entering
the building's drains before the organism and its gametes have been inactivated.
Other types of animals (e.g., nematodes, arthropods, and certain forms of smaller animals) may be
accommodated by using the appropriate BL1 through BL4 or BL1-P through BL4-P containment practices
and procedures as specified in Appendices G and L. Containment for arthropods is described in the
Arthropod Containment Guidelines and Addendum 1 Containment Practices for Arthropods Modified withEngineered Transgenes Capable of Gene Drive (see Appendix G-III-B).