(i) April 2002

NOTICE PERTINENT TO THE APRIL 2002 REVISIONS OF THE 

NIH GUIDELINES FOR RESEARCH INVOLVING RECOMBINANT 

DNA MOLECULES (NIH GUIDELINES)

ROLES AND RESPONSIBILITIES OF THE PRINCIPAL INVESTIGATOR (PI)

Under the amendment to Section IV-B-7, which was published in the Federal Register on November 19, 

2001 (66 FR 57970, specifically: 57975) and became effective on December 19, 2001, a PI may delegate 

the reporting responsibilities set forth in Appendix M-I-C, Reporting Requirements, to another party, with 

written notification of the delegation to OBA. A letter from each PI indicating to whom they have 

delegated the reporting requirements set forth in Appendix M-I-C must be on file with OBA. This 

delegation of reporting responsibility may, if appropriate, be extended to include the material submitted 

under Appendix M-I-A, Requirements for Protocol Submission, of the NIH Guidelines. To that end, a 

letter from the PI should be submitted to OBA, either directly by the investigator or as part of the material 

submitted under Appendix M-I-A. 

Summary of Amendments [Major Actions] 

Page 10 Section I-E: Additions to General Definitions. New sections -- I-E-8, I-E-9, 

I-E-10 

Page 36 Appendix B-1 [Lines 5-8]: New – General definition of an E. coli strain as a RG1 

agent 

Page 97-98 Appendix M-1-C-3: Annual Reports. New – (Harmonized submission 

requirements) 

Page 98 Appendix M-1-C-4: Safety Reporting. New appendix – (Harmonized reporting 

requirements) 

Page 99 Appendix M-1-C-5: Confidentiality. New appendix 

Page 99 Appendix M-1-D: Safety Assessment. New appendix 

Page 106 Appendix M-IV: Privacy; deleted “...and Confidentiality” from heading; 

clarification of protection measures.
Effective June 24, 1994, Published in Federal Register, July 5, 1994 (59 FR 34496) 

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) 

NIH GUIDELINES FOR RESEARCH 

INVOLVING RECOMBINANT 

DNA MOLECULES 

(NIH GUIDELINES) 

April 2002

*********************************************************************************************************************** 

Visit the OBA Web site at: 

http://www4.od.nih.gov/oba

For current information on Guidelines, Protocols, Principal Investigators, Meetings, 

and information about upcoming Gene Therapy Policy Conferences 

*********************************************************************************************************************** 

DEPARTMENT OF HEALTH AND HUMAN SERVICES 

National Institutes of Health 

Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) 

These NIH Guidelines supersede all earlier versions and shall be in effect until further notice. 

*********************************************************************************************************************** 

TABLE OF CONTENTS 

SECTION I. SCOPE OF THE NIH GUIDELINES .......................................................................... 8

Section I-A. Purpose .......................................................................................................................8

Section I-B. Definition of Recombinant DNA Molecules .................................................................9

Section I-C. General Applicability....................................................................................................9

Section I-D. Compliance with the NIH Guidelines.........................................................................10

Section I-E. General Definitions....................................................................................................10 
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SECTION II. SAFETY CONSIDERATIONS.................................................................................. 11

Section II-A. Risk Assessment.......................................................................................................11

Section II-A-1. Risk Groups...............................................................................................................11

Section II-A-2. Criteria for Risk Groups.............................................................................................11

Section II-A-3. Comprehensive Risk Assessment ............................................................................11

Section II-B. Containment ..............................................................................................................12

SECTION III. EXPERIMENTS COVERED BY THE NIH GUIDELINES ........................................ 14

Section III-A. Experiments that Require Institutional Biosafety Committee (IBC) Approval, 

RAC Review, and NIH Director Approval Before Initiation........................................14

Section III-A-1. Major Actions under the NIH Guidelines ...................................................................14

Section III-B. Experiments That Require NIH/OBA and IBC Approval Before Initiation .................14

Section III-B-1. Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than 

100 ng/Kg Body Weight.............................................................................................15

Section III-C. Experiments that Require IBC and Institutional Review Board (IRB) 

Approvals and RAC Review Before Research Participant Enrollment .....................15

Section III-C-1. Deliberate Transfer of Recombinant DNA (rDNA), or DNA or RNA Derived from 

rDNA, into One or More Human Research Participants ...........................................15

Section III-D. Experiments that Require IBC Approval Before Initiation .........................................16

Section III-D-1. Experiments Using Risk Group (RG) 2, RG3, RG4, or Restricted Agents as 

Host-Vector Systems.................................................................................................16

Section III-D-2. DNA From RG2, RG3, RG4, or Restricted Agents Cloned into Nonpathogenic 

Prokaryotic or Lower Eukaryotic Host-Vector Systems ............................................16

Section III-D-3. Use of Infectious DNA or RNA Viruses or Defective DNA or RNA Viruses in the 

Presence of Helper Virus in Tissue Culture Systems ...............................................17

Section III-D-4. Experiments Involving Whole Animals ......................................................................17

Section III-D-5. Experiments Involving Whole Plants.........................................................................18

Section III-D-6. Experiments Involving More than 10 Liters of Culture ..............................................19

Section III-E. Experiments that Require IBC Notice Simultaneous with Initiation...........................19

Section III-E-1. Experiments Involving the Formation of Recombinant DNA Molecules Containing 

No More than Two-Thirds of the Genome of any Eukaryotic Virus...........................19

Section III-E-2. Experiments Involving Whole Plants.........................................................................19

Section III-E-3. Experiments Involving Transgenic Rodents..............................................................20

Section III-F. Exempt Experiments .................................................................................................20

SECTION IV. ROLES AND RESPONSIBILITIES.......................................................................... 21

Section IV-A. Policy .........................................................................................................................21

Section IV-B. Responsibilities of the Institution...............................................................................21

Section IV-B-1. General Information...................................................................................................21

Section IV-B-2. Institutional Biosafety Committee (IBC) ....................................................................22

Section IV-B-2-a. Membership and Procedures ....................................................................................22

Section IV-B-2-b. Functions ...................................................................................................................23

Section IV-B-3. Biological Safety Officer (BSO) .................................................................................24

Section IV-B-4. Plant, Plant Pathogen, or Plant Pest Containment Expert........................................25

Section IV-B-5. Animal Containment Expert.......................................................................................25

Section IV-B-6. Human Gene Therapy Expertise...............................................................................25

Section IV-B-7. Principal Investigator (PI) ..........................................................................................25

Section IV-B-7-a. General Responsibilities............................................................................................26

Section IV-B-7-b. Information to Be Submitted by the PI to NIH OBA ..................................................26

Section IV-B-7-c. Submissions by the PI to the IBC..............................................................................27

Section IV-B-7-d. Responsibilities of the PI Prior to Initiating Research ...............................................27

Section IV-B-7-e. Responsibilities of the PI During the Conduct of the Research ................................27

Section IV-C. Responsibilities of the NIH ........................................................................................28

Section IV-C-1. NIH Director...............................................................................................................28

Section IV-C-1-a. General Responsibilities............................................................................................28

Section IV-C-1-b. Specific Responsibilities............................................................................................28 

Section IV-C-1-b-(1). Major Actions.............................................................................................................28

Section IV-C-1-b-(2). Minor Actions.............................................................................................................29 
Page 3 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section IV-C-2. Recombinant DNA Advisory Committee (RAC) ........................................................30

Section IV-C-3. Office of Biotechnology Activities (OBA)...................................................................31

Section IV-C-4. Other NIH Components.............................................................................................31

Section IV-D. Voluntary Compliance ...............................................................................................32

Section IV-D-1. Basic Policy - Voluntary Compliance ........................................................................32

Section IV-D-2. Institutional Biosafety Committee Approval - Voluntary Compliance........................32

Section IV-D-3. Certification of Host-Vector Systems - Voluntary Compliance..................................32

Section IV-D-4. Requests for Exemptions and Approvals - Voluntary Compliance ...........................32

Section IV-D-5. Protection of Proprietary Data - Voluntary Compliance ............................................32

Section IV-D-5-a. General......................................................................................................................32

Section IV-D-5-b. Pre-submission Review.............................................................................................33

SECTION V. FOOTNOTES AND REFERENCES OF SECTIONS I THROUGH IV ..................... 33

APPENDIX A. EXEMPTIONS UNDER SECTION III-F-5--SUBLISTS OF NATURAL 

EXCHANGERS........................................................................................................ 35

Appendix A-I. Sublist A ....................................................................................................................35

Appendix A-II. Sublist B ....................................................................................................................35

Appendix A-III. Sublist C ....................................................................................................................35

Appendix A-IV. Sublist D ....................................................................................................................35

Appendix A-V. Sublist E ....................................................................................................................35

Appendix A-VI. Sublist F.....................................................................................................................36

APPENDIX B. CLASSIFICATION OF HUMAN ETIOLOGIC AGENTS ON THE BASIS OF 

HAZARD .................................................................................................................. 36

Appendix B-I. Risk Group 1 (RG1) Agents ......................................................................................36

Appendix B-II. Risk Group 2 (RG2) Agents ......................................................................................37

Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia ...................................37

Appendix B-II-B. Risk Group 2 (RG2) - Fungal Agents ........................................................................38

Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents .....................................................................38

Appendix B-II-D. Risk Group 2 (RG2) - Viruses ...................................................................................38

Appendix B-III. Risk Group 3 (RG3) Agents ......................................................................................40

Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia.....................................40

Appendix B-III-B. Risk Group 3 (RG3) - Fungal Agents ........................................................................40

Appendix B-III-C. Risk Group 3 (RG3) - Parasitic Agents .....................................................................40

Appendix B-III-D. Risk Group 3 (RG3) - Viruses and Prions .................................................................40

Appendix B-IV. Risk Group 4 (RG4) Agents ......................................................................................41

Appendix B-IV-A. Risk Group 4 (RG4) - Bacterial Agents .....................................................................41

Appendix B-IV-B. Risk Group 4 (RG4) - Fungal Agents ........................................................................41

Appendix B-IV-C. Risk Group 4 (RG4) - Parasitic Agents .....................................................................41

Appendix B-IV-D. Risk Group 4 (RG4) - Viral Agents............................................................................41

Appendix B-V. Animal Viral Etiologic Agents in Common Use .........................................................42

Appendix B-V-1. Murine Retroviral Vectors..........................................................................................42

APPENDIX C. EXEMPTIONS UNDER SECTION III-F-6 ................................................................ 42

Appendix C-I. Recombinant DNA in Tissue Culture ........................................................................42

Appendix C-I-A. Exceptions .................................................................................................................43

Appendix C-II. Escherichia coli K-12 Host-Vector Systems..............................................................43

Appendix C-II-A. Exceptions .................................................................................................................43

Appendix C-III. Saccharomyces Host-Vector Systems......................................................................43

Appendix C-III-A. Exceptions .................................................................................................................44

Appendix C-IV. Bacillus subtilis or Bacillus licheniformis Host-Vector Systems ................................44

Appendix C-IV-A. Exceptions .................................................................................................................44

Appendix C-V. Extrachromosomal Elements of Gram Positive Organisms ......................................44

Appendix C-V-A. Exceptions .................................................................................................................45

Appendix C-VI. The Purchase or Transfer of Transgenic Rodents....................................................45

Appendix C-VII. Footnotes and References of Appendix C ................................................................45 
Page 4 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

APPENDIX D. MAJOR ACTIONS TAKEN UNDER THE NIH GUIDELINES ................................. 46

APPENDIX E. CERTIFIED HOST-VECTOR SYSTEMS................................................................. 65

Appendix E-I. Bacillus subtilis ..........................................................................................................65

Appendix E-I-A. Bacillus subtilis Host-Vector 1 Systems ....................................................................65

Appendix E-I-B. Bacillus subtilis Host-Vector 2 Systems ....................................................................65

Appendix E-II. Saccharomyces cerevisiae........................................................................................65

Appendix E-II-A. Saccharomyces cerevisiae Host-Vector 2 Systems..................................................65

Appendix E-III. Escherichia coli (E. coli) ............................................................................................66

Appendix E-III-A. Escherichia coli (EK2) Plasmid Systems ..................................................................66

Appendix E-III-B. Escherichia coli (EK2) Bacteriophage Systems........................................................66

Appendix E-IV. Neurospora crassa ....................................................................................................66

Appendix E-IV-A. Neurospora crassa Host-Vector 1 Systems ..............................................................66

Appendix E-V. Streptomyces.............................................................................................................66

Appendix E-V-A. Streptomyces Host-Vector 1 Systems ......................................................................66

Appendix E-VI. Pseudomonas putida.................................................................................................66

Appendix E-VI-A. Pseudomonas putida Host-Vector 1 Systems...........................................................66

APPENDIX F. CONTAINMENT CONDITIONS FOR CLONING OF GENES CODING FOR 

THE BIOSYNTHESIS OF MOLECULES TOXIC FOR VERTEBRATES................ 67

Appendix F-I. General Information...................................................................................................67

Appendix F-II. Cloning of Toxin Molecule Genes in Escherichia coli K-12.......................................67

Appendix F-III. Cloning of Toxic Molecule Genes in Organisms Other Than E. coli K-12 ................67

Appendix F-IV. Specific Approvals.....................................................................................................67

APPENDIX G. PHYSICAL CONTAINMENT.................................................................................... 68

Appendix G-I. Standard Practices and Training...............................................................................68

Appendix G-II. Physical Containment Levels ....................................................................................68

Appendix G-II-A. Biosafety Level 1 (BL1) .............................................................................................69

Appendix G-II-A-1. Standard Microbiological Practices (BL1) .................................................................69

Appendix G-II-A-2. Special Practices (BL1).............................................................................................69

Appendix G-II-A-3. Containment Equipment (BL1)..................................................................................69

Appendix G-II-A-4. Laboratory Facilities (BL1) ........................................................................................69

Appendix G-II-B. Biosafety Level 2 (BL2) .............................................................................................69

Appendix G-II-B-1. Standard Microbiological Practices (BL2) .................................................................70

Appendix G-II-B-2. Special Practices (BL2).............................................................................................70

Appendix G-II-B-3. Containment Equipment (BL2)..................................................................................71

Appendix G-II-B-4. Laboratory Facilities (BL2) ........................................................................................71

Appendix G-II-C. Biosafety Level 3 (BL3) .............................................................................................71

Appendix G-II-C-1. Standard Microbiological Practices (BL3) .................................................................71

Appendix G-II-C-2. Special Practices (BL3).............................................................................................72

Appendix G-II-C-2-t. Alternative Selection of Containment Equipment (BL3)............................................73

Appendix G-II-C-3. Containment Equipment (BL3)..................................................................................74

Appendix G-II-C-4. Laboratory Facilities (BL3) ........................................................................................74

Appendix G-II-D. Biosafety Level 4 (BL4) .............................................................................................75

Appendix G-II-D-1. Standard Microbiological Practices (BL4) .................................................................75

Appendix G-II-D-2. Special Practices (BL4).............................................................................................75

Appendix G-II-D-2-m. Alternative Selection of Containment Equipment (BL4)............................................76

Appendix G-II-D-3. Containment Equipment (BL4)..................................................................................76

Appendix G-II-D-4. Laboratory Facilities (BL4) ........................................................................................76

Appendix G-III. Footnotes and References of Appendix G................................................................78

APPENDIX H. SHIPMENT............................................................................................................... 80

Appendix H-III. Footnotes and References of Appendix H ................................................................81 
Page 5 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

APPENDIX I. BIOLOGICAL CONTAINMENT ............................................................................... 81

Appendix I-I. Levels of Biological Containment ..............................................................................81

Appendix I-I-A. Host-Vector 1 Systems..............................................................................................81

Appendix I-I-A-1. Escherichia coli K-12 Host-Vector 1 Systems (EK1).................................................81

Appendix I-I-A-2. Other Host-Vector 1 Systems....................................................................................81

Appendix I-I-B. Host-Vector 2 Systems (EK2) ...................................................................................81

Appendix I-II. Certification of Host-Vector Systems ........................................................................82

Appendix I-II-A. Responsibility ............................................................................................................82

Appendix I-II-B. Data to be Submitted for Certification .......................................................................82

Appendix I-II-B-1. Host-Vector 1 Systems Other than Escherichia coli K-12.........................................82

Appendix I-II-B-2. Host-Vector 2 Systems..............................................................................................82

Appendix I-III. Footnotes and References of Appendix I..................................................................83

APPENDIX J. BIOTECHNOLOGY RESEARCH SUBCOMMITTEE.............................................. 84

APPENDIX K. PHYSICAL CONTAINMENT FOR LARGE SCALE USES OF ORGANISMS 

CONTAINING RECOMBINANT DNA MOLECULES.............................................. 84

Appendix K-I. Selection of Physical Containment Levels ................................................................85

Appendix K-II. Good Large Scale Practice (GLSP) ..........................................................................85

Appendix K-III. Biosafety Level 1 (BL1) - Large Scale.......................................................................86

Appendix K-IV. Biosafety Level 2 (BL2) - Large Scale.......................................................................86

Appendix K-V. Biosafety Level 3 (BL3) - Large Scale.......................................................................88

Appendix K-VI. Footnotes of Appendix K...........................................................................................92

Appendix K-VII. Definitions to Accompany Containment Grid and Appendix K..................................92

APPENDIX L. GENE THERAPY POLICY CONFERENCES (GTPCS).......................................... 93

APPENDIX M. POINTS TO CONSIDER IN THE DESIGN AND SUBMISSION OF 

PROTOCOLS FOR THE TRANSFER OF RECOMBINANT DNA MOLECULES 

INTO ONE OR MORE HUMAN RESEARCH PARTICIPANTS (POINTS TO 

CONSIDER) ............................................................................................................. 93

Appendix M-I. Requirements for Protocol Submission, Review, and Reporting – Human Gene 

Transfer Experiments ................................................................................................95

Appendix M-I-A. Requirements for Protocol Submission.....................................................................95

Appendix M-I-B. RAC Review Requirements.......................................................................................96

Appendix M-I-B-1. Initial RAC Review.....................................................................................................96

Appendix M-I-B-2. Public RAC Review and Discussion..........................................................................96

Appendix M-I-C. Reporting Requirements ...........................................................................................97

Appendix M-I-C-1. Initiation of the Clinical Investigation .........................................................................97

Appendix M-I-C-2. Additional Clinical Trial Sites.....................................................................................97

Appendix M-I-C-3. Annual Reports..........................................................................................................97

Appendix M-I-C-4. Safety Reporting........................................................................................................98

Appendix M-I-C-4-a. Safety Reporting: Content and Format .....................................................................98

Appendix M-I-C-4-b. Safety Reporting: Time frames for Expedited Reports .............................................99

Appendix M-I-C-5. Confidentiality............................................................................................................99

Appendix M-I-D. Safety Assessment in Human Gene Transfer Research ..........................................99

Appendix M-II. Description of the Proposal .....................................................................................100

Appendix M-II-A. Objectives and Rationale of the Proposed Research .............................................100

Appendix M-II-A-1. Use of Recombinant DNA for Therapeutic Purposes .............................................100

Appendix M-II-A-2. Transfer of DNA for Other Purposes.......................................................................100

Appendix M-II-B. Research Design, Anticipated Risks and Benefits..................................................100

Appendix M-II-B-1. Structure and Characteristics of the Biological System ..........................................100

Appendix M-II-B-2. Preclinical Studies, Including Risk-Assessment Studies ........................................101

Appendix M-II-B-2-a. Delivery System ......................................................................................................101

Appendix M-II-B-2-b. Gene Transfer and Expression ...............................................................................101

Appendix M-II-B-2-c. Retrovirus Delivery Systems ...................................................................................102

Appendix M-II-B-2-d. Non-Retrovirus Delivery/Expression Systems ........................................................102

Appendix M-II-B-3. Clinical Procedures, Including Research Participant Monitoring ............................102 
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Appendix M-II-B-4. Public Health Considerations ..................................................................................103

Appendix M-II-B-5. Qualifications of Investigators and Adequacy of Laboratory and 

Clinical Facilities......................................................................................................103

Appendix M-II-C. Selection of the Human Subjects ............................................................................103

Appendix M-III. Informed Consent....................................................................................................104

Appendix M-III-A. Communication About the Study to Potential Participants......................................104

Appendix M-III-B. Informed Consent Document ..................................................................................104

Appendix M-III-B-1. General Requirements of Human Subjects Research ............................................104

Appendix M-III-B-1-a. Description/Purpose of the Study............................................................................104

Appendix M-III-B-1-b. Alternatives..............................................................................................................105

Appendix M-III-B-1-c. Voluntary Participation ............................................................................................105

Appendix M-III-B-1-d. Benefits....................................................................................................................105

Appendix M-III-B-1-e. Possible Risks, Discomforts, and Side Effects........................................................105

Appendix M-III-B-1-f. Costs .......................................................................................................................105

Appendix M-III-B-2. Specific Requirements of Gene Transfer Research................................................105

Appendix M-III-B-2-a. Reproductive Considerations ..................................................................................105

Appendix M-III-B-2-b. Long-Term Follow-Up..............................................................................................106

Appendix M-III-B-2-c. Request for Autopsy................................................................................................106

Appendix M-III-B-2-d. Interest of the Media and Others in the Research ..................................................106

Appendix M-IV. Privacy.....................................................................................................................106

Appendix M-V. Special Issues .........................................................................................................106

Appendix M-VI. Footnotes of Appendix M ........................................................................................107

APPENDIX P. PHYSICAL AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT DNA 

RESEARCH INVOLVING PLANTS....................................................................... 107

Appendix P-I. General Plant Biosafety Levels ...............................................................................107

Appendix P-II. Physical Containment Levels ..................................................................................108

Appendix P-II-A. Biosafety Level 1 - Plants (BL1-P) ..........................................................................108

Appendix P-II-A-1. Standard Practices (BL1-P).....................................................................................108

Appendix P-II-A-1-a. Greenhouse Access (BL1-P)...................................................................................108

Appendix P-II-A-1-b. Records (BL1-P)......................................................................................................108

Appendix P-II-A-1-c. Decontamination and Inactivation (BL1-P)..............................................................108

Appendix P-II-A-1-d. Control of Undesired Species and Motile Macroorganisms (BL1-P).......................108

Appendix P-II-A-1-e. Concurrent Experiments Conducted in the Greenhouse (BL1-P)...........................108

Appendix P-II-A-2. Facilities (BL1-P).....................................................................................................108

Appendix P-II-A-2-a. Definitions (BL1-P) ..................................................................................................108

Appendix P-II-A-2-b. Greenhouse Design (BL1-P)...................................................................................109

Appendix P-II-B. Biosafety Level 2 - Plants (BL2-P) ..........................................................................109

Appendix P-II-B-1. Standard Practices (BL2-P).....................................................................................109

Appendix P-II-B-1-a. Greenhouse Access (BL2-P)...................................................................................109

Appendix P-II-B-1-b. Records (BL2-P)......................................................................................................109

Appendix P-II-B-1-c. Decontamination and Inactivation (BL2-P)..............................................................109

Appendix P-II-B-1-d. Control of Undesired Species and Motile Macroorganisms (BL2-P).......................109

Appendix P-II-B-1-e. Concurrent Experiments Conducted in the Greenhouse (BL2-P)...........................110

Appendix P-II-B-1-f. Signs (BL2-P) ..........................................................................................................110

Appendix P-II-B-1-g. Transfer of Materials (BL2-P)..................................................................................110

Appendix P-II-B-1-h. Greenhouse Practices Manual (BL2-P) ..................................................................110

Appendix P-II-B-2. Facilities (BL2-P).....................................................................................................110

Appendix P-II-B-2-a. Definitions (BL2-P) ..................................................................................................110

Appendix P-II-B-2-b. Greenhouse Design (BL2-P)...................................................................................110

Appendix P-II-B-2-c. Autoclaves (BL2-P) .................................................................................................111

Appendix P-II-B-2-d. Supply and Exhaust Air Ventilation Systems (BL2-P).............................................111

Appendix P-II-B-2-e. Other (BL2-P) ..........................................................................................................111

Appendix P-II-C. Biosafety Level 3 - Plants (BL3-P) ..........................................................................111

Appendix P-II-C-1. Standard Practices (BL3-P).....................................................................................111

Appendix P-II-C-1-a. Greenhouse Access (BL3-P)...................................................................................111

Appendix P-II-C-1-b. Records (BL3-P)......................................................................................................111

Appendix P-II-C-1-c. Decontamination and Inactivation (BL3-P)..............................................................111 
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Appendix P-II-C-1-d. Control of Undesired Species and Motile Macroorganisms (BL3-P).......................111

Appendix P-II-C-1-e. Concurrent Experiments Conducted in the Greenhouse (BL3-P)...........................112

Appendix P-II-C-1-f. Signs (BL3-P) ..........................................................................................................112

Appendix P-II-C-1-g. Transfer of Materials (BL3-P)..................................................................................112

Appendix P-II-C-1-h. Greenhouse Practices Manual (BL3-P) ..................................................................112

Appendix P-II-C-1-i. Protective Clothing (BL3-P).....................................................................................112

Appendix P-II-C-1-j. Other (BL3-P) ..........................................................................................................112

Appendix P-II-C-2. Facilities (BL3-P).....................................................................................................113

Appendix P-II-C-2-a. Definitions (BL3-P) ..................................................................................................113

Appendix P-II-C-2-b. Greenhouse Design (BL3-P)...................................................................................113

Appendix P-II-C-2-c. Autoclaves (BL3-P) .................................................................................................113

Appendix P-II-C-2-d. Supply and Exhaust Air Ventilation Systems (BL3-P).............................................113

Appendix P-II-C-2-e. Other (BL3-P) ..........................................................................................................114

Appendix P-II-D. Biosafety Level 4 - Plants (BL4-P) ..........................................................................114

Appendix P-II-D-1. Standard Practices (BL4-P).....................................................................................114

Appendix P-II-D-1-a. Greenhouse Access (BL4-P)...................................................................................114

Appendix P-II-D-1-b. Records (BL4-P)......................................................................................................114

Appendix P-II-D-1-c. Decontamination and Inactivation (BL4-P)..............................................................115

Appendix P-II-D-1-d. Control of Undesired Species and Motile Macroorganisms (BL4-P).......................115

Appendix P-II-D-1-e. Concurrent Experiments Conducted in the Greenhouse (BL4-P)...........................115

Appendix P-II-D-1-f. Signs (BL4-P) ..........................................................................................................115

Appendix P-II-D-1-g. Transfer of Materials (BL4-P)..................................................................................115

Appendix P-II-D-1-h. Greenhouse Practices Manual (BL4-P) ..................................................................116

Appendix P-II-D-1-i. Protective Clothing (BL4-P).....................................................................................116

Appendix P-II-D-2. Facilities (BL4-P).....................................................................................................116

Appendix P-II-D-2-a. Greenhouse Design (BL4-P)...................................................................................116

Appendix P-II-D-2-b. Autoclaves (BL4-P) .................................................................................................116

Appendix P-II-D-2-c. Supply and Exhaust Air Ventilation Systems (BL4-P).............................................117

Appendix P-II-D-2-d. Other (BL4-P) ..........................................................................................................117

Appendix P-III. Biological Containment Practices............................................................................117

Appendix P-III-A. Biological Containment Practices (Plants) ..............................................................117

Appendix P-III-B. Biological Containment Practices (Microorganisms)...............................................118

Appendix P-III-C. Biological Containment Practices (Macroorganisms) .............................................118

APPENDIX Q. PHYSICAL AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT DNA 

RESEARCH INVOLVING ANIMALS..................................................................... 118

Appendix Q-I. General Considerations...........................................................................................118

Appendix Q-I-A. Containment Levels.................................................................................................118

Appendix Q-I-B. Disposal of Animals (BL1-N through BL4-N)...........................................................119

Appendix Q-II. Physical and Biological Containment Levels...........................................................119

Appendix Q-II-A. Biosafety Level 1 - Animals (BL1-N) .......................................................................119

Appendix Q-II-A-1. Standard Practices (BL1-N) ....................................................................................119

Appendix Q-II-A-1-a. Animal Facility Access (BL1-N)...............................................................................119

Appendix Q-II-A-1-b. Other (BL1-N)..........................................................................................................119

Appendix Q-II-A-2. Animal Facilities (BL1-N).........................................................................................119

Appendix Q-II-B. Biosafety Level 2 - Animals (BL2-N) .......................................................................119

Appendix Q-II-B-1. Standard Practices (BL2-N) ....................................................................................119

Appendix Q-II-B-1-a. Animal Facility Access (BL2-N)...............................................................................119

Appendix Q-II-B-1-b. Decontamination and Inactivation (BL2-N) .............................................................120

Appendix Q-II-B-1-c. Signs (BL2-N)..........................................................................................................120

Appendix Q-II-B-1-d. Protective Clothing (BL2-N).....................................................................................120

Appendix Q-II-B-1-e. Records (BL2-N) .....................................................................................................120

Appendix Q-II-B-1-f. Transfer of Materials (BL2-N)..................................................................................120

Appendix Q-II-B-1-g. Other (BL2-N)..........................................................................................................121

Appendix Q-II-B-2. Animal Facilities (BL2-N).........................................................................................121

Appendix Q-II-C. Biosafety Level 3 - Animals (BL3-N) .......................................................................122

Appendix Q-II-C-1. Standard Practices (BL3-N) ....................................................................................122

Appendix Q-II-C-1-a. Animal Facility Access (BL3-N)...............................................................................122 
Page 8 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Appendix Q-II-C-1-b. Decontamination and Inactivation (BL3-N) .............................................................122

Appendix Q-II-C-1-c. Signs (BL3-N)..........................................................................................................122

Appendix Q-II-C-1-d. Protective Clothing (BL3-N).....................................................................................122

Appendix Q-II-C-1-e. Records (BL3-N) .....................................................................................................123

Appendix Q-II-C-1-f. Transfer of Materials (BL3-N)..................................................................................123

Appendix Q-II-C-1-g. Other (BL3-N)..........................................................................................................123

Appendix Q-II-C-2. Animal Facilities (BL3-N).........................................................................................124

Appendix Q-II-D. Biosafety Level 4 - Animals (BL4-N) .......................................................................125

Appendix Q-II-D-1. Standard Practices (BL4-N) ....................................................................................125

Appendix Q-II-D-1-a. Animal Facility Access (BL4-N)...............................................................................125

Appendix Q-II-D-1-b. Decontamination and Inactivation (BL4-N) .............................................................126

Appendix Q-II-D-1-c. Signs (BL4-N)..........................................................................................................127

Appendix Q-II-D-1-d. Protective Clothing (BL4-N).....................................................................................127

Appendix Q-II-D-1-e. Records (BL4-N) .....................................................................................................127

Appendix Q-II-D-1-f. Transfer of Materials (BL4-N)..................................................................................127

Appendix Q-II-D-1-g. Other (BL4-N)..........................................................................................................128

Appendix Q-II-D-2. Animal Facilities (BL4-N).........................................................................................129

Appendix Q-III. Footnotes and References for Appendix Q.............................................................130

*********************************************************************************************************************** 

LIST OF TABLES 

Appendix B - Table 1. Basis for the Classification of Biohazardous Agents by Risk Group.........................36

Appendix G - Table 1. Possible Alternate Combinations Of Physical And Biological Containment 

Safeguards ................................................................................................................78

Appendix K - Table 1. Comparison of Good Large Scale Practice (GLSP) and Biosafety Level (BL) - Large 

Scale (LS) Practice....................................................................................................91

*********************************************************************************************************************** 

SECTION I. SCOPE OF THE NIH GUIDELINES

Section I-A. Purpose 

The purpose of the NIH Guidelines is to specify practices for constructing and handling: (i) recombinant 

deoxyribonucleic acid (DNA) molecules, and (ii) organisms and viruses containing recombinant DNA molecules. 

Section I-A-1. Any recombinant DNA 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. (See exception in Section I-A-1-a regarding 

requirement for human gene transfer protocol registration.) 

Section I-A-1-a. For experiments involving the deliberate transfer of recombinant DNA, or DNA or RNA derived 

from recombinant DNA, into human research participants (human gene transfer), no research participant shall 

be enrolled (see definition of enrollment in Section I-E-7) until the RAC review process has been completed (see 

Appendix M-I-B, RAC Review Requirements); Institutional Biosafety Committee (IBC) approval (from the clinical 

trial site) has been obtained; Institutional Review Board approval has been obtained; and all applicable 

regulatory authorization(s) have been obtained. 
Page 9 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

For a clinical trial site that is added after the RAC review process, no research participant shall be enrolled (see 

definition of enrollment in Section I-E-7) at the clinical trial site until the following documentation has been 

submitted to NIH OBA: (1) IBC approval (from the clinical trial site); (2) Institutional Review Board approval; (3) 

Institutional Review Board-approved informed consent document; and (4) curriculum vitae of the principal 

investigator(s) (no more than two pages in biographical sketch format); and (5) NIH grant number(s) if 

applicable. 

Section I-B. Definition of Recombinant DNA Molecules 

In the context of the NIH Guidelines, recombinant DNA molecules are defined as either: (i) molecules that are 

constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can 

replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. 

Synthetic DNA segments which are likely to yield a potentially harmful polynucleotide or polypeptide (e.g., a 

toxin or a pharmacologically active agent) are considered as equivalent to their natural DNA counterpart. If the 

synthetic DNA segment is not expressed in vivo as a biologically active polynucleotide or polypeptide product, it 

is exempt from the NIH Guidelines. 

Genomic DNA of plants and bacteria that have acquired a transposable element, even if the latter was donated 

from a recombinant vector no longer present, are not subject to the NIH Guidelines unless the transposon itself 

contains recombinant DNA. 

Section I-C. General Applicability 

Section I-C-1. The NIH Guidelines are applicable to: 

Section I-C-1-a. All recombinant DNA 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 DNA research from NIH, including research performed directly by NIH. An individual who receives 

support for research involving recombinant DNA 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 DNA 

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 DNA 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. 

Section I-C-1-b-(2). Research that involves testing in humans of materials containing recombinant DNA 

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 DNA research, 

then the research must be in compliance with those rules. If the host country does not have such rules, the 

proposed research must be reviewed and approved by an NIH-approved Institutional Biosafety Committee or 

equivalent review body and accepted in writing by an appropriate national governmental authority of the host 

country. The safety practices that are employed abroad must be reasonably consistent with the NIH Guidelines. 
Page 10 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section I-D. Compliance with the NIH Guidelines

As a condition for NIH funding of recombinant DNA research, institutions shall ensure that such research 

conducted at or sponsored by the institution, irrespective of the source of funding, shall comply with the NIH 

Guidelines. 

Information concerning noncompliance with the NIH Guidelines may be brought forward by any person. It 

should be delivered to both NIH/OBA and the relevant institution. The institution, generally through the 

Institutional Biosafety Committee, shall take appropriate action. The institution shall forward a complete report 

of the incident recommending any further action to the Office of Biotechnology Activities, National Institutes of 

Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985, 301-496-9838/301-496-9839 

(fax) (for non-USPS mail, use zip code 20817). 

In cases where NIH proposes to suspend, limit, or terminate financial assistance because of noncompliance 

with the NIH Guidelines, applicable DHHS and Public Health Service procedures shall govern. 

The policies on compliance are as follows: 

Section I-D-1. All NIH-funded projects involving recombinant DNA techniques must comply with the NIH 

Guidelines. Non-compliance may result in: (i) suspension, limitation, or termination of financial assistance for 

the noncompliant NIH-funded research project and of NIH funds for other recombinant DNA research at the 

institution, or (ii) a requirement for prior NIH approval of any or all recombinant DNA projects at the institution. 

Section I-D-2. All non-NIH funded projects involving recombinant DNA techniques conducted at or sponsored 

by an institution that receives NIH funds for projects involving such techniques must comply with the NIH 

Guidelines. Noncompliance may result in: (i) suspension, limitation, or termination of NIH funds for 

recombinant DNA research at the institution, or (ii) a requirement for prior NIH approval of any or all 

recombinant DNA 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 for 

membership 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). 

Section I-E-3. The "Office of Biotechnology Activities (OBA)" 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-3, Office of Biotechnology Activities (OBA). 

Section I-E-4. The "Recombinant DNA Advisory Committee" is the public advisory committee that advises the 

Department of Health and Human Services (DHHS) Secretary, the DHHS Assistant Secretary for Health, and 

the NIH Director concerning recombinant DNA research. The RAC shall be constituted as specified in Section 

IV-C-2, Recombinant DNA Advisory Committee (RAC). 

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 DNA-containing 

microorganisms, plants, or animals into the environment. 

Section I-E-7. “Enrollment” is the process of obtaining informed consent from a potential research participant, 

or a designated legal guardian of the participant, to undergo a test or procedure associated with the gene 

transfer experiment. 
Page 11 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section I-E-8. A “serious adverse event” is any event occurring at any dose that results in any of the following 

outcomes: death, a life-threatening event, in-patient hospitalization or prolongation of existing hospitalization, a 

persistent or significant disability/incapacity, or a congenital anomaly/birth defect. Important medical events that 

may not result in death, be life-threatening, or require hospitalization also may be considered a serious adverse 

event when, upon the basis of appropriate medical judgment, they may jeopardize the human gene transfer 

research subject and may require medical or surgical intervention to prevent one of the outcomes listed in this 

definition. 

Section I-E-9. An adverse event is “associated with the use of a gene transfer product” when there is a 

reasonable possibility that the event may have been caused by the use of that product. 

Section I-E-10. An “unexpected serious adverse event” is any serious adverse event for which the specificity 

or severity is not consistent with the risk information available in the current investigator’s brochure. 

*********************************************************************************************************************** 

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 to their 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 are associated with human disease which is rarely 

serious and for which preventive or therapeutic interventions 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 therapeutic interventions 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 initial risk assessment from Appendix B, 

Classification of Human Etiologic Agents on the Basis of Hazard, should be followed by a thorough 

consideration of the agent itself and how it is to be manipulated. Factors to be considered in determining the 

level of containment include agent factors such as: virulence, pathogenicity, infectious dose, environmental 

stability, route of spread, communicability, operations, quantity, availability of 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 for handling 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 level compared to the Risk Group assigned to the parent 

strain (see Section V-B, Footnotes and References of Sections I-IV). 
Page 12 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 containment level required may be 

equivalent to the Risk Group classification of the agent or it may be raised or lowered as a result of the above 

considerations. The Institutional Biosafety Committee must approve the risk assessment and the biosafety 

containment level for recombinant DNA experiments described in Sections III-A, Experiments that Require 

Institutional Biosafety Committee Approval, RAC Review, and NIH Director Approval Before Initiation; III-B, 

Experiments that Require NIH/OBA and Institutional Biosafety Committee Approval Before Initiation; III-C, 

Experiments that Require Institutional Biosafety Committee and Institutional Review Board Approvals and 

NIH/OBA Registration Before Initiation; III-D, Experiments that Require Institutional Biosafety Committee 

Approval Before Initiation. 

Careful consideration should be given to the types of manipulation planned for some higher Risk Group agents. 

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 or transmission 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 is recommended 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, are performed 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 selection of 

laboratory procedures applicable to organisms carrying recombinant DNA (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 estimated biohazard. Four biosafety levels are described in Appendix G, Physical 

Containment. These biosafety levels 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 the agents 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 DNA lend themselves to a third containment 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 DNA and/or host cell replication, can be genetically designed 

to decrease, by many orders of magnitude, the probability of dissemination of recombinant DNA outside the 

laboratory (see Appendix I, Biological Containment). 
Page 13 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Since these three means of containment are complementary, different levels of containment can be established 

that apply various combinations of the physical and biological barriers along with a constant use of standard 

practices. Categories of containment are considered separately in order that such combinations can be 

conveniently expressed in the NIH Guidelines. 

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, biological containment 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 recombinant DNA that is 

either integrated into the genome or into 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 of genetic 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 P, 

Physical and Biological Containment for Recombinant DNA 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 dissemination of recombinant DNA into the environment, or in 

which there is no recognizable and predictable risk to the environment in the event of accidental release. BL2-P 

is designed to provide a greater level of containment for experiments involving plants and certain associated 

organisms in which there is a recognized possibility of survival, transmission, or dissemination of recombinant 

DNA 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 most ordinary 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 protected environment for the propagation of plants and microorganisms associated with the 

plants and a degree of containment that adequately controls the potential for release of biologically viable 

plants, plant parts, and microorganisms 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 that 

minimizes 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 Q, Physical and Biological Containment for Recombinant DNA 

Research Involving Animals. BL1-N describes containment for animals that have been modified by stable 

introduction of recombinant DNA, or DNA derived therefrom, into the germ-line (transgenic animals) and 

experiments involving viable recombinant DNA-modified microorganisms and is designed to eliminate the 

possibility of sexual transmission of the modified genome or transmission of recombinant DNA-derived viruses 

known to be transmitted from animal parent to offspring only by sexual 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 DNA-derived organisms and is designed to 

eliminate 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. 
Page 14 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

In constructing the NIH Guidelines, it was necessary to define boundary conditions for the different levels of 

physical and biological containment and for the classes of experiments to which they apply. These definitions 

do not take into account all existing and anticipated information on special procedures that will allow 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 GUIDELINES 

This section describes six categories of experiments involving recombinant DNA: (i) those that require 

Institutional Biosafety Committee (IBC) approval, RAC review, and NIH Director approval before initiation (see 

Section III-A), (ii) those that require NIH/OBA and Institutional Biosafety Committee approval before initiation 

(see Section III-B), (iii) those that require Institutional Biosafety Committee and Institutional Review Board 

approvals and RAC review before research participant enrollment (see Section III-C), (iv) those that require 

Institutional Biosafety Committee approval before initiation (see Section III-D), (v) those that require Institutional 

Biosafety Committee notification simultaneous with initiation (see Section III-E), and (vi) those that are exempt 

from the NIH Guidelines (see Section III-F). 

Note: If an experiment falls into Sections III-A, III-B, or III-C and one of the other sections, the rules pertaining 

to Sections III-A, III-B, or III-C shall be followed. If an experiment falls into Section III-F and into either Sections 

III-D or III-E as well, the experiment is considered exempt from the NIH Guidelines. 

Any change in containment level, which is different from those specified in the NIH Guidelines, may not be 

initiated without the express approval of NIH/OBA (see Section IV-C-1-b-(2) and its subsections, Minor Actions). 

Section III-A. Experiments that Require Institutional Biosafety Committee Approval, RAC Review, and 

NIH Director Approval Before Initiation (See Section IV-C-1-b-(1), Major Actions).

Section III-A-1. Major Actions under the NIH Guidelines

Experiments considered as Major Actions under the NIH Guidelines cannot be initiated without submission of 

relevant information on the proposed experiment to the Office of Biotechnology Activities, National Institutes of 

Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 

301-496-9838, 301-496-9839 (fax), the publication of the proposal in the Federal Register for 15 days of 

comment, review by RAC, and specific approval by NIH. The containment conditions or stipulation 

requirements for such experiments will be recommended by RAC and set by NIH at the time of approval. Such 

experiments require Institutional Biosafety Committee approval before initiation. Specific experiments already 

approved are included in Appendix D, Major Actions Taken under the NIH Guidelines, which may be obtained 

from the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 

7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

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 such acquisition could 

compromise the use of the drug to control disease agents in humans, veterinary medicine, or agriculture, will be 

reviewed by RAC. 

Section III-B. Experiments That Require NIH/OBA 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/OBA. The containment conditions for such experiments will be determined by NIH/OBA 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). 
Page 15 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 DNA containing genes for the biosynthesis 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, and Shigella dysenteriae neurotoxin). Specific approval has 

been given for the cloning in Escherichia coli K-12 of DNA containing genes coding for the biosynthesis of toxic 

molecules which are lethal to vertebrates at 100 nanograms to 100 micrograms per kilogram body weight. 

Specific experiments already approved under this section may be obtained from the Office of Biotechnology 

Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 

20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

Section III-C. Experiments that Require Institutional Biosafety Committee and Institutional Review 

Board Approvals and RAC Review Before Research Participant Enrollment 

Section III-C-1. Experiments Involving the Deliberate Transfer of Recombinant DNA, or DNA or RNA 

Derived from Recombinant DNA, into One or More Human Research Participants 

For an experiment involving the deliberate transfer of recombinant DNA, or DNA or RNA derived from 

recombinant DNA, into human research participants (human gene transfer), no research participant shall be 

enrolled (see definition of enrollment in Section I-E-7) until the RAC review process has been completed (see 

Appendix M-I-B, RAC Review Requirements). 

In its evaluation of human gene transfer proposals, the RAC will consider whether a proposed human gene 

transfer experiment presents characteristics that warrant public RAC review and discussion (See Appendix M-IB-2). The process of public RAC review and discussion is intended to foster the safe and ethical conduct of 

human gene transfer experiments. Public review and discussion of a human gene transfer experiment (and 

access to relevant information) also serves to inform the public about the technical aspects of the proposal, 

meaning and significance of the research, and any significant safety, social, and ethical implications of the 

research. 

Public RAC review and discussion of a human gene transfer experiment may be: (1) initiated by the NIH 

Director; or (2) initiated by the NIH OBA Director following a recommendation to NIH OBA by: (a) three or more 

RAC members; or (b) a Federal agency other than NIH. After a human gene transfer experiment is reviewed by 

the RAC at a regularly scheduled meeting, NIH OBA will send a letter, unless NIH OBA determines that there 

are exceptional circumstances, within 10 working days to the NIH Director, the Principal Investigator, the 

sponsoring institution, and other DHHS components, as appropriate, summarizing the RAC recommendations. 

For a clinical trial site that is added after the RAC review process, no research participant shall be enrolled (see 

definition of enrollment in Section I-E-7) at the clinical trial site until the following documentation has been 

submitted to NIH OBA: (1) Institutional Biosafety Committee approval (from the clinical trial site); (2) Institutional 

Review Board approval; (3) Institutional Review Board-approved informed consent document; (4) curriculum 

vitae of the principal investigator(s) (no more than two pages in biographical sketch format); and (5) NIH grant 

number(s) if applicable. 

In order to maintain public access to information regarding human gene transfer protocols (including protocols 

that are not publicly reviewed by the RAC), NIH OBA will maintain the documentation described in Appendices 

M-I through M-V. The information provided in response to Appendix M should not contain any confidential 

commercial information or trade secrets, enabling all aspects of RAC review to be open to the public. 

Note: For specific directives concerning the use of retroviral vectors for gene delivery, consult Appendix B-V-1, 

Murine Retroviral Vectors.
Page 16 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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

Prior to the initiation of an experiment that falls into this category, the Principal Investigator must submit a 

registration document to the Institutional Biosafety Committee which contains the following information: (i) the 

source(s) of DNA; (ii) the nature of the inserted DNA sequences; (iii) the host(s) and vector(s) to be used; (iv) if 

an attempt will be made to obtain expression of a foreign gene, and if so, indicate the protein that will be 

produced; and (v) the containment conditions that will be implemented as specified in the NIH Guidelines. For 

experiments in this category, the registration document shall be dated, signed by the Principal Investigator, and 

filed with the Institutional Biosafety Committee. The Institutional Biosafety Committee shall review and approve 

all experiments in this category prior to their initiation. Requests to decrease the level of containment specified 

for experiments in this category will be considered by NIH (see Section 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 DNA into Risk Group 2 agents will 

usually be conducted at Biosafety Level (BL) 2 containment. Experiments with such agents will usually be 

conducted with whole animals at BL2 or BL2-N (Animals) containment. 

Section III-D-1-b. Experiments involving the introduction of recombinant DNA into Risk Group 3 agents will 

usually be conducted at BL3 containment. Experiments with such agents will usually be conducted with whole 

animals at BL3 or BL3-N containment. 

Section III-D-1-c. Experiments involving the introduction of recombinant DNA into Risk Group 4 agents shall be 

conducted at BL4 containment. Experiments with such agents shall be conducted with whole animals at BL4 or 

BL4-N containment. 

Section III-D-1-d. Containment conditions for experiments involving the introduction of recombinant DNA into 

restricted agents shall be set on a case-by-case basis following NIH/OBA review. A U.S. Department of 

Agriculture 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 with whole 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, or 

Restricted Agents is Cloned into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector Systems 

Section III-D-2-a. Experiments in which DNA from Risk Group 2 or Risk Group 3 agents (see Section II-A, Risk 

Assessment) is transferred into nonpathogenic prokaryotes or lower eukaryotes may be performed under BL2 

containment. Experiments in which DNA from Risk Group 4 agents is transferred into nonpathogenic 

prokaryotes or lower eukaryotes may be performed under BL2 containment after demonstration that only a 

totally and irreversibly defective fraction of the agent's genome is present in a given recombinant. In the 

absence of such a demonstration, BL4 containment shall be used. The Institutional Biosafety Committee may 

approve the specific lowering of containment for particular experiments 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 DNA for certain genes coding for molecules toxic for vertebrates require NIH/OBA 

approval (see Section 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 is transferred 

into nonpathogenic prokaryotes or lower eukaryotes shall be determined by NIH/OBA 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 IIV). 
Page 17 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section III-D-3. Experiments Involving the Use of Infectious DNA or RNA Viruses or Defective DNA or 

RNA Viruses in the Presence of Helper Virus in Tissue Culture Systems 

Caution: Special care should be used in the evaluation of containment levels for experiments which are likely 

to either enhance the pathogenicity (e.g., insertion of a host oncogene) or to extend the host range (e.g., 

introduction of novel control elements) of viral vectors under conditions that permit a productive infection. In 

such cases, serious consideration should be given to increasing physical containment by at least one level. 

Note: Recombinant DNA or RNA molecules derived 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 V-K, Footnotes and References of Sections I-IV), are 

considered defective and may be used in the absence of helper under the conditions specified in Section III-E-1, 

Experiments Involving the Formation of Recombinant DNA Molecules Containing No More than Two-Thirds of 

the Genome of any Eukaryotic Virus. 

Section III-D-3-a. Experiments involving the use of infectious or defective Risk Group 2 viruses (see Appendix 

B-II, Risk Group 2 Agents) in the presence of helper virus may be conducted at BL2. 

Section III-D-3-b. Experiments involving the use of infectious or defective Risk Group 3 viruses (see Appendix 

B-III-D, Risk Group 3 (RG3) - Viruses and Prions) in the presence of helper virus may be conducted at BL3. 

Section III-D-3-c. Experiments involving the use of infectious or defective Risk Group 4 viruses (see Appendix 

B-IV-D, Risk Group 4 (RG4) - Viral Agents) in the presence of helper virus may be conducted at BL4. 

Section III-D-3-d. Experiments involving the use of infectious or defective restricted poxviruses (see Sections 

V-A and V-L, Footnotes and References of Sections I-IV) in the presence of helper virus shall be determined on 

a case-by-case basis following NIH/OBA review. A U.S. Department of Agriculture permit is required for work 

with plant or animal pathogens (see Section V-G, Footnotes and References of Sections I-IV). 

Section III-D-3-e. Experiments involving the use of infectious or defective viruses in the presence of helper 

virus which are not covered in Sections III-D-3-a through III-D-3-d may be conducted at BL1. 

Section III-D-4. Experiments Involving Whole Animals 

This section covers experiments involving whole animals in which the animal's genome has been altered by 

stable introduction of recombinant DNA, or DNA derived therefrom, into the germ-line (transgenic animals) and 

experiments involving viable recombinant DNA-modified microorganisms tested on whole animals. For the 

latter, other than viruses which are only vertically transmitted, the experiments may not be conducted at BL1-N 

containment. A minimum containment of BL2 or BL2-N is required. 

Caution - Special care should be used in the evaluation of containment conditions for some experiments with 

transgenic animals. For example, such experiments might lead to the creation of novel mechanisms or 

increased transmission of a recombinant pathogen or production of undesirable traits in the host animal. In 

such cases, serious consideration should be given to increasing the containment conditions. 

Section III-D-4-a. Recombinant DNA, or DNA or RNA molecules derived therefrom, from any source except for 

greater than two-thirds of eukaryotic viral genome may be transferred to any non-human vertebrate or any 

invertebrate organism and propagated under conditions of physical containment comparable to BL1 or BL1-N 

and appropriate to the organism under study (see Section V-B, Footnotes and References of Sections I-IV). 

Animals that contain sequences from viral vectors, which do not lead to transmissible infection either directly or 

indirectly as a result of complementation or recombination in animals, may be propagated under conditions of 

physical containment comparable to 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 DNA-modified 

Risk Groups 2, 3, 4, or restricted organisms, see Sections V-A, V-G, and V-L, Footnotes and References of 

Sections I-IV. It is important that the investigator demonstrate that the fraction of the viral genome being utilized 

does not lead to productive infection. A U.S. Department of Agriculture permit is required for work with plant or 

animal pathogens (see Section V-G, Footnotes and References of Sections I-IV). 
Page 18 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section III-D-4-b. For experiments involving recombinant DNA, or DNA or RNA derived therefrom, involving 

whole animals, including transgenic animals, and not covered by Sections III-D-1, Experiments Using Human or 

Animal Pathogens (Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as Host-Vector Systems, or 

III-D-4-a, Experiments Involving Whole Animals, the appropriate containment shall be determined by the 

Institutional Biosafety Committee. 

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

Section III-D-4-c-(1). Experiments involving the generation of transgenic rodents that require 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-VI, The Purchase or Transfer of Transgenic Rodents). 

Section III-D-5. Experiments Involving Whole Plants 

Experiments to genetically engineer plants by recombinant DNA methods, to use such plants for other 

experimental purposes (e.g., response to stress), to propagate such plants, or to use plants together with 

microorganisms or insects containing recombinant DNA, may be conducted under the containment conditions 

described in Sections III-D-5-a through III-D-5-e. If experiments involving whole plants are not described in 

Section III-D-5 and do not fall under Sections III-A, III-B, III-D or III-F, they are included in Section III-E. 

NOTE - For recombinant DNA experiments falling under Sections III-D-5-a through III-D-5-d, physical 

containment requirements may be reduced to the next lower level by appropriate biological containment 

practices, such as conducting experiments on a virus with an obligate insect vector in the absence of that vector 

or using a genetically attenuated strain. 

Section III-D-5-a. BL3-P (Plants) or BL2-P + biological containment is recommended for experiments involving 

most exotic (see Section V-M, Footnotes and References of Sections I-IV) infectious agents with recognized 

potential for serious detrimental impact on managed or natural ecosystems when recombinant DNA techniques 

are associated with whole plants. 

Section III-D-5-b. BL3-P or BL2-P + biological containment is recommended for experiments involving plants 

containing cloned genomes of readily transmissible exotic (see Section V-M, Footnotes and References of 

Sections I-IV) infectious agents with recognized potential for serious detrimental effects on managed or natural 

ecosystems in which there exists the possibility of reconstituting the complete and functional genome of the 

infectious agent by genomic complementation in planta.

Section III-D-5-c. BL4-P containment is recommended for experiments with a small number of readily 

transmissible exotic (see Section V-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 the presence 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 DNA 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 the Cloning of Toxin Molecules with LD50 of Less than 100 

Nanograms Per Kilogram Body Weight, and require NIH/OBA and Institutional Biosafety Committee approval 

before initiation. 

Section III-D-5-e. BL3-P or BL2-P + biological containment is recommended for experiments with microbial 

pathogens of insects or small animals associated with plants if the recombinant DNA-modified organism has a 

recognized potential for serious detrimental impact on managed or natural ecosystems. 
Page 19 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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

The appropriate containment will be decided by the Institutional Biosafety Committee. Where appropriate, 

Appendix K, Physical Containment for Large Scale Uses of Organisms Containing Recombinant DNA 

Molecules, shall be used. Appendix K describes containment conditions Good Large Scale Practice through 

BL3-Large Scale. 

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

Initiation 

Experiments not included in Sections III-A, III-B, III-C, III-D, III-F, and their subsections are considered in 

Section III-E. All such experiments may be conducted at BL1 containment. For experiments in this category, a 

registration document (see Section III-D, Experiments that Require Institutional Biosafety Committee Approval 

Before Initiation) shall be dated and signed by the investigator and filed with the local Institutional Biosafety 

Committee at the time the experiment is initiated. The Institutional Biosafety Committee reviews and approves 

all such proposals, but Institutional Biosafety Committee review and approval prior to initiation of the experiment 

is not required (see Section IV-A, Policy). For example, experiments in which all components derived from 

non-pathogenic prokaryotes and non-pathogenic lower eukaryotes fall under Section III-E and may be 

conducted at BL1 containment. 

Section III-E-1. Experiments Involving the Formation of Recombinant DNA Molecules Containing No 

More than Two-Thirds of the Genome of any Eukaryotic Virus 

Recombinant DNA molecules containing no more than two-thirds of the genome of any eukaryotic virus (all 

viruses from a single Family being considered identical [see Section V-J, Footnotes and References of Sections 

I-IV]) may be propagated and maintained in cells in tissue culture using BL1 containment. For such 

experiments, it must be demonstrated that the cells lack helper virus for the specific Families of defective 

viruses being used. If helper virus is present, procedures specified under Section III-D-3, Experiments Involving 

the Use of Infectious Animal or Plant DNA or RNA Viruses or Defective Animal or Plant DNA or RNA Viruses in 

the Presence of Helper Virus in Tissue Culture Systems, should be used. The DNA may contain fragments of 

the genome of viruses from more than one Family but each fragment shall be less than two-thirds of a genome. 

Section III-E-2. Experiments Involving Whole Plants 

This section covers experiments involving recombinant DNA-modified whole plants, and/or experiments 

involving recombinant DNA-modified organisms associated with whole plants, except those that fall under 

Section III-A, III-B, III-D, or III-F. It should be emphasized that knowledge of the organisms and judgment based 

on accepted scientific practices should be used in all cases in selecting the appropriate level of containment. 

For example, if the genetic modification has the objective of increasing pathogenicity or converting a nonpathogenic organism into a pathogen, then a higher level of containment may be appropriate depending on the 

organism, its mode of dissemination, and its target organisms. By contrast, a lower level of containment may be 

appropriate for small animals associated with many types of recombinant DNA-modified plants. 

Section III-E-2-a. BL1-P is recommended for all experiments with recombinant DNA-containing plants and 

plant-associated microorganisms not covered in Section III-E-2-b or other sections of the NIH Guidelines. 

Examples of such experiments are those involving recombinant DNA-modified plants that are not noxious 

weeds or that cannot interbreed with noxious weeds in the immediate geographic area, and experiments 

involving whole plants and recombinant DNA-modified non-exotic (see Section V-M, Footnotes and References 

of Sections I-IV) microorganisms that have no recognized potential for rapid and widespread dissemination or 

for serious detrimental impact on managed or natural ecosystems (e.g., Rhizobium spp. and Agrobacterium 

spp.). 

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

Section III-E-2-b-(1). Plants modified by recombinant DNA that are noxious weeds or can interbreed with 

noxious weeds in the immediate geographic area. 
Page 20 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section III-E-2-b-(2). Plants in which the introduced DNA represents the complete genome of a non-exotic 

infectious agent (see Section V-M, Footnotes and References of Sections I-IV). 

Section III-E-2-b-(3). Plants associated with recombinant DNA-modified non-exotic microorganisms that have a 

recognized potential for serious detrimental impact on managed or natural ecosystems (see Section V-M, 

Footnotes and References of Sections I-IV). 

Section III-E-2-b-(4). Plants associated with recombinant DNA-modified exotic microorganisms that have no 

recognized potential for serious 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 DNA-modified arthropods or small animals associated with 

plants, or with arthropods or small animals with recombinant DNA-modified microorganisms associated with 

them if the recombinant DNA-modified microorganisms have no recognized potential for serious detrimental 

impact on managed or natural ecosystems (see Section V-M, Footnotes and References of Sections I-IV). 

Section III-E-3. Experiments Involving Transgenic Rodents 

This section covers experiments involving the generation of rodents in which the animal's genome has been 

altered by stable introduction of recombinant DNA, or DNA derived 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 are covered under Section III-D-4, Experiments Involving Whole Animals. 

Section III-F. Exempt Experiments 

The following recombinant DNA molecules are exempt from the NIH Guidelines and registration with the 

Institutional Biosafety Committee is not required: 

Section III-F-1. Those that are not in organisms or viruses. 

Section III-F-2. Those that consist entirely of DNA segments from a single nonchromosomal or viral DNA 

source, though one or more of the segments may be a synthetic equivalent. 

Section III-F-3. Those that consist entirely of DNA from a prokaryotic host including its indigenous plasmids or 

viruses when propagated only in that host (or a closely related strain of the same species), or when transferred 

to another host by well established physiological means. 

Section III-F-4. Those that consist entirely of DNA from an eukaryotic host including its chloroplasts, 

mitochondria, or plasmids (but excluding viruses) when propagated only in that host (or a closely related strain 

of the same species). 

Section III-F-5. Those that consist entirely of DNA segments from different species that exchange DNA by 

known physiological processes, though one or more of the segments may be a synthetic equivalent. A list of 

such exchangers will be prepared and periodically revised by the NIH Director with advice of the RAC after 

appropriate notice and opportunity for public comment (see Section IV-C-1-b-(1)-(c), Major Actions). See 

Appendices A-I through A-VI, Exemptions Under Section III-F-5--Sublists of Natural Exchangers, for a list of 

natural exchangers that are exempt from the NIH Guidelines. 

Section III-F-6. Those that do not present a significant risk to health or the environment (see Section IV-C-1-b-

(1)-(c), Major Actions), as determined by the NIH Director, with the advice of the RAC, and following appropriate 

notice and opportunity for public comment. See Appendix C, Exemptions under Section III-F-6 for other classes 

of experiments which are exempt from the NIH Guidelines. 

***********************************************************************************************************************
Page 21 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

SECTION IV. ROLES AND RESPONSIBILITIES 

Section IV-A. Policy 

The safe conduct of experiments involving recombinant DNA 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 DNA cannot be foreseen. Therefore, 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. Each institution (and the Institutional Biosafety Committee acting on its behalf) is responsible for 

ensuring that all recombinant DNA research conducted at or sponsored by that institution is conducted in 

compliance with the NIH Guidelines. General recognition of institutional authority and responsibility properly 

establishes accountability for safe conduct of the research at the local level. The following roles and 

responsibilities constitute an administrative framework in which safety is an essential and integral part of 

research involving recombinant DNA molecules. 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 DNA research which is 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: 

Section IV-B-1-a. Establish and implement policies that provide for the safe conduct of recombinant DNA 

research and that ensure compliance with the NIH Guidelines. As part of its 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 DNA research at Biosafety Level (BL) 3 or BL4, or (ii) 

engages in large-scale (greater than 10 liters) research. 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 DNA research that requires Institutional Biosafety Committee approval in accordance with 

Appendix P, Physical and Biological Containment for Recombinant DNA Research Involving Plants. 

Section IV-B-1-e. Appoint at least one individual with expertise in animal containment principles (who is a 

member of the Institutional Biosafety Committee) if the institution conducts recombinant DNA research that 

requires Institutional Biosafety Committee approval in accordance with Appendix Q, Physical and Biological 

Containment for Recombinant DNA Research Involving Animals. 

Section IV-B-1-f. Ensure that when the institution participates in or sponsors recombinant DNA research 

involving human subjects: (i) the Institutional Biosafety Committee has adequate expertise and training (using 

ad hoc consultants as deemed necessary), (ii) all aspects of Appendix M have been appropriately addressed by 

the Principal Investigator; and (iii) no research participant shall be enrolled (see definition of enrollment in 

Section I-E-7) in a human gene transfer experiment until the RAC review process has been completed (see 

Appendix M-I-B, RAC Review Requirements), Institutional Biosafety Committee approval has been obtained, 
Page 22 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Institutional Review Board approval has been obtained, and all applicable regulatory authorizations have been 

obtained. Institutional Biosafety Committee approval must be obtained from each institution at which 

recombinant DNA material will be administered to human subjects (as opposed to each institution involved in 

the production of vectors for human application and each institution at which there is ex vivo transduction of 

recombinant DNA material into target cells for human application). 

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 and members, 

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 that laboratory 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 DNA projects; and if appropriate, conduct a health surveillance program for such 

projects. 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 DNA 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 DNA-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 agents handled, active investigation of 

relevant illnesses, and the maintenance of serial serum samples for monitoring 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 are handled. Examples cited in the Laboratory Safety 

Monograph include gastrointestinal disorders and treatment with steroids, immunosuppressive drugs, or 

antibiotics. Workers with such disorders or treatment should be evaluated to determine whether they should be 

engaged in research with potentially hazardous organisms during their treatment or illness. Copies of the 

Laboratory Safety Monograph are available from the Office of Biotechnology Activities, National Institutes of 

Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 

301-496-9838, 301-496-9839 (fax). 

Section IV-B-1-j. Report any significant problems, violations of the NIH Guidelines, or any significant 

research-related accidents and illnesses to NIH/OBA within thirty days, unless the institution determines that a 

report has already been filed by the Principal Investigator or Institutional Biosafety Committee. Reports shall be 

sent to the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, 

MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

Section IV-B-2. Institutional Biosafety Committee (IBC) 

The institution shall establish an Institutional Biosafety Committee whose responsibilities need not be restricted 

to recombinant DNA. 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 be comprised of no fewer than five members 

so selected that they collectively have experience and expertise in recombinant DNA technology and the 

capability to assess the safety of recombinant DNA 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 health and 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 when 

experiments utilizing Appendix P, Physical and Biological Containment for Recombinant DNA Research 
Page 23 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Involving Plants, require prior approval by the Institutional Biosafety Committee. The Institutional Biosafety 

Committee shall include at least one scientist with expertise in animal containment principles when experiments 

utilizing Appendix Q, Physical and Biological Containment for Recombinant DNA Research Involving Animals, 

require Institutional Biosafety Committee prior approval. When the institution conducts recombinant DNA 

research at BL3, BL4, or Large Scale (greater than 10 liters), a Biological Safety Officer is mandatory and shall 

be a member of the Institutional Biosafety Committee (see Section IV-B-3, Biological Safety Officer). When the 

institution participates in or sponsors recombinant DNA research involving human research participants, the 

institution must ensure that: (i) the Institutional Biosafety Committee has adequate expertise and training (using 

ad hoc consultants as deemed necessary); (ii) all aspects of Appendix M have been appropriately addressed by 

the Principal Investigator; (iii) no research participant shall be enrolled (see definition of enrollment in Section IE-7) in a human gene transfer experiment until the RAC review process has been completed (see Appendix M-IB, RAC Review Requirements); and (iv) final IBC approval is granted only after the RAC review process has

been completed (see Appendix M-I-B, RAC Review Requirements). Institutional Biosafety Committee approval 

must be obtained from the institution at which recombinant DNA material will be administered to human 

research participants (rather than the site involved in manufacturing gene transfer products). 

Note: Individuals, corporations, and institutions not otherwise covered by the NIH Guidelines, are encouraged 

to adhere to the standards and procedures set forth in Sections I through IV (see Section IV-D, 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 recombinant DNA 

activities, it is recommended that the Institutional Biosafety Committee: (i) include persons with expertise in 

recombinant DNA technology, biological safety, and physical containment; (ii) include or have available as 

consultants persons knowledgeable in institutional commitments and policies, applicable law, standards of 

professional conduct and practice, community attitudes, and the environment, and (iii) include at least one 

member representing the laboratory technical staff. 

Section IV-B-2-a-(3). The institution shall file an annual report with NIH/OBA which includes: (i) a roster of 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 gene therapy 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 to provide 

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 the Institutional Biosafety Committee's 

response to the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 

750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

Section IV-B-2-b. Functions 

On behalf of the institution, the Institutional Biosafety Committee is responsible for: 
Page 24 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section IV-B-2-b-(1). Reviewing recombinant DNA research conducted at or 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 the containment 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 DNA research; (iii) ensuring that all aspects of Appendix M have been appropriately addressed by 

the Principal Investigator; (iv) ensuring that no research participant is enrolled (see definition of enrollment in 

Section I-E-7) in a human gene transfer experiment until the RAC review process has been completed (see 

Appendix M-I-B, RAC Review Requirements), Institutional Biosafety Committee approval (from the clinical trial 

site) has been obtained, Institutional Review Board approval has been obtained, and all applicable regulatory 

authorizations have been obtained; (v) for human gene transfer protocols selected for public RAC review and 

discussion, consideration of the issues raised and recommendations made as a result of this review and 

consideration of the Principal Investigator’s response to the RAC recommendations; (vi) ensuring that final IBC 

approval is granted only after the RAC review process has been completed (see Appendix M-I-B, RAC Review 

Requirements); and (vii) ensuring compliance with all surveillance, data reporting, and adverse event reporting 

requirements set forth in the NIH Guidelines.

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-2-a, 

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 DNA 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 DNA 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 and references about 

decontamination and emergency plans. The NIH and the Centers for Disease Control and Prevention 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/OBA within 30 

days, unless the Institutional Biosafety Committee determines that a report has already been filed by the 

Principal Investigator. Reports to NIH/OBA shall be sent to the Office of Biotechnology Activities, National 

Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for 

non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

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 (with the advice of the RAC when required) 

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 DNA molecules. 
Page 25 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section IV-B-3-b. The institution shall appoint a Biological Safety Officer if it engages in recombinant DNA 

research at BL3 or BL4. The Biological Safety Officer shall be a member of the Institutional Biosafety 

Committee. 

Section IV-B-3-c. The Biological Safety Officer's duties include, but are not be limited to: 

Section IV-B-3-c-(1). Periodic inspections to ensure that laboratory standards are rigorously followed; 

Section IV-B-3-c-(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 which 

the Biological Safety Officer becomes aware unless the Biological Safety Officer determines that a report has 

already been filed by the Principal Investigator; 

Section IV-B-3-c-(3). Developing emergency plans for handling accidental spills and personnel contamination 

and investigating laboratory accidents involving recombinant DNA research; 

Section IV-B-3-c-(4). Providing advice on laboratory security; 

Section IV-B-3-c-(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 DNA research that requires Institutional Biosafety Committee 

approval in accordance with Appendix P, Physical and Biological Containment for Recombinant DNA Research 

Involving Plants, the institution 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 DNA research that requires Institutional Biosafety Committee 

approval in accordance with Appendix Q, Physical and Biological Containment for Recombinant DNA Research 

Involving Animals, the institution shall appoint at least one individual with expertise in animal containment 

principles (who is a member of the Institutional Biosafety Committee). 

Section IV-B-6. Human Gene Therapy Expertise 

When the institution participates in or sponsors recombinant DNA research involving human subjects, the 

institution must ensure that: (i) the Institutional Biosafety Committee has adequate expertise and training (using 

ad hoc consultants as deemed necessary) and (ii) all aspects of Appendix M, Points to Consider in the Design 

and Submission of Protocols for the Transfer of Recombinant DNA Molecules into One or More Human Subjects 

(Points to Consider) , have been appropriately addressed by the Principal Investigator prior to submission to 

NIH/OBA. 

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 DNA research. A Principal Investigator engaged in human gene transfer research 

may delegate to another party, such as a corporate sponsor, the reporting functions set forth in Appendix M, 

with written notification to the NIH OBA of the delegation and of the name(s), address, telephone, and fax 

numbers of the contact. The Principal Investigator is responsible for ensuring that the reporting requirements 

are fulfilled and will be held accountable for any reporting lapses. 
Page 26 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 DNA 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 the appropriate 

procedures are followed; 

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/OBA, and other appropriate 

authorities (if applicable) within 30 days. Reports to NIH/OBA shall be sent to the Office of Biotechnology 

Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 

20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax); 

Section IV-B-7-a-(4). Report any new information bearing on the NIH Guidelines to the Institutional Biosafety 

Committee and to NIH/OBA (reports to NIH/OBA shall be sent to the Office of Biotechnology Activities, National 

Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for 

non-USPS mail), 301-496-9838, 301-496-9839 (fax); 

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 for handling 

accidental spills and personnel contamination; and 

Section IV-B-7-a-(7). Comply with shipping requirements for recombinant DNA molecules (see Appendix H, 

Shipment, for shipping requirements and the Laboratory Safety Monograph for technical recommendations). 

Section IV-B-7-b. Information to Be Submitted by the Principal Investigator to NIH OBA 

The Principal Investigator shall: 

Section IV-B-7-b-(1). Submit information to NIH/OBA for certification of new host-vector systems;

Section IV-B-7-b-(2). Petition NIH/OBA, with notice to the Institutional Biosafety Committee, for proposed 

exemptions to the NIH Guidelines; 

Section IV-B-7-b-(3). Petition NIH/OBA, with concurrence of the Institutional Biosafety Committee, for approval 

to conduct experiments specified in Sections III-A-1, Major Actions Under the NIH Guidelines, and III-B, 

Experiments that Require NIH/OBA and Institutional Biosafety Committee Approval Before Initiation; 

Section IV-B-7-b-(4). Petition NIH/OBA for determination of containment for experiments requiring 

case-by-case review; and 

Section IV-B-7-b-(5). Petition NIH/OBA for determination of containment for experiments not covered by the 

NIH Guidelines. 

Section IV-B-7-b-(6). Ensure that all aspects of Appendix M have been appropriately addressed prior to 

submission of a human gene transfer experiment to NIH OBA, and provide a letter signed by the Principal 

Investigator(s) on institutional letterhead acknowledging that the documentation being submitted to NIH OBA 

complies with the requirements set forth in Appendix M. No research participant shall be enrolled (see definition 

of enrollment in Section I-E-7) in a human gene transfer experiment until the RAC review process has been 
Page 27 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

completed (see Appendix M-I-B, RAC Review Requirements); IBC approval (from the clinical trial site) has been 

obtained; Institutional Review Board (IRB) approval has been obtained; and all applicable regulatory 

authorization(s) have been obtained. 

For a clinical trial site that is added after the RAC review process, no research participant shall be enrolled (see 

definition of enrollment in Section I-E-7) at the clinical trial site until the following documentation has been 

submitted to NIH OBA: (1) IBC approval (from the clinical trial site); (2) IRB approval; (3) IRB-approved 

informed consent document; (4) curriculum vitae of the principal investigator(s) (no more than two pages in 

biographical sketch format); and (5) NIH grant number(s) if applicable.

Section IV-B-7-c. Submissions by the Principal Investigator to the Institutional Biosafety Committee 

The Principal Investigator shall: 

Section IV-B-7-c-(1). Make an initial determination of the required levels of physical and biological containment 

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 in the 

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 approval or disapproval; 

and 

Section 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 to ensure 

safety, and (ii) the procedures for dealing with accidents; and 

Section 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. Responsibilities of the Principal Investigator During the Conduct of the Research 

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 and 

implementation of containment practices and procedures in writing to the Biological Safety Officer (where 

applicable), Greenhouse/Animal Facility Director (where applicable), Institutional Biosafety Committee, 

NIH/OBA, and other appropriate authorities (if applicable) (reports to NIH/OBA shall be sent to the Office of 

Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, 

MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax); 

Section IV-B-7-e-(3). Correct work errors and conditions that may result in the release of recombinant DNA 

materials; and 
Page 28 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section 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-B-7-e-(5). Comply with reporting requirements for human gene transfer experiments conducted in 

compliance with the NIH Guidelines (see Appendix M-I-C, Reporting Requirements). 

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 their implementation, and 

(iii) their final interpretation. The NIH Director has responsibilities under the NIH Guidelines that involve OBA 

and RAC. OBA's responsibilities under the NIH Guidelines are administrative. Advice from RAC is primarily 

scientific, technical, and ethical. 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 RAC to carry out the responsibilities set forth in Section 

IV-C-2, Recombinant DNA Advisory Committee (RAC membership is specified in its charter and in Section 

IV-C-2); 

Section IV-C-1-a-(3). Establishing and maintaining NIH/OBA to carry out the responsibilities defined in Section 

IV-C-3, Office of Biotechnology Activities; 

Section IV-C-1-a-(4). 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-a-(5). Establishing and convening Gene Therapy Policy Conferences as described in Appendix 

L, Gene Therapy Policy Conferences.

Section IV-C-1-b. Specific Responsibilities 

In carrying out the responsibilities set forth in this section, the NIH Director, or a designee shall weigh each 

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 seek the advice of RAC and provide an opportunity for public 

and Federal agency comment. Specifically, the Notice of Meeting and Proposed Actions shall be published in 

the Federal Register at least 15 days before the RAC meeting. The NIH Director's decision/recommendation (at 

his/her discretion) may be published in the Federal Register for 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 the Federal Register. The RAC and 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; 
Page 29 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section IV-C-1-b-(1)-(c). Promulgating and amending a list of classes of recombinant DNA 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 not present 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 

Institutional Biosafety Committee Approval, RAC Review, and NIH Director Approval Before Initiation; 

Section IV-C-1-b-(1)-(e). Certifying new host-vector systems with the exception of minor modifications of 

already certified systems (the standards and procedures for certification are described in Appendix I-II, 

Certification of Host-Vector Systems). Minor modifications constitute (e.g., those of minimal or no consequence 

to the properties relevant to containment); and 

Section IV-C-1-b-(1)-(f). Adopting other changes in the NIH Guidelines. 

Section IV-C-1-b-(2). Minor Actions 

NIH/OBA shall carry out certain functions as delegated to it by the NIH Director (see Section IV-C-3, Office of 

Biotechnology Activities). Minor Actions (as determined by NIH/OBA in consultation with the RAC Chair and 

one or more RAC members, as necessary) will be transmitted to RAC and 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 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 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-C-1-b-(2)-(f). Approving minor modifications of already certified host-vector systems (the standards 

and procedures for such modifications are described in Appendix I-II, Certification of Host-Vector 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); 

and 

Section IV-C-1-b-(2)-(i). Determining appropriate containment conditions for experiments according to case 

precedents developed under Section IV-C-1-b-(2)-(c). 
Page 30 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section IV-C-2. Recombinant DNA Advisory Committee (RAC) 

The RAC is responsible for carrying out the functions specified in the NIH Guidelines, as well as others specified 

in its charter or assigned by the Secretary of Health and Human Services or the NIH Director. The RAC 

membership and procedures, in addition to those set forth in the NIH Guidelines, are specified in the charter for 

the RAC which is filed as provided in the General Services Administration Federal Advisory Committee 

Management regulations, 41 CFR part 101-6, and is available on the OBA web site, 

http://www4.od.nih.gov/oba/rac/RACcharter2002.pdf. In the event of a conflict between the NIH Guidelines and 

the charter, the charter shall control. 

The RAC will consist of not less than 15 voting members, including the Chair, appointed under the procedures 

of the NIH and the Department of Health and Human Services. The maximum number of voting members will 

be established in the charter of the RAC. At least a majority of the voting members must be knowledgeable in 

relevant scientific fields, e.g., molecular genetics, molecular biology, recombinant DNA research, including 

clinical gene transfer research. At least 4 members of the RAC must be knowledgeable in fields such as public 

health, laboratory safety, occupational health, protection of human subjects of research, the environment, ethics, 

law, public attitudes or related fields. Representatives of the Federal agencies listed in the charter shall serve 

as non-voting members. Nominations for RAC members may be submitted to the Office of Biotechnology 

Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-

7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

All meetings of the RAC shall be announced in the Federal Register, including tentative agenda items, 15 days 

before the meeting. Final agendas, if modified, shall be available at least 72 hours before the meeting. No item 

defined as a Major Action under Section IV-C-1-b-(1) may be added to an agenda following Federal Register

publication. 

RAC shall be responsible for: 

Section IV-C-2-a. Advising the NIH Director on the following actions: (1) Adopting changes in the NIH 

Guidelines. (2) Assigning containment levels, changing containment levels, and approving experiments 

considered as Major Actions under the NIH Guidelines, i.e., the deliberate transfer of a drug resistance trait to 

microorganisms that are not known to acquire the trait naturally, if such acquisition could compromise the use of 

the drug to control disease agents in humans, veterinary medicine, or agriculture. (3) Promulgating and 

amending lists of classes of recombinant DNA 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 not present a significant risk to health or the environment. (4) Certifying new host-vector systems. 

Section IV-C-2-b. Identifying novel human gene transfer experiments deserving of public discussion by the full 

RAC; 

Section IV-C-2-c. Transmitting to the NIH Director specific comments/ recommendations about: (i) a specific 

human gene transfer experiment, or (ii) a category of human gene transfer experiments; 

Section IV-C-2-d. Publicly reviewing human gene transfer clinical trial data and relevant information evaluated 

and summarized by NIH/OBA in accordance with the annual data reporting requirements; 

Section IV-C-2-e. Identifying broad scientific, safety, social, and ethical issues relevant to gene therapy 

research as potential Gene Therapy Policy Conference topics; 

Section IV-C-2-f. Identifying novel social and ethical issues relevant to specific human applications of gene 

transfer and recommending appropriate modifications to the Points to Consider that will provide guidance in the 

preparation of relevant Informed Consent documents; and 

Section IV-C-2-g. Identifying novel scientific and safety issues relevant to specific human applications of gene 

transfer and recommending appropriate modifications to the Points to Consider that will provide guidance in the 

design and submission of human gene transfer clinical trials. 
Page 31 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section IV-C-3. Office of Biotechnology Activities (OBA) 

OBA shall serve as a focal point for information on recombinant DNA 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. OBA shall carry out such other functions as may 

be delegated to it by the NIH Director. OBA's responsibilities include (but are not limited to) the following: 

Section IV-C-3-a. Serving as the focal point for public access to summary information pertaining to human 

gene transfer experiments; 

Section IV-C-3-b. Serving as the focal point for data management of human gene transfer experiments; 

Section IV-C-3-c. Administering the annual data reporting requirements (and subsequent review) for human 

gene transfer experiments (see Appendix M-I-C, Reporting Requirements); 

Section IV-C-3-d. Transmitting comments/recommendations arising from public RAC discussion of a novel 

human gene transfer experiment to the NIH Director. RAC recommendations shall be forwarded to the Principal 

Investigator, the sponsoring institution, and other DHHS components, as appropriate. 

Section IV-C-3-e. Collaborating with Principal Investigators, Institutional Biosafety Committees, Institutional 

Review Boards, and other DHHS components (including FDA and the Office for Human Research Protections), 

to ensure human gene transfer experiment registration compliance in accordance with Appendix M-I, 

Requirements for Protocol Submission, Review, and Reporting-Human Gene Transfer Experiments of the NIH 

Guidelines. 

Section IV-C-3-f. Administering Gene Therapy Policy Conferences as deemed appropriate by the NIH Director 

(see Appendix L, Gene Therapy Policy Conferences). 

Section IV-C-3-g. Reviewing and approving experiments in conjunction with ad hoc experts involving the 

cloning of genes encoding for toxin molecules that are lethal for vertebrates at an LD50 of less than or equal to 

100 nanograms per kilogram body weight in organisms other than Escherichia coli K-12 (see Section 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-3-h. Serving as the executive secretary of RAC; 

Section IV-C-3-i. Publishing in the Federal Register: 

Section IV-C-3-i-(1). Announcements of RAC meetings and tentative agendas at least 15 days in advance 

(Note: If the agenda for a RAC meeting is modified, OBA shall make the revised agenda available to anyone 

upon request in advance of the meeting); 

Section IV-C-3-i-(2). Announcements of Gene Therapy Policy Conferences and tentative agendas at least 15 

days in advance; 

Section IV-C-3-i-(3). Proposed Major Actions (see Section IV-C-1-b-(1), Major Actions) at least 15 days prior to 

the RAC meeting; and 

Section IV-C-3-j. 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-4. Other NIH Components 

Other NIH components shall be responsible for certifying maximum containment (BL4) facilities, inspecting them 

periodically, and inspecting other recombinant DNA facilities as deemed necessary. 
Page 32 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Section IV-D. Voluntary Compliance 

Section IV-D-1. Basic Policy - Voluntary Compliance 

Individuals, corporations, and institutions not otherwise covered by the NIH Guidelines are encouraged to follow 

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 no organizational 

affiliation. For purposes of complying with the NIH Guidelines, an individual intending to carry out research 

involving recombinant DNA 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, some 

modifications 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 the review and approval of a project in 

which the member has a direct financial interest so long as the member 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 the procedures 

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 the institution 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 proprietary data, any public 

notice regarding a request for an exemption or other approval which is designated by the institution 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. General 

In general, the Freedom of Information Act requires Federal agencies to make their records available to the 

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 or confidential.” 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. Members of RAC are “special Government 

employees.” 
Page 33 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 of Information 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 this information (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, 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/OBA along with the records involved to the 

Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, 

Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). These records 

shall be clearly marked as being the property of the institution on loan to NIH solely for the purpose of making a 

determination under the Freedom on Information Act. NIH/OBA will seek a determination from the responsible 

official under DHHS regulations (45 CFR Part 5) as to whether the records involved, (or some portion) will be 

made available to members of the public under the Freedom of Information Act. Pending such a determination, 

the records will be kept separate from NIH/OBA files, will be considered records of the institution and not 

NIH/OBA, and will not be received as part of NIH/OBA files. No copies will be made of such records. 

NIH/OBA 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 the institution or 

to become a part of NIH/OBA files. If the institution instructs NIH/OBA to return the records, no copies or 

summaries of the records will be made or retained by DHHS, NIH, or OBA. The NIH Freedom of Information 

Officer's determination will represent that official's judgment at the time of the determination as to whether the 

records involved (or some portion) would be exempt from disclosure under the Freedom on Information Act if at 

the time of the determination the records were in NIH/OBA 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, with advice of the RAC, may revise the classification for the purposes of the 

NIH Guidelines (see Section IV-C-1-b-(2)-(e), Minor Actions). The revised list of organisms in each risk group is 

reprinted in Appendix 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 judgment on these 

matters as part of his/her responsibility to "make the initial determination of the required levels of physical 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, this judgment 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/OBA as part of NIH/OBA's functions to "provide advice to all within and outside NIH" (see Section IV-C-3, 

). NIH/OBA may request advice from the RAC as part of the RAC's responsibility for "interpreting the NIH 

Guidelines for experiments to which the NIH Guidelines do not specifically assign containment levels" (see 

Section IV-C-1-b-(2)-(f), Minor Actions). 

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 and Biomedical 

Laboratories, 4th Edition, 1999. Copies are available from: Superintendent of Documents, U.S. Government 

Printing Office, Washington, D.C. 20402-9371 (stock # 017-040-00547-4), Phone (202) 512-1800. 
Page 34 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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, Office of Biosafety, 

Atlanta, Georgia 30333. 

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 of plant 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, are restricted 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 a pathogen 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 the geographic area of the research. 

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APPENDIX A. EXEMPTIONS UNDER SECTION III-F-5--SUBLISTS OF NATURAL EXCHANGERS 

Certain specified recombinant DNA molecules that consist entirely of DNA segments from different species that 

exchange DNA by known physiological processes, though one or more of the segments may be a synthetic 

equivalent are exempt from these NIH Guidelines (see Section III-F-5, Exempt Experiments). Institutional 

Biosafety Committee registration is not required for these exempt experiments. A list of such exchangers will be 

prepared and periodically revised by the NIH Director with advice from the RAC after appropriate notice and 

opportunity for public comment (see Section IV-C-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-5 Sublists of Natural Exchangers. Section III-F-5, Exempt Experiments, describes

recombinant DNA 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 Classification 

of Bergey's Manual of Determinative Bacteriology; 8th edition, R. E. Buchanan and N. E. Gibbons, editors, 

Williams and Wilkins Company; Baltimore, Maryland 1984). Although these experiments are exempt, it is 

recommended that they be performed at the appropriate biosafety level for the host or recombinant organism 

(see Biosafety in Microbiological and Biomedical Laboratories, 3rd edition, May 1993, U.S. DHHS, Public Health 

Service, Centers for Disease Control and Prevention, Atlanta, Georgia, and NIH Office of Biosafety, Bethesda, 

Maryland). 

Appendix A-I. Sublist A 

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

Appendix A-V. Sublist E 

One way transfer of Streptococcus mutans or Streptococcus lactis DNA into Streptococcus sanguis
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Appendix A-VI. Sublist F 

Streptococcus sanguis 

Streptococcus pneumoniae

Streptococcus faecalis

Streptococcus pyogenes

Streptococcus mutans

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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 agents that 

may pose theoretical risks if inoculated into humans. Included are lists of representative genera and species 

known to be pathogenic; mutated, recombined, and non-pathogenic species and strains are not considered. 

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; National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 50010, Phone: 

(515) 862-8258. 

A special committee of the American Society for Microbiology will conduct an annual review of this appendix and 

its recommendation for changes will be presented to the Recombinant DNA Advisory Committee as proposed 

amendments to the NIH Guidelines. 

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 for 

which 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) types 1 through 4; and 

recombinant AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene 

product or a toxin molecule and are produced in the absence of a helper virus. A strain of 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. 
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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 agents and 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 or therapeutic 

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, Cl. chauvoei, Cl. haemolyticum, Cl. histolyticum, Cl. novyi, Cl. septicum, Cl. tetani

--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 

--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. chelonei, 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 

--Neisseria gonorrhoeae, N. meningitidis 

--Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. transvalensis

--Rhodococcus equi

--Salmonella including S. arizonae, S. cholerasuis, 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. parahemolyticus, V. vulnificus

--Yersinia enterocolitica
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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. peruvania, L. tropica 

--Loa loa filaria worms

--Microsporidium 

--Naegleria fowleri 

--Necator human hookworms including N. americanus 

--Onchocerca filaria worms including, O. volvulus

--Plasmodium including simian species, P. cynomologi, 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 

--Eastern equine encephalomyelitis virus 

--Venezuelan equine encephalomyelitis vaccine strain TC-83 

--Western equine encephalomyelitis virus 
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Arenaviruses 

--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 (Togaviruses) - Group B Arboviruses 

--Dengue virus serotypes 1, 2, 3, and 4 

--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 

--Human herpesvirus types 6 and 7 

Orthomyxoviruses 

--Influenza viruses types A, B, and C 

--Other tick-borne orthomyxoviruses as listed in the reference source (see Section V-C, Footnotes and 

References of Sections I through IV) 

Papovaviruses 

--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 and Prions) 

and restricted poxviruses including Alastrim, Smallpox, and Whitepox (see Section V-L, Footnotes and 

References of Sections I through IV) 
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Reoviruses - all types including Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus) 

Rhabdoviruses 

--Rabies virus - all strains 

--Vesicular stomatitis virus - laboratory adapted strains including VSV-Indiana, San Juan, and Glasgow 

Togaviruses (see Alphaviruses and Flaviviruses) 

--Rubivirus (rubella) 

Appendix B-III. Risk Group 3 (RG3) Agents 

RG3 agents are associated with serious or lethal human disease for which preventive or therapeutic 

interventions 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

--Francisella tularensis

--Mycobacterium bovis (except BCG strain, see Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents 

Including Chlamydia), M. tuberculosis

--Pasteurella multocida type B -"buffalo" and other virulent strains 

--Rickettsia akari, R. australis, R. canada, R. conorii, R. prowazekii, R. rickettsii, R, siberica, R. tsutsugamushi, 

R. typhi (R. mooseri) 

--Yersinia pestis

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 

--Semliki Forest virus 

--St. Louis encephalitis virus 

--Venezuelan equine encephalomyelitis virus (except the vaccine strain TC-83, see Appendix B-II-D (RG2)) 

--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 

Flaviviruses (Togaviruses) - Group B Arboviruses 

--Japanese encephalitis virus 

--Yellow fever virus 

--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I 

through IV) 
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Poxviruses 

--Monkeypox virus 

Prions 

--Transmissible spongioform encephalopathies (TME) agents (Creutzfeldt-Jacob disease and kuru agents)(see 

Section V-C, Footnotes and References of Sections I through IV, for containment 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 

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

RG4 agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions 

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 

--Machupo virus 

--Sabia 

Bunyaviruses (Nairovirus) 

--Crimean-Congo hemorrhagic fever virus 

Filoviruses 

--Ebola virus 

--Marburg virus 

Flaviruses (Togaviruses) - 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 
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Hemorrhagic fever agents and viruses as yet undefined 

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 of these 

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 that are 

infectious to human cells, e.g., amphotropic and xenotropic strains of murine leukemia virus, a containment level 

appropriate for RG2 human agents is recommended. 

Baculoviruses 

Herpesviruses 

--Herpesvirus ateles 

--Herpesvirus saimiri 

--Marek's disease virus 

--Murine cytomegalovirus 

Papovaviruses 

--Bovine papilloma virus 

--Polyoma virus 

--Shope papilloma 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 less than 

50% of their respective parental viral genome and that have been demonstrated to be free of detectable 

replication competent retrovirus can be maintained, handled, and administered, under BL1 containment. 

*********************************************************************************************************************** 

APPENDIX C. EXEMPTIONS UNDER SECTION III-F-6 

Section III-F-6 states that exempt from these NIH Guidelines are "those that do not present a significant risk to 

health or the environment (see Section IV-C-1-b-(1)-(c), NIH Director--Specific Responsibilities), as determined 

by the NIH Director, with the advice of the RAC, and following appropriate notice and opportunity for public

comment. See Appendix C, Exemptions under Sections III-F-6, for other classes of experiments which are 

exempt from the NIH Guidelines." The following classes of experiments are exempt under Section III-F-6: 

Appendix C-I. Recombinant DNA in Tissue Culture 

Recombinant DNA molecules containing less than one-half of any eukaryotic viral genome (all viruses from a 

single family being considered identical -- see Appendix C-VII-E, Footnotes and References of Appendix C),
Page 43 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

that are propagated and maintained in cells in tissue culture are exempt from these NIH Guidelines with the 

exceptions listed in Appendix C-I-A. 

Appendix C-I-A. Exceptions 

The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section III-A

which require Institutional Biosafety Committee approval, RAC review, and NIH Director approval before 

initiation, (ii) experiments described in Section III-B which require NIH/OBA and Institutional Biosafety 

Committee approval before initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or restricted 

organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G 

and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents, 

(iv) experiments involving the deliberate introduction of genes coding for the biosynthesis of molecules that are 

toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis 

of Molecules Toxic for Vertebrates), and (v) whole plants regenerated from plant cells and tissue cultures are 

covered by the exemption provided they remain axenic cultures even though they differentiate into embryonic 

tissue and regenerate into plantlets. 

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

Experiments which use Escherichia coli K-12 host-vector systems, with the exception of those experiments 

listed in Appendix C-II-A, are exempt from the NIH Guidelines provided that: (i) the Escherichia coli host does 

not contain conjugation proficient plasmids or generalized transducing phages; or (ii) lambda or lambdoid or Ff 

bacteriophages or non-conjugative plasmids (see Appendix C-VII-B, Footnotes and References of Appendix C) 

shall be used as vectors. However, experiments involving the insertion into Escherichia coli K-12 of DNA from 

prokaryotes that exchange genetic information (see Appendix C-VII-C, Footnotes and References of Appendix

C) with Escherichia coli may be performed with any Escherichia coli K-12 vector (e.g., conjugative plasmid). 

When a non-conjugative vector is used, the Escherichia coli K-12 host may contain conjugation-proficient 

plasmids either autonomous or integrated, or generalized transducing phages. For these exempt laboratory 

experiments, Biosafety Level (BL) 1 physical containment conditions are recommended. For large-scale 

fermentation experiments, the appropriate physical containment conditions need be no greater than those for 

the host organism unmodified by recombinant DNA techniques; the Institutional Biosafety Committee can 

specify higher containment if deemed necessary. 

Appendix C-II-A. Exceptions 

The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section III-A

which require Institutional Biosafety Committee approval, RAC review, and NIH Director approval before 

initiation, (ii) experiments described in Section III-B which require NIH/OBA and Institutional Biosafety 

Committee approval before initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or restricted 

organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G 

and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents 

may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety 

Committee review and approval, (iv) large-scale experiments (e.g., more than 10 liters of culture), and (v) 

experiments involving the cloning of toxin molecule genes coding for the biosynthesis of molecules toxic for 

vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of 

Molecules Toxic for Vertebrates). 

Appendix C-III. Saccharomyces Host-Vector Systems 

Experiments involving Saccharomyces cerevisiae and Saccharomyces uvarum host-vector systems, with the 

exception of experiments listed in Appendix C-III-A, are exempt from the NIH Guidelines. For these exempt 

experiments, BL1 physical containment is recommended. For large-scale fermentation experiments, the 

appropriate physical containment conditions need be no greater than those for the host organism unmodified by 

recombinant DNA techniques; the Institutional Biosafety Committee can specify higher containment if deemed 

necessary. 
Page 44 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Appendix C-III-A. Exceptions 

The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section III-A

which require Institutional Biosafety Committee approval, RAC review, and NIH Director approval before 

initiation, (ii) experiments described in Section III-B which require NIH/OBA and Institutional Biosafety 

Committee approval before initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or restricted 

organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G 

and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents 

may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety 

Committee review and approval, (iv) large-scale experiments (e.g., more than 10 liters of culture), and (v) 

experiments involving the deliberate cloning of genes coding for the biosynthesis of molecules toxic for 

vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of 

Molecules Toxic for Vertebrates). 

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

Any asporogenic Bacillus subtilis or asporogenic Bacillus licheniformis strain which does not revert to a sporeformer with a frequency greater than 10-7 may be used for cloning DNA with the exception of those experiments 

listed in Appendix C-IV-A, Exceptions. For these exempt laboratory experiments, BL1 physical containment 

conditions are recommended. For large-scale fermentation experiments, the appropriate physical containment 

conditions need be no greater than those for the host organism unmodified by recombinant DNA techniques; the 

Institutional Biosafety Committee can specify higher containment if it deems necessary. 

Appendix C-IV-A. Exceptions 

The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section III-A

which require Institutional Biosafety Committee approval, RAC review, and NIH Director approval before 

initiation, (ii) experiments described in Section III-B which require NIH/OBA and Institutional Biosafety 

Committee approval before initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or restricted 

organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G 

and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents 

may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety 

Committee review and approval, (iv) large-scale experiments (e.g., more than 10 liters of culture), and (v) 

experiments involving the deliberate cloning of genes coding for the biosynthesis of molecules toxic for 

vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of 

Molecules Toxic for Vertebrates). 

Appendix C-V. Extrachromosomal Elements of Gram Positive Organisms 

Recombinant DNA molecules derived entirely from extrachromosomal elements of the organisms listed below 

(including shuttle vectors constructed from vectors described in Appendix C), propagated and maintained in 

organisms listed below are exempt from these NIH Guidelines. 

Bacillus amyloliquefaciens

Bacillus amylosacchariticus 

Bacillus anthracis 

Bacillus aterrimus 

Bacillus brevis 

Bacillus cereus 

Bacillus globigii 

Bacillus licheniformis 

Bacillus megaterium 

Bacillus natto 

Bacillus niger 

Bacillus pumilus 

Bacillus sphaericus 

Bacillus stearothermophilis 

Bacillus subtilis 

Bacillus thuringiensis 
Page 45 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Clostridium acetobutylicum 

Lactobacillus casei 

Listeria grayi 

Listeria monocytogenes

Listeria murrayi

Pediococcus acidilactici 

Pediococcus damnosus 

Pediococcus pentosaceus 

Staphylococcus aureus 

Staphylcoccus carnosus 

Staphylococcus epidermidis 

Streptococcus agalactiae 

Streptococcus anginosus 

Streptococcus avium 

Streptococcus cremoris 

Streptococcus dorans 

Streptococcus equisimilis 

Streptococcus faecalis 

Streptococcus ferus 

Streptococcus lactis 

Streptococcus ferns 

Streptococcus mitior 

Streptococcus mutans 

Streptococcus pneumoniae 

Streptococcus pyogenes 

Streptococcus salivarious 

Streptococcus sanguis 

Streptococcus sobrinus 

Streptococcus thermophylus 

Appendix C-V-A. Exceptions 

The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section III-A

which require Institutional Biosafety Committee approval, RAC review, and NIH Director approval before 

initiation, (ii) experiments described in Section III-B which require NIH/OBA and Institutional Biosafety 

Committee approval before initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or restricted 

organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G 

and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents 

may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety 

Committee review and approval, (iv) large-scale experiments (e.g., more than 10 liters of culture), and (v) 

experiments involving the deliberate cloning of genes coding for the biosynthesis of molecules toxic for 

vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of 

Molecules Toxic for Vertebrates). 

Appendix C-VI. The Purchase or Transfer of Transgenic Rodents 

The purchase or transfer of transgenic rodents for experiments that require BL1 containment (See Appendix GIII-M, Footnotes and References of Appendix G) are exempt from the NIH Guidelines. 

Appendix C-VII. Footnotes and References of Appendix C 

Appendix C-VII-A. The NIH Director, with advice of the RAC, may revise the classification for the purposes of 

these NIH Guidelines (see Section IV-C-1-b-(2)-(b), Minor Actions). The revised list of organisms in each Risk 

Group is located in Appendix B. 
Page 46 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Appendix C-VII-B. A subset of non-conjugative plasmid vectors are poorly mobilizable (e.g., pBR322, 

pBR313). Where practical, these vectors should be employed. 

Appendix C-VII-C. Defined as observable under optimal laboratory conditions by transformation, transduction, 

phage infection, and/or conjugation with transfer of phage, plasmid, and/or chromosomal genetic information. 

Note that this definition of exchange may be less stringent than that applied to exempt organisms under Section 

III-F-5, Exempt Experiments. 

Appendix C-VII-D. As classified in the Third Report of the International Committee on Taxonomy of Viruses: 

Classification and Nomenclature of Viruses, R. E. F. Matthews (ed.), Intervirology 12 (129-296), 1979. 

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

*********************************************************************************************************************** 

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 with regard to 

the NIH Guidelines after the issues have been considered by the 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 under BL1 + EK1 

conditions. 

Appendix D-3. The Rd strain of Hemophilus influenzae can be used as a host for the propagation of the cloned 

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 disease virus 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 RAC. 

Appendix D-7. Attenuated laboratory strains of Salmonella typhimurium may be used under BL1 physical 

containment conditions to screen for the Saccharomyces cerevisiae pseudouridine synthetase gene. The 

plasmid YEp13 will be employed as the vector. 

Appendix D-8. Permission is granted to transfer certain clones of subgenomic segments of foot and mouth 

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 RAC. 

Appendix D-9. Permission is granted to Dr. John Sanford of Cornell University to field test tomato and tobacco 

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 University of 

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. 
Page 47 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Appendix D-11. Agracetus of Middleton, Wisconsin, may field test under specified conditions disease 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, and reinfused 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 of patients 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 conduct certain 

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 of laboratory 

personnel. 

Appendix D-15. Drs. R. Michael Blaese and W. French Anderson of the NIH, Bethesda, Maryland, can conduct 

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 will then 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 CD-34+-

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 to transduce 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, can conduct 

experiments on patients with advanced melanoma who have failed all effective therapy. These patients 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 infiltrating lymphocytes (TIL) transduced with the 

gene coding for TNF. (Protocol #9007-003) 
Page 48 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 for neomycin resistance. The purpose of 

this gene marking experiment is to determine whether the source of 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 source of 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 marrow for 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 the source of relapse after autologous bone marrow 

transplantation is residual malignant cells in the harvested 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, can conduct 

experiments on patients with chronic myelogenous leukemia who have been reinduced into a second chronic 

phase or blast cells. The patients in these studies will receive autologous bone marrow transplantation. 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 bone marrow 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 transduced with 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 of engraftment. (Protocol #9105-008) 

Appendix D-21. Dr. Steven A. Rosenberg of the National Institutes of Health, Bethesda, Maryland, can conduct 

experiments on patients with advanced melanoma, renal cell cancer, and colon carcinoma who have failed all 

effective therapy. In an attempt to increase these patients' immune responses to the tumor, 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 the thigh 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 #9110-010/9110-011) 

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 for engraftment of the these treated hepatocytes 

through a series of metabolic studies; three months after gene therapy, a liver biopsy will be taken and analyzed 

for the presence of recombinant derived RNA and DNA to document the presence of the gene coding for the 

normal LDL receptor. (Protocol #9110-012) 
Page 49 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 gene marking 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 and survival 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 the expression of this antigen will be 

characterized. These experiments will be analyzed for their efficacy in treating cancer. (Protocol #9202-013) 

Appendix D-25. Kenneth Cornetta of Indiana University, Indianapolis, Indiana, can conduct gene transfer 

experiments on up to 10 patients with acute myelogenous leukemia (AML) and up to 10 patients with acute 

lymphocytic leukemia (ALL). The patient population will be limited to persons between 18 and 65 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 bone marrow transplantation. Patients 

will then be monitored for evidence of the neomycin resistance gene in peripheral blood and bone marrow cells 

in order to determine whether relapse of their disease is a result of 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 the bone marrow is a necessary procedure for these 

leukemia patients. Further studies will be performed in order to determine the percentage of leukemic cells that 

contain the LNL-6 vector and the clonality of the marked cells. (Protocol #9202-014) 

Appendix D-26. Dr. James S. Economou of the University of California, Los Angeles, can conduct gene 

transfer 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 alpha interferon 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 tumor-infiltrating lymphocytes and peripheral 

blood lymphocytes is designed to answer questions about the trafficking 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 Research Center, 

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 marrow transplantation 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 the hygromycin 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 the event that patients develop side effects as a 

consequence of CTL therapy. Data will be correlated over time, 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-specific CTL clones, and determining if ganciclovir therapy can eradicate 

genetically modified, adoptively transferred CTL cells. (Protocol #9202-017) 
Page 50 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 who have 

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, and these gene-modified 

cells will be injected subcutaneously in a Phase 1 dose escalation trial. Patients will be evaluated for an antitumor 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 with primary 

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 the age of 18. 

Patients will be divided into two groups based on the surgical accessibility of their lesions. Both surgically 

accessible 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 the tumor bed will be 

infiltrated with the HS-tk producer cells. The removed tumor will be evaluated for the efficiency of transduction. 

Ganciclovir (GCV) will be administered beginning on the fifth postoperative day. In the case of surgically 

inaccessible lesions, the patients will receive intravenous therapy with GCV seven days after receiving the intratumoral 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 conduct gene 

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 resistance will 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 the leukemic 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, can conduct 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 of growth factors to examine 

hematopoietic reconstitution after bone marrow transplantation. The efficiency of 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 cell 

transplantation. 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 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 metastatic melanoma 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 human Interleukin-2 (IL-2) can be injected 

subcutaneously and used to create a potent tumor specific immune response without producing toxicity. By 
Page 51 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

allowing the tumor cells to present the MHC Class I molecule as well 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 human Interleukin-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 as well 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 cell line 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 gene coding for neomycin resistance (neoR

) using 

the retroviral vector, LN. Patients will be continuously monitored for neoR

 to determine the relative contribution 

of autologous PBRCs to long-term hematopoietic reconstitution. Demonstration of long-term contribution of 

autologous PBRC to hematopoiesis will enable the use of PBRC alone for autologous transplants and suggest 

the use of PBRC as long-term carriers of therapeutically relevant genes. (Protocol #9209-027/9209-028) 

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 twin that 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 long-term bone marrow engraftment. Demonstration of long-term 

contribution of allogeneic PBRC to hematopoiesis 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) 

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 resistance into 

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 of recurrent, 

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 
Page 52 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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-TK- cells when exposed to 

ganciclovir therapy. Patients will be evaluated for safety and side effects of this treatment. (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 initial administration 

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) in vivo safety and efficacy of the administration of 

AdCFTR into the respiratory epithelium; (2) efficacy of the correction 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 of age 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 be limited 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 is observed 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 this treatment will be eligible for 

additional VPC injections and subsequent GCV therapy. (Protocol #9303-037) 

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 of the 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 who will be 

recipients of mismatched-related or phenotypically similar unrelated donor marrow grafts for leukemia. In this 

Phase I dose escalation study, spontaneous lymphoblastoid cell lines will be established 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 of bone marrow transplant. This study will 

determine: (1) survival and expansion of these EBV-specific cell lines 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 
Page 53 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

(CFTR) mRNA in vivo, (2) synthesis of CFTR protein, and (3) correction of epithelial cell cAMP dependent Clpermeability. 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 recombination or 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-incompetent adenovirus 

vector, Ad.CΒ-CFTR using a bronchoscope for gene delivery. Ad.CB-CFTR contains the human 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, immunological responses 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 will be 

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 of generating 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 of tumor-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 human glucocerebrosidase and 

administered intravenously. Patients will be monitored for toxicity and glucocerebrosidase 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(+) 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 gene transfer of plasmid DNA. Patients will be 

monitored for survival of the transduced CD4(+) cells by polymerase 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 vivo transduction 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 be administered 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 #9306-045) 

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 monitored by 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 and interleukin-2. 
Page 54 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

The enriched population of cells will be transduced with either LNL6 or G1Na, which contain the neoR

 gene. 

The transduced cells will be expanded in tissue culture and administered to the HIV-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 will be treated. Patients with 

either surgically accessible or non-accessible tumors will be treated with the vector 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 with unresectable 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 injections of 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 of Physicians 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 be monitored for the presence and 

expression of the MDR-1 gene. The investigators will determine whether MDR-1 expression increases following 

chemotherapy. (Protocol #9306-051) 

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 have achieved a partial 

or complete response to induction chemotherapy. This study will determine the feasibility 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 be instilled 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. 
Page 55 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 to determine the efficiency of bone 

marrow purging and the origin of relapse following autologous bone marrow 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 the retrovirus 

vector, MFG, containing the human granulocyte-macrophage colony stimulating factor gene. Acute and longterm 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 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 and University 

Hospital of Cleveland, Cleveland, Ohio, may conduct experiments on 12 patients (≥ 18 years of age) 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 injected subcutaneously into 

patients in an attempt to express antisense Insulin growth factor-1. Patients will be monitored 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 metastatic melanoma. 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 tumor-specific 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-hIL2 vector (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 tumor-specific 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 antitumor response by magnetic resonance imaging (MRI) and cerebral 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 of 

Medicine, 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 with the human 
Page 56 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 of antitumor 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 will administer increasing 

doses of either of the two adenovirus vectors, AD2/CFTR-1 or AD2-ORF6/PGK-CFTR, to the nasal epithelium of 

10 patients (1 nostril) or maxillary sinus epithelium of 10 patients (1 maxillary 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 the safety 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 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 and antibody responses in order to eliminate HIVinfected 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 by 

lipofection with the plasmid DNA vector, CMV-B7, derived from bovine papilloma virus to express the human B7 

antigen. The B7 antigen, which binds to the CD28 receptor of T cells, will serve as a co-stimulatory signal to 

elicit an antitumor immune response. Subjects will be monitored for induction of cytotoxic 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 β-2 microglobulin. 

Subjects must be HLA-B7 negative. The objective of this study is to determine a safe and 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 conduct gene 

marking experiments on 9 subjects with ovarian carcinoma or peritoneal carcinomatosis (≥ 16 years of age). 

Autologous CD3(+)/CD8(+) tumor infiltrating lymphocyte derived T cells will be transduced with the 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) 
Page 57 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 of age). The distinguishable 

retroviral vectors, LNL6 and G1Na (both encoding neoR

), will be used to determine 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 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 the safety 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 to determine whether 

autologous FACC transduced hematopoietic progenitor cells can be safely administered to subjects, the extent 

of engraftment, and correction of cell phenotype. (Protocol #9406-078) 

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 multi-forme (≥ 18 years 

of age). Subjects will receive subcutaneous injections of autologous tumor cells that have been lethally 

irradiated and transduced with the retroviral vector, G1NaCvi2.23, which encodes for interleukin-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 that have 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 immunological reactivities 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 conduct gene 

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

 HIVspecific CD8(+) cells. The investigator will: (1) Assess the safety and tolerance of the adoptive transfer of antiHIV cytotoxic, syngeneic, CD8(+) peripheral blood lymphocytes that 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 immunized with autologous or allogeneic 

neuroblastoma cells transduced to express γ interferon. Cells will be transduced 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 and immunocytologic evaluation of marrow metastases. (Protocol 

#9403-068) 
Page 58 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Appendix D-82. Dr. Kenneth L. Brigham of Vanderbilt University, Nashville, Tennessee, may conduct gene 

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 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 course of 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 will 

assess 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 irradiated tumor 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 of California 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 ribozyme that cleaves the HIV-1 RNA 

in the 5' leader sequence. The transduced cells will be expanded and reinfused into the patients. The 

objectives of the study are: (1) to evaluate safety of reinfusing the transduced lymphocytes, (2) to compare (in 

vivo) the kinetics and survival of ribozyme-transduced cells with 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 gene therapy on in vivo HIV mRNA expression, viral burden and CD4(+) 

lymphocyte levels. (Protocol #9309-057) 

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 with recombinant 

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 human glucocerebrosidase 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 adenoassociated virus (AAV) derived vector, encoding cystic fibrosis transmembrane conductance regulator (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 of age) with 

peripheral artery disease (PAD). A plasmid DNA vector, phVEGF165, encoding the human gene for vascular 

endothelial growth factor (VEGF) will be used to express VEGF to induce collateral neovascularization. 
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Percutaneous arterial gene transfer will be achieved using an angioplasty catheter with a hydrogel coated 

balloon to deliver the plasmid DNA vector to the artery. The objectives of the study 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 the safety 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 determined by 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 gene transfer 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) with metastatic 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 c-myc 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 sequences in 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 gene transfer 

experiments on 14 non-small cell lung cancer subjects (≥ 18 and ≤ 80 years of age) who have failed 

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 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) with metastatic 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 
Page 60 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

subcutaneous injections of lethally irradiated autologous tumor cells. The objectives of 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 conduct gene 

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 injections of 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 and systemic 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 human gene 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-specific immunity. 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 and pharmacokinetics; and (4) 

determine whether IL-2 expression stimulates tumor regression in subjects with metastatic 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, transduced with 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/or transdominant 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 treatment using identical or different combinations of control and anti-HIV 

retrovirus vectors. The relative survival of these transduced T cell populations will be monitored by vectorspecific 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 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) with rheumatoid 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 the rheumatoid 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 the human 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 joint material 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) 
Page 61 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 and transduced 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 with interleukin-2 and anti-CD3 

monoclonal antibody. T cell depleted lymphocytes will be transduced with the retroviral 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 cell-mediated 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 human immunodeficiency 

virus type-1 (HIV-1). Autologous lymphocytes from asymptomatic subjects will be transduced 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 survival of 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 E4-deleted, replication-deficient, 

type 2 adenovirus vector, AD2/CFTR-2, will be used to deliver the human cystic fibrosis transmembrane 

conductance regulator (CFTR) gene by aerosol administration (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 Research Institute, 

Des Moines, Iowa, may conduct gene transfer experiments on 24 female subjects (≥ 18 years of age) with 

refractory or recurrent ovarian cancer. Subjects will undergo intraperitoneal delivery (via Tenkhoff 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 antiviral drug, 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) 
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Appendix D-103. Dr. David T. Curiel of the University of Alabama, Birmingham, Alabama, may conduct gene 

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 of the 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 be transduced 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 for potential 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 of California, Los 

Angeles, California, may conduct gene transfer experiments on 25 subjects (≥ 18 years of age) with metastatic 

melanoma. The protocol is an open label, Phase I trial to evaluate the safety and immunological 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 IL7 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 #9503-101) 

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). 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 to demonstrate 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 (human osteosarcoma cell line with 

homozygous p53 deletion) at an MOI of 50:1 (titer > 5 x 1010) on day 5 of culture. 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 luciferase gene) 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) 
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Appendix D-107A. Dr. Gary Clayman. M.D. Anderson Cancer Center, Houston, Texas, may conduct gene 

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 human cytomegalovirus promoter (CMV). 

Subjects will be divided into 2 treatment groups: (1) those with non-resectable 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 (human osteosarcoma cell line with 

homozygous p53 deletion) at an MOI of 50:1 (titer > 5x1010) on day 5 of culture. 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 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 #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 be surgically 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 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 antitumor effector T-cells 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 of this Phase I study are to: (1) quantitatively 

assess the uptake and expression of XM6:MMTV-antisense c-myc by prostate cancer cells in vivo, (2) 

determine whether c-myc gene expression is prostate tumor-specific, (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 HospitalCornell 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. 5-fluorocytosine (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 
Page 64 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

administration of AdGVCD.10 to hepatic metastases combined with oral 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 will be 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 or abdomen) 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 of age) 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 a palliative debulking procedure 

will receive the same treatment followed by resection on day 7, and a second dose of the vector intraoperatively. Brain tissues removed by resection will be analyzed for adenovirus infection, transgene expression, 

and signs of inflammation. The size and metabolic activity of tumors 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 

refractive central nervous system malignancies. Subjects will receive stereotaxic injections of a replicationdefective, 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 a Rous 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 ovarian carcinoma. 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 the HER-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 the intraperitoneal or intrapleural route. The objectives of the study are: (1) to 

determine E1A gene transduction 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 

the toxicity and tolerance of E1A/lipid complex administered into the pleural or peritoneal space, and to assess 

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 at the time of clinically indicated 
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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 third cohort, 2 x 107 cells. The results of this Phase I trial 

will be used to assess the safety of this form of gene therapy and may provide preliminary data to evaluate the 

potential utility of TGF- β2 antisense gene therapy 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 distant compartment (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. (Protocol #9701-171) 

*********************************************************************************************************************** 

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 of these 

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 on these 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. 
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Appendix E-III. Escherichia coli

Appendix E-III-A. 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 the sterile 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: 

Vector Host

λgt WESλB' DP50supF 

λgt WESλB* DP50supF 

λgt ZJ virλB' Escherichia coli K-12 

λgtALO·λB DP50supF 

Charon 3A DP50 or DP50supF 

Charon 4A DP50 or DP50supF 

Charon 16A DP50 or DP50supF 

Charon 21A DP50supF 

Charon 23A DP50 or DP50supF 

Charon 24A 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 VERTEBRATES 

Appendix F-I. General Information 

Appendix F specifies the containment to be used for the deliberate cloning of genes coding for the biosynthesis 

of molecules toxic for vertebrates. The cloning of genes coding for molecules toxic for vertebrates that have an 

LD50 of < 100 nanograms per kilograms body weight (e.g., microbial toxins such as 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/OBA approval 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/OBA prior to initiating the experiments. A list of toxin molecules classified as to LD50 is 

available from NIH/OBA. Testing procedures for determining toxicity of toxin molecules not on the list are 

available from the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 

750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

The results of such tests shall be forwarded to NIH/OBA, 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 under BL1 + 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, and certain neurotoxins 

present in snake venoms and other venoms). 

Appendix F-II-C. Some enterotoxins are substantially more toxic when administered enterally than 

parenterally. 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 of Escherichia 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/OBA 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 DNA containing genes coding 

for toxins lethal for vertebrates at an LD50 of <100 nanograms per kilogram body weight is available from the 

Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, 

Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

*********************************************************************************************************************** 
Page 68 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 DNA Molecules) supersedes 

Appendix G. Appendix K defines Good Large Scale Practice through Biosafety Level 3 - Large Scale. For 

certain work with plants, Appendix P (Physical and Biological Containment for Recombinant DNA Research 

Involving Plants) supersedes Appendix G. Appendix P defines Biosafety Levels 1 through 4 - Plants. For 

certain work with animals, Appendix Q (Physical and Biological Containment for Recombinant DNA Research 

Involving Animals) supersedes Appendix G. Appendix Q 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 G-III-A

through G-III-J, Footnotes and References of Appendix G). Consequently, all personnel directly or indirectly

involved in experiments using recombinant DNA shall receive adequate instruction (see Sections IV-B-1-h, 

Responsibilities of the Institution--General Information, and IV-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 the experiments 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 the environment. The 

Principal Investigator shall ensure that everyone in the laboratory is familiar with both the 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. Serological monitoring, when clearly appropriate, will be 

provided (see Section IV-B-1-f, Responsibilities of the Institution--General Information). 

The Laboratory Safety Monograph (see Appendix G-III-O, Footnotes and References of Appendix G) and 

Biosafety in Microbiological and Biomedical Laboratories (see Appendix G-III-B, Footnotes and References 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 DNA molecules and to 

reduce the potential for exposure of the laboratory worker, persons outside of the laboratory, and the 

environment to organisms containing recombinant DNA molecules. Physical containment is achieved through 

the use of laboratory practices, containment equipment, and special laboratory design. Emphasis is placed on 

primary means of physical containment which are provided by laboratory practices and containment equipment. 

Special laboratory design provides a secondary 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 of moderate to high potential hazard are performed. 

Combinations of laboratory practices, containment equipment, and special laboratory design can be made to 

achieve different levels of physical containment. Four levels of physical containment, which are designated as 

BL1, BL2, BL3, and BL4 are described. It should be emphasized that the descriptions and assignments of 

physical containment detailed below are based on existing approaches to containment of pathogenic organisms 

(see Appendix G-III-B, Footnotes and References of Appendix G). The National Cancer Institute describes 

three levels for research on oncogenic viruses which roughly correspond to our BL2, 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, and special 

laboratory design may be appropriate for containment of specific research activities. The NIH Guidelines, 

therefore, allow alternative selections of primary containment equipment within facilities that have been 

designed to provide BL3 and BL4 levels of physical containment. The selection of alternative methods of 
Page 69 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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

Appendix 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 viable material. 

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 work area. 

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 DNA molecules and animals, and (ii) before exiting the laboratory. 

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 that are appropriate 

for the risk of exposure to viable organisms containing recombinant DNA 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 the laboratory 

are placed in a durable leak-proof container which is closed before being removed from the laboratory. 

Appendix G-II-A-2-b. An insect and rodent control program is in effect. 

Appendix G-II-A-3. Containment Equipment (BL1) 

Appendix G-II-A-3-a. Special containment equipment is generally not required for manipulations of agents 

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. Biosafety Level 2 (BL2) (See Appendix G-III-N, Footnotes and References of Appendix G) 
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Appendix 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 when work 

with organisms containing recombinant DNA molecules is in progress. 

Appendix G-II-B-1-b. Work surfaces are decontaminated at least once a day and after any spill of viable 

material. 

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 work area. 

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 DNA molecules and animals, and (ii) when exiting the laboratory. 

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 the laboratory 

are placed in a durable leak-proof container which is closed before being removed from the laboratory. 

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 enter or 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., immunization) 

may enter the laboratory or animal rooms. 

Appendix G-II-B-2-d. When the organisms containing recombinant DNA 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 the laboratory. 

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), this protective 

clothing is removed and left in the laboratory or covered with a clean coat not used in the laboratory. 

Appendix G-II-B-2-g. Animals not involved in the work being performed are not permitted in the laboratory. 

Appendix G-II-B-2-h. Special care is taken to avoid skin contamination with organisms containing recombinant 

DNA 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 decontaminated before 

disposal. 
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Appendix G-II-B-2-j. Hypodermic needles and syringes are used only for parenteral injection and aspiration of 

fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable 

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 DNA molecules. Extreme caution should be used when 

handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. 

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 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 

DNA molecules are immediately reported to the Institutional Biosafety Committee and NIH/OBA. Reports to 

NIH/OBA shall be sent to the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge 

Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-

9839 (fax). Medical evaluation, surveillance, and treatment 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 for 

laboratory and other at-risk personnel are collected and stored. Additional serum specimens may be collected 

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 and References 

of Appendix G) or other appropriate personal protective or physical containment devices are used whenever: 

Appendix G-II-B-3-a-(1). Procedures with a high potential for creating aerosols are conducted (see Appendix 

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 internal pressures 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 DNA 

molecules are used. Such materials may be centrifuged in the open laboratory if sealed 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, and equipment 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. Biosafety Level 3 (BL3) (See Appendix G-III-P, Footnotes and References of Appendix G)

Appendix G-II-C-1. Standard Microbiological Practices (BL3) 
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Appendix G-II-C-1-a. Work surfaces are decontaminated at least once a day and after any spill of viable 

material. 

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 DNA 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 physical containment, 

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. 

Appendix G-II-C-2-b. Contaminated materials that are to be decontaminated at a site away from the laboratory 

are placed in a durable leak-proof container which is closed before being removed from the laboratory. 

Appendix G-II-C-2-c. The Principal Investigator controls access to the laboratory and restricts access to 

persons 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 the laboratory. 

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 animal rooms. 

Appendix G-II-C-2-e. When organisms containing recombinant DNA molecules or experimental 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 the laboratory such as the need for immunizations, respirators, or other 

personal protective measures. 

Appendix G-II-C-2-f. All activities involving organisms containing recombinant DNA molecules are conducted 

in biological safety cabinets or other physical containment devices within the containment 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 DNA molecules is finished. Plastic-backed 

paper toweling used on non-perforated work surfaces within biological 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-around gowns, 

scrub suits, coveralls) is worn in the laboratory. Laboratory clothing is not worn outside the laboratory, 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. 
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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 the 

laboratory. 

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 or solid-wall and -bottom 

cages placed on holding racks equipped with ultraviolet in radiation lamps and reflectors. 

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 devices are 

required. 

Appendix G-II-C-2-n. All wastes from laboratories and animal rooms are appropriately decontaminated before 

disposal. 

Appendix G-II-C-2-o. Vacuum lines are protected with high efficiency particulate air/HEPA filters and liquid 

disinfectant traps. 

Appendix G-II-C-2-p. Hypodermic needles and syringes are used only for parenteral injection and aspiration of 

fluids from laboratory animals and diaphragm bottles. Only needle locking syringes or disposable 

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 DNA molecules. Extreme caution should be used when 

handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. 

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 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 DNA molecules are immediately reported to the Biological Safety Officer, Institutional Biosafety 

Committee, and NIH/OBA. Reports to NIH/OBA shall be sent to the Office of Biotechnology Activities, National 

Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for 

non-USPS mail), 301-496-9838, 301-496-9839 (fax). Appropriate medical evaluation, surveillance, and 

treatment are provided and written records are maintained. 

Appendix G-II-C-2-r. Baseline serum samples for all laboratory and other at-risk personnel should be collected 

and stored. 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 biological 

containment 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 physical containment. 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. Containment Equipment (BL3) 

Appendix G-II-C-3-a. Biological safety cabinets (Class I, II, or III) (see Appendix G-III-L, Footnotes and 

References of Appendix G) or other appropriate combinations of personal protective or physical containment 

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 

DNA molecules which pose a threat of aerosol exposure. These include: manipulation of cultures and of those 

clinical or environmental materials which may be a source 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 the laboratory 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-doored 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, and equipment 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 through the entry area. The exhaust air is not recirculated to any other 

area of the building, is discharged to the outside, and is dispersed away from the occupied areas and air 

intakes. Personnel shall verify that the direction of the airflow (into the laboratory) is proper. The exhaust air 

from the laboratory room may be discharged to the outside without being filtered or otherwise treated. 

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. Exhaust 

air from Class I or II biological safety cabinets may be recirculated within the laboratory if the cabinet 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 or building exhaust system. 
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Appendix G-II-D. Biosafety Level 4 (BL4) 

Appendix G-II-D-1. Standard Microbiological Practices (BL4) 

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 this purpose. 

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 high temperatures 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 is required 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 other applicable entry and exit procedures. A logbook signed by all 

personnel indicates the date and time of each 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 the laboratory 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 jump suits, shoes, and 

gloves, is provided and used by all personnel entering the facility. Head covers are provided 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 a locker or hamper in the inner change 

room. Protective clothing shall be decontaminated prior to laundering or disposal. 

Appendix G-II-D-2-f. When materials that contain organisms containing recombinant DNA 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 the agent, 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). 

Appendix G-II-D-2-g. Supplies and materials needed in the facility are brought in by way of the double-doored 

autoclave, fumigation chamber, or airlock which is appropriately decontaminated between each use. After 

securing the outer doors, personnel within the facility retrieve the materials by opening the interior 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. 
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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 and aspiration of 

fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable 

syringe-needle units (i.e., needle is integral part of unit) are used for the injection or aspiration of fluids 

containing organisms that contain recombinant DNA 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 and decontaminated, preferably by autoclaving before discard or reuse. 

Whenever possible, cannulas are used 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 and accidents which result in 

overt exposures to organisms containing recombinant DNA molecules are immediately reported to the Biological 

Safety Officer, Institutional Biosafety Committee, and NIH/OBA. Reports to the NIH/OBA shall be sent to the 

Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, 

Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). Written records 

are prepared and maintained. An essential adjunct to such a reporting-surveillance system is the availability of 

a facility for quarantine, isolation, and medical care of personnel with potential or known laboratory associated 

illnesses. 

Appendix G-II-D-2-l. Laboratory animals involved in experiments requiring BL4 level physical containment 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 in which all personnel are required to wear one-piece 

positive pressure suits. 

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 biological 

containment than that specified may be conducted in the BL4 facility using containment equipment requirements 

specified for the BL3 level of physical containment. Alternative combinations of containment safeguards are 

shown in Appendix G-Table 1, Possible Alternate Combinations of Physical and 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 are conducted 

in the Class III biological safety cabinet or in Class I or II biological safety cabinets used in conjunction 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 a clearly 

demarcated and isolated zone within a building. Outer and inner change rooms separated by a shower 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 with a 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. 

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. 
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Appendix G-II-D-4-d. Bench tops have seamless surfaces which are impervious to water and resistant to 

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 the door 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. In-line 

high efficiency particulate air/HEPA filters are placed as near as practicable to each use point or service cock. 

Filters are installed to permit in-place decontamination and replacement. Other liquid and gas 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 the facility 

corridors outside the laboratory. The water service to the fountain is not connected to the back-flow 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-doored 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 can be safely 

removed from the facility. 

Appendix G-II-D-4-m. Liquid effluent from laboratory sinks, biological safety cabinets, floors, and autoclave 

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 heat decontamination 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 of demonstrated 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 to sense 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 lifetime of the exhaust HEPA filters and to 

protect the supply air system should air pressures become unbalanced in the laboratory. 
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Appendix G-II-D-4-p. The treated exhaust air from Class I and II biological safety cabinets may be discharged 

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 are tested and certified at 

six-month intervals. 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 any of these cabinets is discharged to the outside through the facility 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 or 

the facility exhaust air system. 

Appendix G-II-D-4-q. A specially designed suit area may be provided in the facility. Personnel who enter 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 is through an airlock fitted 

with airtight doors. A chemical shower is provided to decontaminate the surface of the suit before the worker 

exits the area. The exhaust air from the suit area is filtered by two sets of high efficiency particulate air/HEPA 

filters installed in series. A duplicate filtration unit, exhaust fan, and an automatically starting emergency power 

source are provided. The air pressure within the suit area is greater 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 for decontaminating waste materials to be removed from the suit areas. 

Appendix G - Table 1. Possible Alternate Combinations Of Physical And Biological Containment 

Safeguards 

Classification 

of Physical & 

Biological 

Containment

Alternate Physical Containment

 Laboratory Laboratory Laboratory 

 Facilities Practices Equipment 

Alternate 

Biological 

Containment

 BL3/HV2 

 BL3/HV1 

 BL4/HV1 

 BL3 BL3 BL3 

 BL3 BL3 BL4 

 BL3 BL3 BL3 

 BL3 BL3 BL2 

 BL4 BL4 BL4 

 BL4 BL4 BL3 

HV2 

HV1 

HV1 

HV2 

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, 4th edition, May, 1999, DHHS, 

Public Health Service, Centers for Disease Control and Prevention, Atlanta, Georgia, and National Institutes of 

Health, Bethesda, Maryland. 

Appendix G-III-B. Biosafety in Microbiological and Biomedical Laboratories, 3rd edition, May 1993, U.S. 

DHHS, Public Health Service, Centers for Disease Control and Prevention, Atlanta, Georgia, and NIH, 

Bethesda, Maryland. 

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. 1740-00383, 

1974. 
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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 Rubber Co., 

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 air away 

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 flow of 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 is filtered 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 the cabinet. The cabinet is fitted with arm-length rubber gloves and is operated 

under a negative pressure of at least 0.5 inches water gauge. All supply air is filtered through HEPA filters. 

Exhaust air is filtered through two HEPA filters or one HEPA filter and incinerator before being discharged to the 

outside environment. 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 

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 the procedures conducted in the 

laboratory and are supervised by a scientist with general training in microbiology or a related science (see 

Appendix G-III-B, 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 of moderate 

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) access to 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-B, 

Footnotes and References of Appendix G). 
Page 80 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 have specific training in 

handling pathogenic and potentially lethal agents and are supervised by competent scientists 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, and susceptibility testing) in 

laboratories where facility features satisfy BL2 recommendations provided the recommended "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 DNA molecules contained in an organism or in a viral genome shall be shipped under the 

applicable regulations of the U.S. Postal Service (39 Code of Federal Regulations, Part 3); the Public Health 

Service (42 Code of Federal Regulations, Part 72); the U.S. Department of Agriculture (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 the 

procedures 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 the institution 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 DNA, 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). 

Appendix H-II. Host organisms and viruses will be shipped as etiologic agents if they contain recombinant DNA 

when: (i) the recombinant DNA 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 DNA codes for a toxin or other 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 DNA 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. 
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Appendix H-III. Footnotes and References of Appendix H 

For further information on shipping etiologic agents contact: (i) The Centers for Disease Control and Prevention, 

ATTN: Biohazards Control Office, 1600 Clifton Road, Atlanta, Georgia 30333, (404) 639-3883, 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 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. 

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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 DNA 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 to provide biological containment shall be chosen or 

constructed so that the following types of "escape" are minimized: (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-IA through I-II-B describe levels of biological containment (host-vector systems) for prokaryotes. Specific criteria 

will depend on the organisms 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-conjugative 

plasmids (e.g., pSC101, Co1E1, or derivatives thereof (see Appendices I-III-A through G, Footnotes and 

References of Appendix I) and variants of bacteriophage, such as lambda (see Appendices I-III-H through O, 

Footnotes and References of Appendix I). The Escherichia coli K-12 hosts shall not contain 

conjugation-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. 

Appendix I-I-B. Host-Vector 2 Systems (EK2) 

Host-Vector 2 Systems provide a high level of biological containment as demonstrated by data from suitable 

tests performed in the laboratory. Escape of the recombinant DNA either via survival of the organisms or via 

transmission of recombinant DNA 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 non-permissive 

laboratory conditions designed to represent the natural environment, either by survival of the original host or as 

a consequence of transmission of the cloned DNA fragment. 
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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 non-permissive 

laboratory conditions designed to represent the natural environment, either as a prophage (in the inserted or 

plasmid form) in the laboratory host used for phage propagation, or survival in natural environments 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 be designated as 

such until they have been certified by the NIH Director. Requests for certification of host-vector systems may be 

submitted to the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 

750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

Proposed host-vector systems will be reviewed by the RAC (see Section IV-C-1-b-(1)-(f), Major Actions). Initial 

review will based on the construction, properties, and testing of the proposed host-vector system by a 

subcommittee composed of one or more RAC members and/or ad hoc experts. The RAC will evaluate the 

subcommittee's report and any other available information at the next scheduled RAC meeting. The NIH 

Director is responsible for certification of host-vector systems, following advice of the RAC. 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 without prior RAC review (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/OBA to the applicant and to the Institutional Biosafety Committee Chairs. A list of all currently certified 

host-vector systems is available from the Office of Biotechnology Activities, National Institutes of Health, 6705 

Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 

301-496-9839 (fax). The NIH Director may rescind the certification of a host-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 of physical containment level, unless NIH determines that the 

already constructed clones incorporate adequate biological containment. Certification of an host-vector system 

does not extend to modifications of 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 under 

consideration: (i) a description of the organism and vector; the strain's natural habitat and growth requirements; 

its physiological properties, particularly those related to its reproduction, survival, and the mechanisms by which 

it exchanges genetic information; the range of organisms with which this organism normally exchanges genetic 

information and the type of information is exchanged; and any relevant information about its pathogenicity or 

toxicity; (ii) a description of the history of the particular strains and vectors to be used, including data on any 

mutations which render this organism less able to survive or transmit genetic information; and (iii) a general 

description of the range of experiments contemplated with emphasis on the need for developing such an HostVector 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/OBA 

concerning data to be submitted (see Appendices I-III-N and O, Footnotes and References of Appendix I). In 

general, the following types of data are required: (i) description of construction steps with indication of source, 

properties, and manner of introduction of genetic traits; (ii) quantitative data on the stability of genetic traits that 

contribute to the containment of the system; (iii) data on the survival of the host-vector system under nonpermissive laboratory conditions designed to represent the relevant natural environment; (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 affect containment and utility, including 
Page 83 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 12 weeks prior to the RAC meeting at which such data will be 

considered by the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 

750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

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 the RAC and its subcommittee. Specific instructions 

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 Biotechnology 

Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 

20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

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 Plasmid in 

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 and 

Characterization 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 Multi-Purpose 

Cloning System, Gene, 1977, 2, pp. 95-113. 

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 to Form 

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. 
Page 84 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 SUBCOMMITTEE 

The 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 research needs, 

stimulating international cooperation, and assess national and international policy issues concerning 

biotechnology sciences. The primary emphasis will be on scientific issues to increase the overall effectiveness 

and productivity of the Federal investment in biotechnology sciences, especially regarding 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 of Commerce, 

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 and Space Administration, and National 

Science Foundation. The Biotechnology Research Subcommittee will function 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 are developed. 

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 each agency in those 

areas, and delineate where interagency cooperation would enhance progress in the biotechnology sciences, 

with an emphasis on integrated research efforts, where appropriate; (iii) assess 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 and biotechnology 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 on scientific issues, although it 

will still provide scientific support for regulatory responsibilities. 

*********************************************************************************************************************** 

APPENDIX K. PHYSICAL CONTAINMENT FOR LARGE SCALE USES OF ORGANISMS CONTAINING 

RECOMBINANT DNA MOLECULES 

Appendix K specifies physical containment guidelines for large-scale (greater than 10 liters of culture) research 

or production involving viable organisms containing recombinant DNA molecules. 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 DNA. 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 not addressed and shall be considered separately, albeit in conjunction with this appendix. 
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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 liters of 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 DNA 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 DNA molecules which require Biosafety Level (BL) 3 

containment at the laboratory scale. The program shall include: preassignment and periodic physical and 

medical examinations; collection, maintenance, and analysis of serum specimens for monitoring serologic 

changes 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 DNA 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 large-scale 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 posed by the organism, judged by experience with similar organisms unmodified by 

recombinant DNA techniques and consistent with Good Large Scale Practice. The four biosafety levels of largescale 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 large-scale research or production 

involving viable, non-pathogenic, and non-toxigenic recombinant 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-6, which have built-in environmental limitations 

that permit optimum growth in the large-scale setting but limited survival without adverse consequences in the 

environment. BL1-Large Scale is recommended for large-scale research or production of viable organisms 

containing recombinant DNA 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 DNA 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 DNA molecules that require BL3 containment at the laboratory scale. No provisions are made for 

large-scale research or production of viable organisms containing recombinant DNA 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 cultures of 

viable organisms containing recombinant DNA 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 are appropriate for the risk 

of exposure to viable organisms containing recombinant DNA molecules. Eating, drinking, smoking, applying 

cosmetics, and mouth pipetting shall be prohibited in the work area. 

Appendix K-II-D. Cultures of viable organisms containing recombinant DNA molecules 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 organisms shall be handled in accordance with 

applicable governmental environmental regulations. 
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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 DNA molecules at a level 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 

DNA molecules are immediately reported to the Laboratory Director. Medical evaluation, surveillance, and 

treatment are provided as appropriate and written records are maintained. 

Appendix K-III-B. Cultures of viable organisms containing recombinant DNA molecules shall be handled in a 

closed system (e.g., closed vessel used for the propagation and growth of 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 equipment provided all physical containment 

requirements specified in Appendix G-II-A, Physical Containment 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 a closed 

system or other primary containment equipment unless the viable organisms containing recombinant DNA 

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 DNA molecules. Culture fluids that contain viable organisms or viral vectors intended as final 

product may be removed 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 viable organisms containing recombinant 

DNA molecules to the environment. 

Appendix K-III-F. A closed system or other primary containment equipment that has contained viable 

organisms containing recombinant DNA molecules shall not be opened for maintenance 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 in Appendix K-III-C above. A validated sterilization 

procedure is one which has been demonstrated to be effective using the organism that will serve as the host for 

propagating the recombinant DNA 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 

DNA molecules are immediately reported to the Biological Safety Officer, Institutional Biosafety Committee, 

NIH/OBA, and other appropriate authorities (if applicable). Reports to NIH/OBA shall be sent to the Office of 

Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, 

MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). Medical evaluation, 

surveillance, and treatment are provided as appropriate and written records are maintained. 
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Appendix K-IV-B. Cultures of viable organisms containing recombinant DNA molecules shall be handled in a 

closed system (e.g., closed vessel used for the propagation and growth of cultures) or other primary 

containment equipment (e.g., Class III biological safety cabinet containing a centrifuge 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, Physical Containment 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 a closed 

system or other primary containment equipment unless the viable organisms containing recombinant DNA 

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 DNA molecules. Culture fluids that contain viable organisms or viral vectors intended as final 

product may be removed 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 viable organisms containing recombinant 

DNA molecules to the environment. 

Appendix K-IV-F. A closed system or other primary containment equipment that has contained viable 

organisms containing recombinant DNA molecules shall not be opened for maintenance 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 in Appendix K-IV-C above. A validated sterilization

procedure is one which has been demonstrated to be effective using the organisms that will serve as the host 

for propagating the recombinant DNA molecules. 

Appendix K-IV-G. Rotating seals and other mechanical devices directly associated with a closed system used 

for the propagation and growth of viable organisms containing recombinant DNA molecules 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 filters or through other equivalent treatment 

devices. 

Appendix K-IV-H. A closed system used for the propagation and growth of viable organisms containing 

recombinant DNA molecules and other primary containment equipment used to contain operations involving 

viable organisms containing sensing devices that monitor the integrity of containment during operations. 

Appendix K-IV-I. A closed system used for the propagation and growth of viable organisms containing the 

recombinant DNA molecules shall be tested for integrity of the containment features using the organism that will 

serve as the host for propagating recombinant DNA molecules. Testing shall be accomplished prior to the 

introduction of viable organisms containing recombinant DNA molecules and following modification or 

replacement of essential containment features. Procedures and methods used in the testing shall be 

appropriate for the equipment 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 DNA molecules shall be permanently identified. This identification shall be used in all records 

reflecting testing, operation, and maintenance and in all documentation relating to use of this equipment for 

research or production activities involving viable organisms containing recombinant DNA molecules. 

Appendix K-IV-K. The universal biosafety sign shall be posted on each closed system and primary 

containment equipment when used to contain viable organisms containing recombinant DNA molecules. 
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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. 

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 

DNA molecules are immediately reported to the Biological Safety Officer, Institutional Biosafety Committee, 

NIH/OBA, and other appropriate authorities (if applicable). Reports to NIH/OBA shall be sent to the Office of 

Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, 

MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). Medical evaluation, 

surveillance, and treatment are provided as appropriate and written records are maintained. 

Appendix K-V-B. Cultures of viable organisms containing recombinant DNA molecules shall be handled in a 

closed system (e.g., closed vessels used for the propagation and growth of cultures) or other primary 

containment equipment (e.g., Class III biological safety cabinet containing a centrifuge 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 a closed 

system or other primary containment equipment unless the viable organisms containing recombinant DNA 

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 DNA molecules. Culture fluids that contain viable organisms or viral vectors intended as final 

product may be removed 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 viable organisms containing recombinant 

DNA molecules to the environment. 

Appendix K-V-F. A closed system or other primary containment equipment that has contained viable 

organisms containing recombinant DNA molecules shall not be opened for maintenance 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 in Appendix K-V-C above. A validated sterilization 

procedure is one which has been demonstrated to be effective using the organisms that will serve as the host 

for propagating the recombinant DNA molecules. 

Appendix K-V-G. A closed system used for the propagation and growth of viable organisms containing 

recombinant DNA 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 to maintain the integrity of containment 

features. 

Appendix K-V-H. Rotating seals and other mechanical devices directly associated with a closed system used 

to contain viable organisms containing recombinant DNA molecules 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 filters or through other equivalent treatment devices. 

Appendix K-V-I. A closed system used for the propagation and growth of viable organisms containing 

recombinant DNA molecules and other primary containment equipment used to contain operations involving 

viable organisms containing recombinant DNA molecules shall include monitoring or sensing devices that 

monitor the integrity of containment during operations. 
Page 89 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Appendix K-V-J. A closed system used for the propagation and growth of viable organisms containing 

recombinant DNA molecules shall be tested for integrity of the containment features using the organisms that 

will serve as the host for propagating the recombinant DNA molecules. Testing shall be accomplished prior to 

the introduction of viable organisms containing recombinant DNA molecules and following modification or 

replacement of essential containment features. Procedures and methods used in the testing shall be 

appropriate for the equipment 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 DNA molecules shall be permanently identified. This identification shall be used in all records 

reflecting testing, operation, maintenance, and use of this equipment for research production activities involving 

viable organisms containing recombinant DNA molecules. 

Appendix K-V-L. The universal biosafety sign shall be posted on each closed system and primary containment 

equipment when used to contain viable organisms containing recombinant DNA 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 DNA molecules shall be located within a controlled 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 a 

double-doored space such as an air lock, anteroom, or change room that separates the controlled area from 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 to permit 

ready cleaning and decontamination. 

Appendix K-V-N-3. Penetrations into the controlled area shall be sealed to permit liquid or vapor phase space 

decontamination. 

Appendix K-V-N-4. All utilities and service or process piping and wiring entering the controlled area shall be 

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 primary 

containment 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 operate 

in a manner that prevents the reversal of the direction of air movement or shall be equipped with an 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: 
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Appendix K-V-O-1. Personnel entry into the controlled area shall be through the entry area specified in 

Appendix K-V-N-1. 

Appendix K-V-O-2. Persons entering the controlled area shall exchange or cover their personal clothing with 

work garments such as jump suits, laboratory coats, pants and shirts, head cover, and shoes or shoe covers. 

On exit from the controlled area the work clothing may be stored in a locker separate from that used 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 shall be 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 for access, 

while work is in progress. Serve 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 DNA molecules shall be available in the controlled area. 

Appendix K-V-O-14. The controlled area shall be decontaminated in accordance with established procedures 

following spills or other accidental release of viable organisms containing recombinant DNA molecules. 
Page 91 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 for safety of 

personnel and adequate control of hygiene and safety 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 a level that does not adversely affect 

health and safety of employees. 

K-II-B G-I 

3. Provide changing and hand washing facilities as well as protective 

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, and applying 

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 that physically 

separates the process from the external environment (closed 

system or other primary containment). 

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 to minimize 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 to be 

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 system designed 

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 

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 of cultures. 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 
Page 92 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 DNA. 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 not 

addressed and shall be considered separately, albeit in conjunction with this 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 been released 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 and biological barriers and 

inactivation using physical or chemical means. 

Appendix K-VII-E. De minimis Release. De minimis release is the release of: (i) viable microbiological 

agents or eukaryotic cells that does not result in the establishment of disease in healthy people, plants, or 

animals; 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 host organism 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; (ii) the recombinant DNA-engineered organism 

should be non-pathogenic, should be as safe in the large-scale setting as the host organism, and without 

adverse consequences in the 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 

requirement of 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 a 

drug 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 specific 

microbiological 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 in order 

that any incidental release is a de minimis release. 
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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 disease in 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 from a 

containment system. Discharges can be incidental or accidental. Incidental releases are de minimis in nature; 

accidental releases may be de minimis in nature. 

*********************************************************************************************************************** 

APPENDIX L. GENE THERAPY POLICY CONFERENCES (GTPCS) 

In order to enhance the depth and value of public discussion relevant to scientific, safety, social, and ethical 

implications of gene therapy research, the NIH Director will convene GTPCs at regular intervals. As 

appropriate, the NIH Director may convene a GTPC in conjunction with a RAC meeting. GTPCs will be 

administered by NIH/OBA. Conference participation will not involve a standing committee membership but 

rather will offer the unique advantage of assembling numerous participants who possess significant scientific, 

ethical, and legal expertise and/or interest that is directly applicable to a specific gene therapy research issue. 

At least one member of RAC will serve as Co-chair of each GTPC and report the findings of each GTPC to RAC 

at its next scheduled meeting. The RAC representative for each GTPC will be chosen based on the 

participant’s area of expertise relative to the specific gene therapy research issue to be discussed. All RAC 

members will be invited to attend GTPCs. GTPCs will have representation from other Federal agencies, 

including FDA and OHRP. GTPCs will focus on broad overarching policy and scientific issues related to gene 

therapy research. Proposals for GTPC topics may be submitted by members of RAC, representatives of 

academia, industry, patient and consumer advocacy organizations, other Federal agencies, professional 

scientific societies, and the general public. GTPC topics will not be limited to discussion of human applications 

of gene therapy research, i.e., they may include basic research on the use of novel gene delivery vehicles, or 

novel applications of human gene transfer. The RAC, with the Director’s approval, will have the primary 

responsibility for planning GTPC agendas. GTPC findings will be transmitted to the NIH Director and will be 

made publicly available. The NIH Director anticipates that this public policy forum will serve as a model for 

interagency communication and collaboration, concentrated expert discussion of novel scientific issues and their 

potential societal implications, and enhanced opportunity for public discussion of specific issues and potential 

impact of such applications on human health and the environment. 

*********************************************************************************************************************** 

APPENDIX M. POINTS TO CONSIDER IN THE DESIGN AND SUBMISSION OF PROTOCOLS FOR THE 

TRANSFER OF RECOMBINANT DNA MOLECULES INTO ONE OR MORE HUMAN RESEARCH 

PARTICIPANTS (POINTS TO CONSIDER) 

Appendix M applies to research conducted at or sponsored by an institution that receives any support for 

recombinant DNA research from NIH. Researchers not covered by the NIH Guidelines are encouraged to use 

Appendix M (see Section I-C, General Applicability). 

The acceptability of human somatic cell gene transfer has been addressed in several public documents as well 

as in numerous academic studies. In November 1982, the President's Commission for the Study of Ethical 

Problems in Medicine and Biomedical and Behavioral Research published a report, Splicing Life, which resulted 

from a two-year process of public deliberation and hearings. Upon release of that report, a U.S. House of 

Representatives subcommittee held three days of public hearings with witnesses from a wide range of fields 

from the biomedical and social sciences to theology, philosophy, and law. In December 1984, the Office of 

Technology Assessment released a background paper, Human Gene Therapy, which concluded that civic, 

religious, scientific, and medical groups have all accepted, in principle, the appropriateness of gene transfer of 

somatic cells in humans for specific genetic diseases. Somatic cell gene transfer is seen as an extension of 

present methods that might be preferable to other technologies. In light of this public support, RAC is prepared 

to consider proposals for somatic cell gene transfer. 
Page 94 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

RAC will not at present entertain proposals for germ line alterations but will consider proposals involving somatic 

cell gene transfer. The purpose of somatic cell gene transfer is to treat an individual patient, e.g., by inserting a 

properly functioning gene into the subject's somatic cells. Germ line alteration involves a specific attempt to 

introduce genetic changes into the germ (reproductive) cells of an individual, with the aim of changing the set of 

genes passed on to the individual's offspring. 

The RAC continues to explore the issues raised by the potential of in utero gene transfer clinical research. 

However, the RAC concludes that, at present, it is premature to undertake any in utero gene transfer clinical 

trial. Significant additional preclinical and clinical studies addressing vector transduction efficacy, biodistribution, 

and toxicity are required before a human in utero gene transfer protocol can proceed. In addition, a more 

thorough understanding of the development of human organ systems, such as the immune and nervous 

systems, is needed to better define the potential efficacy and risks of human in utero gene transfer. 

Prerequisites for considering any specific human in utero gene transfer procedure include an understanding of 

the pathophysiology of the candidate disease and a demonstrable advantage to the in utero approach. Once 

the above criteria are met, the RAC would be willing to consider well rationalized human in utero gene transfer 

clinical trials. 

Research proposals involving the deliberate transfer of recombinant DNA, or DNA or RNA derived from 

recombinant DNA, into human subjects (human gene transfer) will be considered through a review process 

involving both NIH/OBA and RAC. Investigators shall submit their relevant information on the proposed human 

gene transfer experiments to NIH/OBA. Submission of human gene transfer protocols to NIH will be in the 

format described in Appendix M-I-A, Submission Requirements for Protocol Submission. Submission to NIH 

shall be for registration purposes and will ensure continued public access to relevant human gene transfer 

information conducted in compliance with the NIH Guidelines. Investigational New Drug (IND) applications 

should be submitted to FDA in the format described in 21 CFR, Chapter I, Subchapter D, Part 312, Subpart B, 

Section 23, IND Content and Format. 

Institutional Biosafety Committee approval must be obtained from each institution at which recombinant DNA 

material will be administered to human subjects (as opposed to each institution involved in the production of 

vectors for human application and each institution at which there is ex vivo transduction of recombinant DNA 

material into target cells for human application). 

Factors that may contribute to public discussion of a human gene transfer experiment by RAC include: (i) new 

vectors/new gene delivery systems, (ii) new diseases, (iii) unique applications of gene transfer, and (iv) other 

issues considered to require further public discussion. Among the experiments that may be considered exempt 

from RAC discussion are those determined not to represent possible risk to human health or the environment. 

Full, public RAC review and discussion of a human gene transfer experiment may be (1) initiated by the NIH 

Director; or (2) initiated by the NIH OBA Director following a recommendation to NIH OBA by: (a) three or more 

RAC members, or (b) a Federal agency other than NIH. An individual human gene transfer experiment that is 

recommended for full RAC review should represent novel characteristics deserving of public discussion. If it is 

determined that an experiment will undergo full RAC discussion, NIH/OBA will immediately notify the Principal 

Investigator. RAC members may forward individual requests for additional information relevant to a specific 

protocol through NIH/OBA to the Principal Investigator. In making a determination whether an experiment is 

novel, and thus deserving of full RAC discussion, reviewers will examine the scientific rationale, scientific 

context (relative to other proposals reviewed by RAC), whether the preliminary in vitro and in vivo safety data 

were obtained in appropriate models and are sufficient, and whether questions related to relevant social and 

ethical issues have been resolved. RAC recommendations on a specific human gene transfer experiment shall 

be forwarded to the NIH Director, the Principal Investigator, the sponsoring institution, and other DHHS 

components, as appropriate. Relevant documentation will be included in the material for the RAC meeting at 

which the experiment is scheduled to be discussed. RAC meetings will be open to the public except where 

trade secrets and proprietary information are reviewed (see Section IV-D-5, Protection of Proprietary Data – 

Voluntary Compliance). RAC prefers that information provided in response to Appendix M contain no 

proprietary data or trade secrets, enabling all aspects of the review to be open to the public. 
Page 95 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Note: Any application submitted to NIH/OBA shall not be designated as ‘confidential’ in its entirety. In the event 

that a sponsor determines that specific responses to one or more of the items described in Appendix M should 

be considered as proprietary or trade secret, each item should be clearly identified as such. The cover letter 

(attached to the submitted material) shall: (1) clearly indicate that select portions of the application contain 

information considered as proprietary or trade secret, (2) a brief explanation as to the reason that each of these 

items is determined proprietary or trade secret. 

Public discussion of human gene transfer experiments (and access to relevant information) shall serve to inform 

the public about the technical aspects of the proposals, meaning and significance of the research, and 

significant safety, social, and ethical implications of the research. RAC discussion is intended to ensure safe 

and ethical conduct of gene transfer experiments and facilitate public understanding of this novel area of 

biomedical research. 

In its evaluation of human gene transfer proposals, RAC will consider whether the design of such experiments 

offers adequate assurance that their consequences will not go beyond their purpose, which is the same as the 

traditional purpose of clinical investigation, namely, to protect the health and well being of human subjects being 

treated while at the same time gathering generalizable knowledge. Two possible undesirable consequences of 

the transfer of recombinant DNA would be unintentional: (i) vertical transmission of genetic changes from an 

individual to his/her offspring, or (ii) horizontal transmission of viral infection to other persons with whom the 

individual comes in contact. Accordingly, Appendices M-I through M-V request information that will enable RAC 

and NIH/OBA to assess the possibility that the proposed experiment(s) will inadvertently affect reproductive 

cells or lead to infection of other people (e.g., medical personnel or relatives). 

Appendix M will be considered for revisions as experience in evaluating proposals accumulates and as new 

scientific developments occur. This review will be carried out periodically as needed. 

Appendix M-I. Requirements for Protocol Submission, Review, and Reporting – Human Gene Transfer 

Experiments 

Appendix M-I-A. Requirements for Protocol Submission 

The following documentation must be submitted (see exemption in Appendix M-VI-A, Footnotes of Appendix M) 

in printed or electronic form to the: Office of Biotechnology Activities, National Institutes of Health, 6705 

Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 

301-496-9839 (fax), E-mail: rosenthg@od.nih.gov. NIH OBA will confirm receipt within three working days after 

receiving the submission. Investigators should contact OBA if they do not receive this confirmation. 

1. A cover letter on institutional letterhead, signed by the Principal Investigator(s), that: (1) acknowledges that 

the documentation submitted to NIH OBA complies with the requirements set forth in Appendix M-I-A, 

Requirements for Protocol Submission; (2) identifies the Institutional Biosafety Committee (IBC) and Institutional 

Review Board (IRB) at the proposed clinical trial site(s) responsible for local review and approval of the protocol; 

and (3) acknowledges that no research participant will be enrolled (see definition of enrollment in Section I-E-7) 

until the RAC review process has been completed (see Appendix M-I-B, RAC Review Requirements); IBC 

approval (from the clinical trial site) has been obtained; IRB approval has been obtained; and all applicable 

regulatory authorizations have been obtained. 

2. The scientific abstract. 

3. The non-technical abstract. 

4. The proposed clinical protocol, including tables, figures, and relevant manuscripts. 

5. Responses to Appendices M-II through M-V, Description of the Proposal, Informed Consent, Privacy and 

Confidentiality, and Special Issues. Responses to Appendices M-II through M-V may be provided either as an 

appendix to the clinical protocol or incorporated in the clinical protocol. If responses to Appendices M-II through 

M-V are incorporated in the clinical protocol, each response must refer to the appropriate Appendix M-II through 

M-V. 
Page 96 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

6. The proposed informed consent document (see Appendix M-III, Informed Consent). 

7. Curriculum vitae of the principal investigator(s) (no more than two pages in biographical sketch format). 

 

Note: A human gene transfer experiment submitted to NIH OBA should not contain confidential commercial 

information or trade secrets, enabling all aspects of the review to be open to the public. 

Appendix M-I-B. RAC Review Requirements 

Appendix M-I-B-1. Initial RAC Review 

The initial RAC review process shall include a determination as to whether the human gene transfer experiment 

presents characteristics that warrant public RAC review and discussion. During the RAC’s initial review, 

individual committee members may request additional information relevant to the protocol. NIH OBA will 

immediately notify the Principal Investigator(s) of RAC requests for additional information. In making a 

determination whether an experiment presents characteristics warranting public RAC review and discussion, 

reviewers will examine the scientific rationale, scientific content, whether the preliminary in vitro and in vivo

safety data were obtained in appropriate models and are sufficient, and whether questions related to relevant 

social and ethical issues have been resolved. Other factors that may warrant public review and discussion of a 

human gene transfer experiment by the RAC include: (1) a new vector/new gene delivery system; (2) a new 

clinical application; (3) a unique application of gene transfer; and/or (4) other issues considered to require 

further public discussion. 

Initial RAC review shall be completed within 15 working days of receipt of a complete submission (see Appendix 

M-I-A, Requirements for Protocol Submission). At the end of the 15-day review period, NIH OBA will notify the 

Principal Investigator(s) in writing about the results of the RAC’s initial review. Two outcomes are possible: (1) 

the experiment does not present characteristics that warrant further review and discussion and is therefore 

exempt from public RAC review and discussion; or (2) the experiment presents characteristics that warrant 

public RAC review and discussion. Completion of the RAC review process is defined as: (1) receipt by the 

Principal Investigator(s) of a letter from NIH OBA indicating that the submission does not present characteristics 

that warrant public RAC review and discussion; or (2) receipt by the Principal Investigator(s) of a letter from NIH 

OBA after public RAC review that summarizes the committee’s key comments and recommendations (if any). 

If a human gene transfer protocol is submitted less than eight weeks before a scheduled RAC meeting and is 

subsequently recommended for public RAC review and discussion, the review of the protocol by the RAC will be 

deferred until the next scheduled RAC meeting. This eight-week period is needed to ensure adequate time for 

public notice and comment and thorough review by the committee members. 

No research participant shall be enrolled (see definition of enrollment in Section I-E-7) in the human gene 

transfer experiment until: (1) the RAC review process has been completed; (2) Institutional Biosafety 

Committee (IBC) approval (from the clinical trial site) has been obtained; (3) Institutional Review Board (IRB) 

approval has been obtained; and (4) all applicable regulatory authorization(s) have been obtained. 

For a clinical trial site that is added after the RAC review process, no research participant shall be enrolled (see 

definition of enrollment in Section I-E-7) at the clinical trial site until the following documentation has been 

submitted to NIH OBA: (1) IBC approval (from the clinical trial site); (2) IRB approval; (3) IRB-approved 

informed consent document; (4) curriculum vitae of the principal investigator(s) (no more than two pages in 

biographical sketch format); and (5) NIH grant numbers(s) if applicable. 

Appendix M-I-B-2. Public RAC Review and Discussion 

Public RAC review and discussion of a human gene transfer experiment may be: (1) initiated by the NIH 

Director; or (2) initiated by the NIH OBA Director following a recommendation to NIH OBA by: (a) three or more 

RAC members; or (b) a Federal agency other than NIH. In making a determination whether an experiment 

presents characteristics warranting public RAC review and discussion, reviewers will examine the scientific 

rationale, scientific content, whether the preliminary in vitro and in vivo safety data were obtained in appropriate 

models and are sufficient, and whether questions related to relevant social and ethical issues have been 
Page 97 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

resolved. Other factors that may warrant public review and discussion of a human gene transfer experiment by 

the RAC include: (1) a new vector/new gene delivery system; (2) a new clinical application; (3) a unique 

application of gene transfer; and/or (4) other issues considered to require further public discussion. 

After a human gene transfer experiment is reviewed by the full RAC at a regularly scheduled meeting, NIH OBA 

will send a letter summarizing the RAC key comments and recommendations (if any) regarding the protocol to 

the NIH Director, the Principal Investigator, the sponsoring institution, and other DHHS components, as 

appropriate. Completion of RAC review is defined as receipt by the Principal Investigator(s) of a letter from NIH 

OBA summarizing the committee’s findings. Unless NIH OBA determines that there are exceptional 

circumstances, the RAC summary letter will be sent to the Principal Investigator(s) within 10 working days after 

the completion of the RAC meeting at which the experiment was reviewed. 

RAC meetings will be open to the public except where trade secrets or confidential commercial information are 

reviewed. To enable all aspects of the protocol review process to be open to the public, information provided in 

response to Appendix M should not contain trade secrets or confidential commercial information. No application 

submitted to NIH OBA shall be designated as ‘confidential’ in its entirety. In the event that an investigator 

determines that specific responses to one or more of the items described in Appendix M should be considered 

as confidential commercial information or a trade secret, each item must be clearly identified as such. The 

cover letter (attached to the submitted material) shall: (1) clearly designate the information that is considered as 

confidential commercial information or a trade secret; and (2) explain and justify each designation. 

Appendix M-I-C. Reporting Requirements 

Appendix M-I-C-1. Initiation of the Clinical Investigation 

No later than 20 working days after enrollment (see definition of enrollment in Section I-E-7) of the first research 

participant in a human gene transfer experiment, the Principal Investigator(s) shall submit the following 

documentation to NIH OBA: (1) a copy of the informed consent document approved by the Institutional Review 

Board (IRB); (2) a copy of the protocol approved by the Institutional Biosafety Committee (IBC) and IRB; (3) a 

copy of the final IBC approval from the clinical trial site; (4) a copy of the final IRB approval; (5) a brief written 

report that includes the following information: (a) how the investigator(s) responded to each of the RAC’s 

recommendations on the protocol (if applicable); and (b) any modifications to the protocol as required by FDA; 

(6) applicable NIH grant number(s); (7) the FDA Investigational New Drug Application (IND) number; and (8) the 

date of the initiation of the trial. The purpose of requesting the FDA IND number is for facilitating interagency 

collaboration in the Federal oversight of human gene transfer research. 

Appendix M-I-C-2. Additional Clinical Trial Sites 

No research participant shall be enrolled (see definition of enrollment in Section I-E-7) at a clinical trial site until 

the following documentation has been submitted to NIH OBA: (1) Institutional Biosafety Committee approval 

(from the clinical trial site); (2) Institutional Review Board approval; (3) Institutional Review Board-approved 

informed consent document; (4) curriculum vitae of the principal investigator(s) (no more than two pages in 

biographical sketch format); and (5) NIH grant number(s) if applicable. 

Appendix M-I-C-3. Annual Reports 

Within 60 days after the one-year anniversary of the date on which the investigational new drug (IND) 

application went into effect, and after each subsequent anniversary until the trial is completed, the Principal 

Investigator (or delegate) shall submit the information set forth in (a), (b), and (c). When multiple studies are 

conducted under the single IND, the Principal Investigator (or delegate) may choose to submit a single annual 

report covering all studies, provided that each study is identified by its OBA protocol number. 

(a) Clinical Trial Information. A brief summary of the status of each trial in progress and each trial completed 

during the previous year. The summary is required to include the following information for each trial: (1) the title 

and purpose of the trial; (2) clinical site; (3) the Principal Investigator; (4) clinical protocol identifiers, including 

the NIH OBA protocol number, NIH grant number(s) (if applicable), and the FDA IND application number; (5) 

participant population (such as disease indication and general age group, e.g., adult or pediatric); (6) the total 

number of participants planned for inclusion in the trial; the number entered into the trial to date; the number 
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whose participation in the trial was completed; and the number who dropped out of the trial with a brief 

description of the reasons; (7) the status of the trial, e.g., open to accrual of subjects, closed but data collection 

ongoing, or fully completed, and (8) if the trial has been completed, a brief description of any study results. 

(b) Progress Report and Data Analysis. Information obtained during the previous year's clinical and non-clinical 

investigations, including: (1) a narrative or tabular summary showing the most frequent and most serious 

adverse experiences by body system; (2) a summary of all serious adverse events submitted during the past 

year; (3) a summary of serious adverse events that were expected or considered to have causes not associated 

with the use of the gene transfer product such as disease progression or concurrent medications; (4) if any 

deaths have occurred, the number of participants who died during participation in the investigation and causes 

of death; and (5) a brief description of any information obtained that is pertinent to an understanding of the gene 

transfer product’s actions, including, for example, information about dose-response, information from controlled 

trials, and information about bioavailability. 

(c) A copy of the updated clinical protocol including a technical and non-technical abstract. 

Appendix M-I-C-4. Safety Reporting 

Principal Investigators must submit, in accordance with this section, Appendix M-I-C-4-a and Appendix M-I-C-4-

b, a written report on: (1) any serious adverse event that is both unexpected and associated with the use of the 

gene transfer product (i.e., there is reasonable possibility that the event may have been caused by the use of 

the product; investigators should not await definitive proof of association before reporting such events); and (2) 

any finding from tests in laboratory animals that suggests a significant risk for human research participants 

including reports of mutagenicity, teratogenicity, or carcinogenicity. The report must be clearly labeled as a 

“Safety Report” and must be submitted to the NIH Office of Biotechnology Activities (NIH OBA) and to the local 

Institutional Biosafety Committee within the timeframes set forth in Appendix M-I-C-4-b. 

Principal Investigators should adhere to any other serious adverse event reporting requirements in accordance 

with federal regulations, state laws, and local institutional policies and procedures, as applicable. 

Principal Investigators may delegate to another party, such as a corporate sponsor, the reporting functions set 

forth in Appendix M, with written notification to the NIH OBA of the delegation and of the name(s), address, 

telephone and fax numbers of the contact(s). The Principal Investigator is responsible for ensuring that the 

reporting requirements are fulfilled and will be held accountable for any reporting lapses. 

The three alternative mechanisms for reporting serious adverse events to the NIH OBA are: by e-mail to 

oba@od.nih.gov; by fax to 301-496-9839; or by mail to the Office of Biotechnology Activities, National Institutes 

of Health, MSC 7985, 6705 Rockledge Drive, Suite 750, Bethesda, Maryland 20892-7985. 

Appendix M-I-C-4-a. Safety Reporting: Content and Format 

The serious adverse event report must include, but need not be limited to: (1) the date of the event; (2) 

designation of the report as an initial report or a follow-up report, identification of all safety reports previously 

filed for the clinical protocol concerning a similar adverse event, and an analysis of the significance of the 

adverse event in light of previous similar reports; (3) clinical site; (4) the Principal Investigator; (5) NIH Protocol 

number; (6) FDA’s Investigational New Drug (IND) Application number; (7) vector type , e.g., adenovirus; (8) 

vector subtype, e.g., type 5, relevant deletions; (9) gene delivery method, e.g., in vivo, ex vivo transduction; (10) 

route of administration, e.g., intratumoral, intravenous; (11) dosing schedule; (12) a complete description of the 

event; (13) relevant clinical observations; (14) relevant clinical history; (15) relevant tests that were or are 

planned to be conducted; (16) date of any treatment of the event; and (17) the suspected cause of the event. 

These items may be reported by using the recommended Adverse Event Reporting Template available on NIH 

OBA’s web site at: http://www4.od.nih.gov/oba/rac/documents1.htm, the FDA MedWatch forms, or other means 

provided that all of the above elements are specifically included. 

Reports from laboratory animal studies as delineated in Appendix M-I-C-4 must be submitted in a narrative 

format. 
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Appendix M-I-C-4-b. Safety Reporting: Time frames for Expedited Reports 

Any serious adverse event that is fatal or life-threatening, that is unexpected, and associated with the use of the 

gene transfer product must be reported to the NIH OBA as soon as possible, but not later than 7 calendar days 

after the sponsor’s initial receipt of the information (i.e., at the same time the event must be reported to the 

FDA). 

Serious adverse events that are unexpected and associated with the use of the gene transfer product, but are 

not fatal or life-threatening, must be reported to the NIH OBA as soon as possible, but not later than 15 calendar 

days after the sponsor’s initial receipt of the information (i.e., at the same time the event must be reported to the 

FDA). 

Changes in this schedule are permitted only where, under the FDA IND regulations [21 CFR 312(c)(3)], changes 

in this reporting schedule have been approved by the FDA and are reflected in the protocol. 

If, after further evaluation, an adverse event initially considered not to be associated with the use of the gene 

transfer product is subsequently determined to be associated, then the event must be reported to the NIH OBA 

within 15 days of the determination. 

Relevant additional clinical and laboratory data may become available following the initial serious adverse event 

report. Any follow-up information relevant to a serious adverse event must be reported within 15 calendar days 

of the sponsor’s receipt of the information. If a serious adverse event occurs after the end of a clinical trial and 

is determined to be associated with the use of the gene transfer product, that event shall be reported to the NIH 

OBA within 15 calendar days of the determination. 

Any finding from tests in laboratory animals that suggests a significant risk for human research participants 

including reports of mutagenicity, teratogenicity, or carcinogenicity must be reported as soon as possible, but 

not later than 15 calendar days after the sponsor’s initial receipt of the information (i.e., at the same time the 

event must be reported to the FDA). 

Appendix M-I-C-5. Confidentiality 

Data submitted in accordance with Appendix M-I-C that are claimed to be confidential commercial or trade 

secret information must be clearly labeled as such. Prior to making its determination about the confidentiality of 

data labeled confidential commercial or trade secret, the NIH will contact the Principal Investigator or delegate to 

ascertain the basis for the claim and subsequently will notify the Principal Investigator or delegate of its final 

determination regarding the claim. 

If NIH determines that the data so labeled are confidential commercial or trade secret and that their public 

disclosure would promote an understanding of key scientific or safety issues, the NIH will seek agreement from 

the appropriate party to release such data. Public discussion of scientific and safety issues raised by data 

submitted in accordance with Appendix M-I-C is vital to informing both investigators and human subjects about 

the safety of gene transfer research. 

To protect the privacy of participants in gene transfer research, any serious adverse event or annual reports 

submitted to NIH OBA must not contain any information that would identify the human research participants. 

Appendix M-I-D. Safety Assessment in Human Gene Transfer Research 

A working group of the RAC, the NIH Gene Transfer Safety Assessment Board, with staff support from the NIH 

OBA, will: 1) review in closed session as appropriate safety information from gene transfer trials for the purpose 

of assessing toxicity and safety data across gene transfer trials; 2) identify significant trends or significant single 

events; and 3) report significant findings and aggregated trend data to the RAC. It is expected that this process 

will enhance review of new protocols, improve the development, design, and conduct of human gene transfer 

trials, promote public understanding and awareness of the safety of human gene transfer research studies, and 

inform the decision-making of potential trial participants. 
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Appendix M-II. Description of the Proposal 

Responses to this appendix should be provided in the form of either written answers or references to specific 

sections of the protocol or its appendices. Investigators should indicate the points that are not applicable with a 

brief explanation. Investigators submitting proposals that employ the same vector systems may refer to 

preceding documents relating to the vector sequence without having to rewrite such material. 

Appendix M-II-A. Objectives and Rationale of the Proposed Research 

State concisely the overall objectives and rationale of the proposed study. Provide information on the specific 

points that relate to whichever type of research is being proposed. 

Appendix M-II-A-1. Use of Recombinant DNA for Therapeutic Purposes 

For research in which recombinant DNA is transferred in order to treat a disease or disorder (e.g., genetic 

diseases, cancer, and metabolic diseases), the following questions should be addressed: 

Appendix M-II-A-1-a. Why is the disease selected for experimental treatment by means of gene transfer a 

good candidate for such treatment? 

Appendix M-II-A-1-b. Describe the natural history and range of expression of the disease selected for 

experimental treatment. What objective and/or quantitative measures of disease activity are available? In your 

view, are the usual effects of the disease predictable enough to allow for meaningful assessment of the results 

of gene transfer? 

Appendix M-II-A-1-c. Is the protocol designed to prevent all manifestations of the disease, to halt the 

progression of the disease after symptoms have begun to appear, or to reverse manifestations of the disease in 

seriously ill victims? 

Appendix M-II-A-1-d. What alternative therapies exist? In what groups of subjects are these therapies 

effective? What are their relative advantages and disadvantages as compared with the proposed gene 

transfer? 

Appendix M-II-A-2. Transfer of DNA for Other Purposes 

Appendix M-II-A-2-a. Into what cells will the recombinant DNA be transferred? Why is the transfer of 

recombinant DNA necessary for the proposed research? What questions can be answered by using 

recombinant DNA? 

Appendix M-II-A-2-b. What alternative methodologies exist? What are their relative advantages and 

disadvantages as compared to the use of recombinant DNA? 

Appendix M-II-B. Research Design, Anticipated Risks and Benefits 

Appendix M-II-B-1. Structure and Characteristics of the Biological System 

Provide a full description of the methods and reagents to be employed for gene delivery and the rationale for 

their use. The following are specific points to be addressed: 

Appendix M-II-B-1-a. What is the structure of the cloned DNA that will be used? 

Appendix M-II-B-1-a-(1). Describe the gene (genomic or cDNA), the bacterial plasmid or phage vector, and the 

delivery vector (if any). Provide complete nucleotide sequence analysis or a detailed restriction enzyme map of 

the total construct. 

Appendix M-II-B-1-a-(2). What regulatory elements does the construct contain (e.g., promoters, enhancers, 

polyadenylation sites, replication origins, etc.)? From what source are these elements derived? Summarize 

what is currently known about the regulatory character of each element. 
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Appendix M-II-B-1-a-(3). Describe the steps used to derive the DNA construct. 

Appendix M-II-B-1-b. What is the structure of the material that will be administered to the research participant? 

Appendix M-II-B-1-b-(1). Describe the preparation, structure, and composition of the materials that will be 

given to the human research subject or used to treat the subject’s cells: (i) If DNA, what is the purity (both in 

terms of being a single DNA species and in terms of other contaminants)? What tests have been used and 

what is the sensitivity of the tests? (ii) If a virus, how is it prepared from the DNA construct? In what cell is the 

virus grown (any special features)? What medium and serum are used? How is the virus purified? What is its 

structure and purity? What steps are being taken (and assays used with their sensitivity) to detect and eliminate 

any contaminating materials (for example, VL30 RNA, other nucleic acids, or proteins) or contaminating viruses 

(both replication-competent or replication-defective) or other organisms in the cells or serum used for 

preparation of the virus stock including any contaminants that may have biological effects? (iii) If co-cultivation 

is employed, what kinds of cells are being used for co-cultivation? What steps are being taken (and assays 

used with their sensitivity) to detect and eliminate any contaminating materials? Specifically, what tests are 

being conducted to assess the material to be returned to the subject for the presence of live or killed donor cells 

or other non-vector materials (for example, VL30 sequences) originating from those cells? (iv) If methods other 

than those covered by Appendices M-II-B-1 through M-II-B-3, Research Design, Anticipated Risks and Benefits, 

are used to introduce new genetic information into target cells, what steps are being taken to detect and 

eliminate any contaminating materials? What are possible sources of contamination? What is the sensitivity of 

tests used to monitor contamination? 

Appendix M-II-B-1-b-(2). Describe any other material to be used in preparation of the material to be 

administered to the human research subject. For example, if a viral vector is proposed, what is the nature of the 

helper virus or cell line? If carrier particles are to be used, what is the nature of these? 

Appendix M-II-B-2. Preclinical Studies, Including Risk-Assessment Studies 

Provide results that demonstrate the safety, efficacy, and feasibility of the proposed procedures using animal 

and/or cell culture model systems, and explain why the model(s) chosen is/are most appropriate. 

Appendix M-II-B-2-a. Delivery System 

Appendix M-II-B-2-a-(1). What cells are the intended target cells of recombinant DNA? What target cells are 

to be treated ex vivo and returned to the human subject, how will the cells be characterized before and after 

treatment? What is the theoretical and practical basis for assuming that only the target cells will incorporate the 

DNA? 

Appendix M-II-B-2-a-(2). Is the delivery system efficient? What percentage of the target cells contain the 

added DNA? 

Appendix M-II-B-2-a-(3). How is the structure of the added DNA sequences monitored and what is the 

sensitivity of the analysis? Is the added DNA extrachromosomal or integrated? Is the added DNA 

unrearranged? 

Appendix M-II-B-2-a-(4). How many copies are present per cell? How stable is the added DNA both in terms 

of its continued presence and its structural stability? 

Appendix M-II-B-2-b. Gene Transfer and Expression 

Appendix M-II-B-2-b-(1). What animal and cultured cell models were used in laboratory studies to assess the 

in vivo and in vitro efficacy of the gene transfer system? In what ways are these models similar to and different 

from the proposed human treatment? 

Appendix M-II-B-2-b-(2). What is the minimal level of gene transfer and/or expression that is estimated to be 

necessary for the gene transfer protocol to be successful in humans? How was this level determined? 
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Appendix M-II-B-2-b-(3). Explain in detail all results from animal and cultured cell model experiments which 

assess the effectiveness of the delivery system in achieving the minimally required level of gene transfer and 

expression. 

Appendix M-II-B-2-b-(4). To what extent is expression only from the desired gene (and not from the 

surrounding DNA)? To what extent does the insertion modify the expression of other genes? 

Appendix M-II-B-2-b-(5). In what percentage of cells does expression from the added DNA occur? Is the 

product biologically active? What percentage of normal activity results from the inserted gene? 

Appendix M-II-B-2-b-(6). Is the gene expressed in cells other than the target cells? If so, to what extent? 

Appendix M-II-B-2-c. Retrovirus Delivery Systems 

Appendix M-II-B-2-c-(1). What cell types have been infected with the retroviral vector preparation? Which 

cells, if any, produce infectious particles? 

Appendix M-II-B-2-c-(2). How stable are the retroviral vector and the resulting provirus against loss, 

rearrangement, recombination, or mutation? What information is available on how much rearrangement or 

recombination with endogenous or other viral sequences is likely to occur in the human subject’s cells? What 

steps have been taken in designing the vector to minimize instability or variation? What laboratory studies have 

been performed to check for stability, and what is the sensitivity of the analyses? 

Appendix M-II-B-2-c-(3). What laboratory evidence is available concerning potential harmful effects of the 

transfer (e.g., development of neoplasia, harmful mutations, regeneration of infectious particles, or immune 

responses)? What steps will be taken in designing the vector to minimize pathogenicity? What laboratory 

studies have been performed to check for pathogenicity, and what is the sensitivity of the analyses? 

Appendix M-II-B-2-c-(4). Is there evidence from animal studies that vector DNA has entered untreated cells, 

particularly germ-line cells? What is the sensitivity of these analyses? 

Appendix M-II-B-2-c-(5). Has a protocol similar to the one proposed for a clinical trial been conducted in nonhuman primates and/or other animals? What were the results? Specifically, is there any evidence that the 

retroviral vector has recombined with any endogenous or other viral sequences in the animals? 

Appendix M-II-B-2-d. Non-Retrovirus Delivery/Expression Systems 

If a non-retroviral delivery system is used, what animal studies have been conducted to determine if there are 

pathological or other undesirable consequences of the protocol (including insertion of DNA into cells other than 

those treated, particularly germ-line cells)? How long have the animals been studied after treatment? What 

safety studies have been conducted? (Include data about the level of sensitivity of such assays.) 

Appendix M-II-B-3. Clinical Procedures, Including Research Participant Monitoring 

Describe the experimental treatment that will be administered to the human subjects and the diagnostic 

methods that will be used to monitor the success or failure of the experimental treatment. If previous clinical 

studies using similar methods have been performed by yourself or others, indicate their relevance to the 

proposed study. Specifically: 

Appendix M-II-B-3-a. Will cells (e.g., bone marrow cells) be removed from human subjects and treated ex 

vivo? If so, describe the type, number, and intervals at which these cells will be removed. 

Appendix M-II-B-3-b. Will human subjects be treated to eliminate or reduce the number of cells containing 

malfunctioning genes (e.g., through radiation or chemotherapy)? 

Appendix M-II-B-3-c. What treated cells (or vector/DNA combination) will be given to human subjects? How 

will the treated cells be administered? What volume of cells will be used? Will there be single or multiple 

experimental treatments? If so, over what period of time? 
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Appendix M-II-B-3-d. How will it be determined that new gene sequences have been inserted into the subject’s 

cells and if these sequences are being expressed? Are these cells limited to the intended target cell 

populations? How sensitive are these analyses? 

Appendix M-II-B-3-e. What studies will be conducted to assess the presence and effects of the contaminants? 

Appendix M-II-B-3-f. What are the clinical endpoints of the study? Are there objectives and quantitative 

measurements to assess the natural history of the disease? Will such measurements be used in human subject 

follow-up? How will subjects be monitored to assess specific effects of the treatment on the disease? What is 

the sensitivity of the analyses? How frequently will follow-up studies be conducted? How long will follow-up 

continue? 

Appendix M-II-B-3-g. What are the major beneficial and adverse effects of the experimental treatment that you 

anticipate? What measures will be taken in an attempt to control or reverse these adverse effects if they occur? 

Compare the probability and magnitude of deleterious consequences from the disease if recombinant DNA 

transfer is not used. 

Appendix M-II-B-3-h. If a treated human subject dies, what special post-mortem studies will be performed? 

Appendix M-II-B-4. Public Health Considerations 

Describe any potential benefits and hazards of the proposed gene transfer to persons other than the human 

subjects receiving the experimental treatment. Specifically: 

Appendix M-II-B-4-a. On what basis are potential public health benefits or hazards postulated? 

Appendix M-II-B-4-b. Is there a significant possibility that the added DNA will spread from the human subject 

to other persons or to the environment? 

Appendix M-II-B-4-c. What precautions will be taken against such spread (e.g., subjects sharing a room, 

health-care workers, or family members)? 

Appendix M-II-B-4-d. What measures will be undertaken to mitigate the risks, if any, to public health? 

Appendix M-II-B-4-e. In light of possible risks to offspring, including vertical transmission, will birth control 

measures be recommended to subjects? Are such concerns applicable to health care personnel? 

Appendix M-II-B-5. Qualifications of Investigators and Adequacy of Laboratory and Clinical Facilities 

Indicate the relevant training and experience of the personnel who will be involved in the preclinical studies and 

clinical administration of recombinant DNA. Describe the laboratory and clinical facilities where the proposed 

study will be performed. Specifically: 

Appendix M-II-B-5-a. What professional personnel (medical and nonmedical) will be involved in the proposed 

study and what is their relevant expertise? Provide a two-page curriculum vitae for each key professional 

person in biographical sketch format (see Appendix M-I-A, Requirements for Protocol Submission). 

Appendix M-II-B-5-b. At what hospital or clinic will the experimental treatment be given? Which facilities of the 

hospital or clinic will be especially important for the proposed study? Will subjects occupy regular hospital beds 

or clinical research center beds? Where will subjects reside during the follow-up period? What special 

arrangements will be made for the comfort and consideration of the research participants. Will the research 

institution designate an ombudsman, patient care representative, or other individual to help protect the rights 

and welfare of the research participant? 

Appendix M-II-C. Selection of the Human Subjects 

Estimate the number of human subjects to be involved in the proposed study. Describe recruitment procedures 

and eligibility requirements, paying particular attention to whether these procedures and requirements are fair 

and equitable. Specifically: 
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Appendix M-II-C-1. How many subjects do you plan to involve in the proposed study? 

Appendix M-II-C-2. How many eligible subjects do you anticipate being able to identify each year? 

Appendix M-II-C-3. What recruitment procedures do you plan to use? 

Appendix M-II-C-4. What selection criteria do you plan to employ? What are the exclusion and inclusion 

criteria for the study? 

Appendix M-II-C-5. How will subjects be selected if it is not possible to include all who desire to participate? 

Appendix M-III. Informed Consent 

In accordance with the Protection of Human Subjects (45 CFR Part 46), investigators should indicate how 

subjects will be informed about the proposed study and the manner in which their consent will be solicited. They 

should indicate how the Informed Consent document makes clear the special requirements of gene transfer 

research. If a proposal involves children, special attention should be paid to the Protection of Human Subjects 

(45 CFR Part 46), Subpart D, Additional Protections for Children Involved as Subjects in Research. 

Appendix M-III-A. Communication About the Study to Potential Participants 

Appendix M-III-A-1. Which members of the research group and/or institution will be responsible for contacting 

potential participants and for describing the study to them? What procedures will be used to avoid possible 

conflicts of interest if the investigator is also providing medical care to potential subjects? 

Appendix M-III-A-2. How will the major points covered in Appendix M-II, Description of Proposal, be disclosed 

to potential participants and/or their parents or guardians in language that is understandable to them? 

Appendix M-III-A-3. What is the length of time that potential participants will have to make a decision about 

their participation in the study? 

Appendix M-III-A-4. If the study involves pediatric or mentally handicapped subjects, how will the assent of 

each person be obtained? 

Appendix M-III-B. Informed Consent Document 

Submission of a human gene transfer experiment to NIH OBA must include a copy of the proposed informed 

consent document. A separate Informed Consent document should be used for the gene transfer portion of a 

research project when gene transfer is used as an adjunct in the study of another technique, e.g., when a gene 

is used as a "marker" or to enhance the power of immunotherapy for cancer. 

Because of the relative novelty of the procedures that are used, the potentially irreversible consequences of the 

procedures performed, and the fact that many of the potential risks remain undefined, the Informed Consent 

document should include the following specific information in addition to any requirements of the DHHS 

regulations for the Protection of Human Subjects (45 CFR 46). Indicate if each of the specified items appears in 

the Informed Consent document or, if not included in the Informed Consent document, how those items will be 

presented to potential subjects. Include an explanation if any of the following items are omitted from the 

consent process or the Informed Consent document. 

Appendix M-III-B-1. General Requirements of Human Subjects Research 

Appendix M-III-B-1-a. Description/Purpose of the Study 

The subjects should be provided with a detailed explanation in non-technical language of the purpose of the 

study and the procedures associated with the conduct of the proposed study, including a description of the gene 

transfer component. 
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Appendix M-III-B-1-b. Alternatives 

The Informed Consent document should indicate the availability of therapies and the possibility of other 

investigational interventions and approaches. 

Appendix M-III-B-1-c. Voluntary Participation 

The subjects should be informed that participation in the study is voluntary and that failure to participate in the 

study or withdrawal of consent will not result in any penalty or loss of benefits to which the subjects are 

otherwise entitled. 

Appendix M-III-B-1-d. Benefits 

The subjects should be provided with an accurate description of the possible benefits, if any, of participating in 

the proposed study. For studies that are not reasonably expected to provide a therapeutic benefit to subjects, 

the Informed Consent document should clearly state that no direct clinical benefit to subjects is expected to 

occur as a result of participation in the study, although knowledge may be gained that may benefit others. 

Appendix M-III-B-1-e. Possible Risks, Discomforts, and Side Effects 

There should be clear itemization in the Informed Consent document of types of adverse experiences, their 

relative severity, and their expected frequencies. For consistency, the following definitions are suggested: side 

effects that are listed as mild should be ones which do not require a therapeutic intervention; moderate side 

effects require an intervention; and severe side effects are potentially fatal or life-threatening, disabling, or 

require prolonged hospitalization. 

If verbal descriptors (e.g., "rare," "uncommon," or "frequent") are used to express quantitative information 

regarding risk, these terms should be explained. 

The Informed Consent document should provide information regarding the approximate number of people who 

have previously received the genetic material under study. It is necessary to warn potential subjects that, for 

genetic materials previously used in relatively few or no humans, unforeseen risks are possible, including ones 

that could be severe. 

The Informed Consent document should indicate any possible adverse medical consequences that may occur if 

the subjects withdraw from the study once the study has started. 

Appendix M-III-B-1-f. Costs 

The subjects should be provided with specific information about any financial costs associated with their 

participation in the protocol and in the long-term follow-up to the protocol that are not covered by the 

investigators or the institution involved. 

Subjects should be provided an explanation about the extent to which they will be responsible for any costs for 

medical treatment required as a result of research-related injury. 

Appendix M-III-B-2. Specific Requirements of Gene Transfer Research 

Appendix M-III-B-2-a. Reproductive Considerations 

To avoid the possibility that any of the reagents employed in the gene transfer research could cause harm to a 

fetus/child, subjects should be given information concerning possible risks and the need for contraception by 

males and females during the active phase of the study. The period of time for the use of contraception should 

be specified. 

The inclusion of pregnant or lactating women should be addressed. 
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Appendix M-III-B-2-b. Long-Term Follow-Up 

To permit evaluation of long-term safety and efficacy of gene transfer, the prospective subjects should be 

informed that they are expected to cooperate in long-term follow-up that extends beyond the active phase of the 

study. The Informed Consent document should include a list of persons who can be contacted in the event that 

questions arise during the follow-up period. The investigator should request that subjects continue to provide a 

current address and telephone number. 

The subjects should be informed that any significant findings resulting from the study will be made known in a 

timely manner to them and/or their parent or guardian including new information about the experimental 

procedure, the harms and benefits experienced by other individuals involved in the study, and any long-term 

effects that have been observed. 

Appendix M-III-B-2-c. Request for Autopsy 

To obtain vital information about the safety and efficacy of gene transfer, subjects should be informed that at the 

time of death, no matter what the cause, permission for an autopsy will be requested of their families. Subjects 

should be asked to advise their families of the request and of its scientific and medical importance. 

Appendix M-III-B-2-d. Interest of the Media and Others in the Research 

To alert subjects that others may have an interest in the innovative character of the protocol and in the status of 

the treated subjects, the subjects should be informed of the following: (i) that the institution and investigators will 

make efforts to provide protection from the media in an effort to protect the participants' privacy, and (ii) that 

representatives of applicable Federal agencies (e.g., the National Institutes of Health and the Food and Drug 

Administration), representatives of collaborating institutions, vector suppliers, etc., will have access to the 

subjects' medical records. 

Appendix M-IV. Privacy 

Indicate what measures will be taken to protect the privacy of subjects and their families as well as maintain the 

confidentiality of research data. These measures should help protect the confidentiality of information that could 

directly or indirectly identify study participants. 

Appendix M-IV-A. What provisions will be made to honor the wishes of individual human subjects (and the 

parents or guardians of pediatric or mentally handicapped subjects) as to whether, when, or how the identity of a 

subject is publicly disclosed. 

Appendix M-IV-B. What provisions will be made to maintain the confidentiality of research data, at least in 

cases where data could be linked to individual subjects? 

Appendix M-V. Special Issues 

Although the following issues are beyond the normal purview of local Institutional Review Boards, investigators 

should respond to the following questions: 

Appendix M-V-A. What steps will be taken, consistent with Appendix M-IV, Privacy, to ensure that accurate 

and appropriate information is made available to the public with respect to such public concerns as may arise 

from the proposed study? 

Appendix M-V-B. Do you or your funding sources intend to protect under patent or trade secret laws either the 

products or the procedures developed in the proposed study? If so, what steps will be taken to permit as full 

communication as possible among investigators and clinicians concerning research methods and results? 
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Appendix M-VI. Footnotes of Appendix M 

Appendix M-VI-A. Human studies in which induction or enhancement of an immune response to a vectorencoded microbial immunogen is the major goal, such an immune response has been demonstrated in model 

systems, and the persistence of the vector-encoded immunogen is not expected, are exempt from Appendix 

M-I, Requirements for Protocol Submission, Review and Reporting – Human Gene Transfer Experiments. 

*********************************************************************************************************************** 

APPENDIX P. PHYSICAL AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT DNA RESEARCH 

INVOLVING PLANTS 

Appendix P specifies physical and biological containment conditions and practices suitable to the greenhouse 

conduct of experiments involving recombinant DNA-containing plants, plant-associated microorganisms, and 

small animals. All provisions of the NIH Guidelines apply to plant research activities with the following 

modifications: 

Appendix P 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 in Appendix G. 

The plants covered in Appendix P 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 to microorganisms 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. 

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 P require prior approval by the 

Institutional Biosafety Committee. 

Appendix P-I. General Plant Biosafety Levels 

Appendix P-I-A. The principal purpose of plant containment is to avoid the unintentional transmission of a 

recombinant DNA-containing plant genome, including nuclear or organelle hereditary material or release of 

recombinant DNA-derived organisms associated with plants. 

Appendix P-I-B. The containment principles are based on the recognition that the organisms that are used 

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 on organisms 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 and establishment of an organism in a 

new ecosystem. 

Appendix P-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 P-II, Physical Containment Levels. The selection of containment levels 

required for research involving recombinant DNA molecules in plants or associated with plants is specified in 

Appendix P-III, Biological Containment Practices. These biosafety levels are described in Appendix P-II, 

Physical Containment Levels. This appendix describes greenhouse practices and special greenhouse facilities 

for physical containment. 
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Appendix P-I-D. BL1-P through BL4-P are designed to provide differential levels of biosafety for plants in the 

absence or presence of other experimental organisms that contain recombinant DNA. These biosafety levels, in 

conjunction with biological containment conditions described in Appendix P-III, Biological Containment 

Practices, provide flexible approaches to ensure the safe conduct of research. 

Appendix P-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 P-III, Biological 

Containment Practices), if botanical reproductive structures are produced that have the potential of being 

released. 

Appendix P-II. Physical Containment Levels 

Appendix P-II-A. Biosafety Level 1 - Plants (BL1-P) 

Appendix P-II-A-1. Standard Practices (BL1-P) 

Appendix P-II-A-1-a. Greenhouse Access (BL1-P) 

Appendix P-II-A-1-a-(1). Access to the greenhouse shall be limited or restricted, at the discretion of the 

Greenhouse Director, when experiments are in progress. 

Appendix P-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 in accordance 

with accepted greenhouse practices that are appropriate to the experimental organism. 

Appendix P-II-A-1-b. Records (BL1-P) 

Appendix P-II-A-1-b-(1). A record shall be kept of experiments currently in progress in the greenhouse facility. 

Appendix P-II-A-1-c. Decontamination and Inactivation (BL1-P) 

Appendix P-II-A-1-c-(1). Experimental organisms shall be rendered biologically inactive by appropriate 

methods before disposal outside of the greenhouse facility. 

Appendix P-II-A-1-d. Control of Undesired Species and Motile Macroorganisms (BL1-P) 

Appendix P-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 accordance with applicable 

state and Federal laws. 

Appendix P-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 P-II-A-1-e. Concurrent Experiments Conducted in the Greenhouse (BL1-P) 

Appendix P-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 P-II-A-2. Facilities (BL1-P) 

Appendix P-II-A-2-a. Definitions (BL1-P) 
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Appendix P-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 for plant growth. 

Appendix P-II-A-2-a-(2). The term "greenhouse facility" includes the actual greenhouse rooms or 

compartments for growing plants, including all immediately contiguous hallways and head-house areas, and is 

considered part of the confinement area. 

Appendix P-II-A-2-b. Greenhouse Design (BL1-P) 

Appendix P-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 P-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 to contain or exclude 

pollen, microorganisms, or small flying animals (e.g., arthropods and birds); however, screens are 

recommended. 

Appendix P-II-B. Biosafety Level 2 - Plants (BL2-P) 

Appendix P-II-B-1. Standard Practices (BL2-P) 

Appendix P-II-B-1-a. Greenhouse Access (BL2-P) 

Appendix P-II-B-1-a-(1). Access to the greenhouse shall be limited or restricted, at the discretion of the 

Greenhouse Director, to individuals directly involved with the experiments when they are in progress. 

Appendix P-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 P-II-B-1-b. Records (BL2-P) 

Appendix P-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 P-II-B-1-b-(2). A record shall be kept of experiments currently in progress in the greenhouse facility. 

Appendix P-II-B-1-b-(3). The Principal Investigator shall report any greenhouse accident involving the 

inadvertent release or spill of microorganisms to the Greenhouse Director, Institutional Biosafety Committee, 

NIH/OBA and other appropriate authorities immediately (if applicable). Reports to the NIH/OBA shall be sent to 

the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 

7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). 

Documentation of any such accident shall be prepared and maintained. 

Appendix P-II-B-1-c. Decontamination and Inactivation (BL2-P) 

Appendix P-II-B-1-c-(1). Experimental organisms shall be rendered biologically inactive by appropriate 

methods before disposal outside of the greenhouse facility. 

Appendix P-II-B-1-c-(2). Decontamination of run-off water is not necessarily required. If part of the 

greenhouse is composed of gravel or similar material, appropriate treatments should be made periodically to 

eliminate, or render inactive, any organisms potentially entrapped by the gravel. 

Appendix P-II-B-1-d. Control of Undesired Species and Motile Macroorganisms (BL2-P) 

Appendix P-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 accordance with applicable 

state and Federal laws. 
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Appendix P-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 P-II-B-1-e. Concurrent Experiments Conducted in the Greenhouse (BL2-P) 

Appendix P-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 P-II-B-1-f. Signs (BL2-P) 

Appendix P-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 P-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 sign posted on the 

greenhouse access doors. 

Appendix P-II-B-1-f-(3). If there is a risk to human health, a sign shall be posted incorporating the universal 

biosafety symbol. 

Appendix P-II-B-1-g. Transfer of Materials (BL2-P) 

Appendix P-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 P-II-B-1-h. Greenhouse Practices Manual (BL2-P) 

Appendix P-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 P-II-B-2. Facilities (BL2-P) 

Appendix P-II-B-2-a. Definitions (BL2-P) 

Appendix P-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 for plant growth. 

Appendix P-II-B-2-a-(2). The term "greenhouse facility" includes the actual greenhouse rooms or 

compartments for growing plants, including all immediately contiguous hallways and head-house areas and is 

considered part of the confinement area. 

Appendix P-II-B-2-b. Greenhouse Design (BL2-P) 

Appendix P-II-B-2-b-(1). A greenhouse floor composed of an impervious material. Concrete is recommended, 

but gravel or other porous material under benches is acceptable unless propagules of experimental organisms 

are readily disseminated through soil. Soil beds are acceptable unless propagules of experimental organisms 

are readily disseminated through soil. 

Appendix P-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). 
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Appendix P-II-B-2-c. Autoclaves (BL2-P) 

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

materials. 

Appendix P-II-B-2-d. Supply and Exhaust Air Ventilation Systems (BL2-P) 

Appendix P-II-B-2-d-(1). If intake fans are used, measures shall be taken to minimize the ingress of 

arthropods. Louvers or fans shall be constructed such that they can only be opened when the fan is in 

operation. 

Appendix P-II-B-2-e. Other (BL2-P) 

Appendix P-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 the foregoing clauses. 

Appendix P-II-C. Biosafety Level 3 - Plants (BL3-P) 

Appendix P-II-C-1. Standard Practices (BL3-P) 

Appendix P-II-C-1-a. Greenhouse Access (BL3-P) 

Appendix P-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 P-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 with 

accepted greenhouse practices that are appropriate to the experimental organisms. 

Appendix P-II-C-1-b. Records (BL3-P) 

Appendix P-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 P-II-C-1-b-(2). A record shall be kept of experiments currently in progress in the greenhouse facility. 

Appendix P-II-C-1-b-(3). The Principal Investigator shall report any greenhouse accident involving the 

inadvertent release or spill of microorganisms to the Biological Safety Officer, Greenhouse Director, Institutional 

Biosafety Committee, NIH/OBA, and other appropriate authorities immediately (if applicable). Reports to the 

NIH/OBA shall be sent to the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge 

Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-

9839 (fax). Documentation of any such accident shall be prepared and maintained. 

Appendix P-II-C-1-c. Decontamination and Inactivation (BL3-P) 

Appendix P-II-C-1-c-(1). All experimental materials shall be sterilized in an autoclave or rendered biologically 

inactive by appropriate methods before disposal, except those that are to remain in a viable or intact state for 

experimental purposes; including water that comes in contact with experimental microorganisms or with material 

exposed to such microorganisms, and contaminated equipment and supplies. 

Appendix P-II-C-1-d. Control of Undesired Species and Motile Macroorganisms (BL3-P) 

Appendix P-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 accordance with applicable 

state and Federal laws. 
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Appendix P-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 to contain the motile 

organisms. 

Appendix P-II-C-1-e. Concurrent Experiments Conducted in the Greenhouse (BL3-P) 

Appendix P-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 P-II-C-1-f. Signs (BL3-P) 

Appendix P-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 P-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 sign posted on the 

greenhouse access doors. 

Appendix P-II-C-1-f-(3). If there is a risk to human health, a sign shall be posted incorporating the universal 

biosafety symbol. 

Appendix P-II-C-1-g. Transfer of Materials (BL3-P) 

Appendix P-II-C-1-g-(1). Experimental materials that are brought into or removed from the greenhouse facility 

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 a chemical disinfectant or fumigation chamber or by 

an alternative procedure that has demonstrated effective inactivation of the experimental organism. 

Appendix P-II-C-1-h. Greenhouse Practices Manual (BL3-P) 

Appendix P-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 with recognized potential for 

serious detrimental impact. 

Appendix P-II-C-1-i. Protective Clothing (BL3-P) 

Appendix P-II-C-1-i-(1). Disposable clothing (e.g., solid front or wrap-around gowns, scrub suits, or other 

appropriate clothing) shall be worn in the greenhouse if deemed necessary by the Greenhouse Director 

because of potential dissemination of the experimental microorganisms. 

Appendix P-II-C-1-i-(2). Protective clothing shall be removed before exiting the greenhouse and 

decontaminated prior to laundering or disposal. 

Appendix P-II-C-1-j. Other (BL3-P) 

Appendix P-II-C-1-j-(1). Personnel are required to thoroughly wash their hands upon exiting the greenhouse. 

Appendix P-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. 
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Appendix P-II-C-2. Facilities (BL3-P) 

Appendix P-II-C-2-a. Definitions (BL3-P) 

Appendix P-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 are usually 

constructed of transparent or translucent material to allow passage of sunlight for plant growth. 

Appendix P-II-C-2-a-(2). The term "greenhouse facility" includes the actual greenhouse rooms or 

compartments for growing plants, including all immediately contiguous hallways and head-house areas, and is 

considered part of the confinement area. The need to maintain negative pressure should be considered when 

constructing or renovating the greenhouse.

Appendix P-II-C-2-b. Greenhouse Design (BL3-P) 

Appendix P-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 P-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 P-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 P-II-C-2-b-(4). The greenhouse facility shall be surrounded by a security fence or protected by 

equivalent security measures. 

Appendix P-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 these structures and 

surfaces (e.g., plumbing and utilities) shall be sealed. 

Appendix P-II-C-2-b-(6). Bench tops and other work surfaces should have seamless surfaces that are 

impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. 

Appendix P-II-C-2-b-(7). The greenhouse contains a foot, elbow, or automatically operated sink, which is 

located near the exit door for hand washing. 

Appendix P-II-C-2-c. Autoclaves (BL3-P) 

Appendix P-II-C-2-c-(1). An autoclave shall be available for decontaminating materials within the greenhouse 

facility. A double-door autoclave is recommended (not required) for the decontamination of materials passing 

out of the greenhouse facility. 

Appendix P-II-C-2-d. Supply and Exhaust Air Ventilation Systems (BL3-P) 

Appendix P-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 P-II-C-2-d-(2). The exhaust air from the greenhouse facility shall be filtered through high efficiency 

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. 
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Appendix P-II-C-2-e. Other (BL3-P) 

Appendix P-II-C-2-e-(1). BL3-P greenhouse containment requirements may be satisfied using a growth 

chamber or growth room within a building provided that the location, access, airflow patterns, and provisions for 

decontamination of experimental materials and supplies meet the intent of the foregoing clauses. 

Appendix P-II-C-2-e-(2). Vacuum lines shall be protected with high efficiency particulate air/HEPA or 

equivalent filters and liquid disinfectant traps. 

Appendix P-II-D. Biosafety Level 4 - Plants (BL4-P) 

Appendix P-II-D-1. Standard Practices (BL4-P) 

Appendix P-II-D-1-a. Greenhouse Access (BL4-P) 

Appendix P-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 P-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 limited by means of 

secure, locked doors. 

Appendix P-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 applicable entry/exit procedures. 

Appendix P-II-D-1-a-(4). Personnel shall enter and exit the greenhouse facility only through the clothing 

change and shower rooms and shall shower each time they exit the greenhouse facility. Personnel shall use 

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 from containment. 

Appendix P-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 P-II-D-1-b. Records (BL4-P) 

Appendix P-II-D-1-b-(1). A record shall be kept of all experimental materials brought into or removed from the 

greenhouse. 

Appendix P-II-D-1-b-(2). A record shall be kept of experiments currently in progress in the greenhouse facility. 

Appendix P-II-D-1-b-(3). A record shall be kept of all personnel entering and exiting the greenhouse facility, 

including the date and time of each entry. 

Appendix P-II-D-1-b-(4). The Principal Investigator shall report any greenhouse accident involving the 

inadvertent release or spill of microorganisms to the Biological Safety Officer, Greenhouse Director, Institutional 

Biosafety Committee, NIH/OBA, and other appropriate authorities immediately (if applicable). Reports to the 

NIH/OBA shall be sent to the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge 

Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-

9839 (fax). Documentation of any such accident shall be prepared and maintained. 
Page 115 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Appendix P-II-D-1-c. Decontamination and Inactivation (BL4-P) 

Appendix P-II-D-1-c-(1). All materials, except for those that are to remain in a viable or intact state for 

experimental 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 

alternative methods (e.g., gas or vapor sterilization) in an airlock or chamber designed for this purpose. 

Appendix P-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 and decontaminated 

before disposal. 

Appendix P-II-D-1-c-(3). Standard microbiological procedures shall be followed for decontamination of 

equipment and materials. Spray or liquid waste or rinse water from containers used to apply the experimental 

microorganisms shall be decontaminated before disposal. 

Appendix P-II-D-1-d. Control of Undesired Species and Motile Macroorganisms (BL4-P) 

Appendix P-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 P-II-D-1-d-(2). Arthropods and other motile macroorganisms used in conjunction with experiments 

requiring BL4-P level physical containment shall be housed in appropriate cages. When appropriate to the 

organism, experiments shall be conducted within cages designed to contain the motile organisms. 

Appendix P-II-D-1-e. Concurrent Experiments Conducted in the Greenhouse (BL4-P) 

Appendix P-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. When the experimental 

microorganisms in use require a containment level lower than BL4-P, greenhouse practices reflect the level of 

containment required by the highest containment level microorganisms being tested. 

Appendix P-II-D-1-f. Signs (BL4-P) 

Appendix P-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 P-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 sign posted on the 

greenhouse access doors. 

Appendix P-II-D-1-f-(3). If there is a risk to human health, a sign shall be posted incorporating the universal 

biosafety symbol. 

Appendix P-II-D-1-g. Transfer of Materials (BL4-P) 

Appendix P-II-D-1-g-(1). Experimental materials that are brought into or removed from the greenhouse in a 

viable or intact state shall be transferred to a non-breakable, sealed, primary container then enclosed in a nonbreakable, sealed secondary container. These containers shall be removed from the greenhouse facility 

through a chemical disinfectant, fumigation chamber, or an airlock designed for this purpose. 

Appendix P-II-D-1-g-(2). Supplies and materials shall be brought into the greenhouse facility through a doubledoor autoclave, fumigation chamber, or airlock that is appropriately decontaminated between each use. 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 P-II-D-1-h. Greenhouse Practices Manual (BL4-P) 

Appendix P-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 of experimental 

organisms. 

Appendix P-II-D-1-i. Protective Clothing (BL4-P) 

Appendix P-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 P-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 inner change room. 

Appendix P-II-D-1-i-(3). All laboratory clothing shall be autoclaved before laundering. 

Appendix P-II-D-2. Facilities (BL4-P) 

Appendix P-II-D-2-a. Greenhouse Design (BL4-P) 

Appendix P-II-D-2-a-(1). The maximum containment greenhouse facility shall consist of a separate building 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 P-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 P-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 P-II-D-2-a-(4). Access doors to the greenhouse shall be self-closing and locking. 

Appendix P-II-D-2-a-(5). The greenhouse facility shall be surrounded by a security fence or protected by 

equivalent security measures. 

Appendix P-II-D-2-a-(6). The walls, floors, and ceilings of the greenhouse shall be constructed to form a sealed 

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 and decontamination of 

the area. All penetrations into these structures and surfaces (e.g., plumbing and utilities) shall be sealed. 

Appendix P-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 P-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 greenhouse facility through the 

change room. 

Appendix P-II-D-2-b. Autoclaves (BL4-P) 

Appendix P-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 the greenhouse 

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 P-II-D-2-c. Supply and Exhaust Air Ventilation Systems (BL4-P) 

Appendix P-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 sense pressure levels. If a 

system malfunctions, the transducers shall sound an alarm. A backup source of power should be considered. 

The supply and exhaust airflow shall be interlocked to assure inward (or zero) airflow at all times. The integrity 

of the greenhouse shall have an air leak rate (decay rate) not to exceed 7 percent per minute (logarithm of 

pressure against time) over a 20-minute period at 2 inches of water gauge pressure. Nominally, this is 0.05 

inches of water gauge pressure loss in 1 minute at 2 inches water gauge pressure. 

Appendix P-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 to treat air supplied to the 

greenhouse facility. HEPA filters shall be certified annually. 

Appendix P-II-D-2-d. Other (BL4-P) 

Appendix P-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 P-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 P-II-D-2-d-(3). Liquid effluent from sinks, floors, and autoclave chambers shall be decontaminated 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 by appropriate standard procedures for 

autoclaved wastes. Decontamination shall be evaluated mechanically and biologically using a recording 

thermometer and an indicator microorganism with a defined heat susceptibility pattern. If liquid wastes are 

decontaminated with chemical disinfectants, the chemicals used must have demonstrated efficacy against the 

target or indicator microorganisms. 

Appendix P-II-D-2-d-(4). If there is a central vacuum system, it shall not serve areas outside of the greenhouse 

facility. In-line high efficiency particulate air/HEPA filters shall be placed as near as practicable to each use 

point or vacuum service cock. Other liquid and gas services to the greenhouse facility shall be protected by 

devices that prevent back-flow. HEPA filters shall be certified annually. 

Appendix P-III. Biological Containment Practices 

Appropriate selection of the following biological containment practices may be used to meet the containment 

requirements for a given organism. The present list is not exhaustive; there may be other ways 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 P-III-A. Biological Containment Practices (Plants) 

Appendix P-III-A-1. Effective dissemination of plants by pollen or seed can be prevented by one or more of 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 male sterile strains, or harvest the 

plant material prior to the reproductive stage; (iii) ensure that experimental 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. 
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Appendix P-III-B. Biological Containment Practices (Microorganisms) 

Appendix P-III-B-1. Effective dissemination of microorganisms beyond the confines of the greenhouse can 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 can serve as hosts or promote the 

transmission of the virus or microorganism, are not present within the farthest 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 or their 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 of transmission requires injury of the target organism, or assures that inadvertent release is 

unlikely to initiate productive infection of organisms outside of the experimental facility. 

Appendix P-III-C. Biological Containment Practices (Macroorganisms) 

Appendix P-III-C-1. Effective dissemination of arthropods and other small animals can be prevented by using 

one or more of the following procedures: (i) use non-flying, flight-impaired, or sterile arthropods; (ii) use nonmotile 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. 

*********************************************************************************************************************** 

APPENDIX Q. PHYSICAL AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT DNA RESEARCH 

INVOLVING ANIMALS 

Appendix Q specifies containment and confinement practices for research involving whole animals, both those 

in which the animal's genome has been altered by stable introduction of recombinant DNA, or DNA derived 

therefrom, into the germ-line (transgenic animals) and experiments involving viable recombinant DNA-modified 

microorganisms tested on whole animals. The appendix applies to animal research activities with the following 

modifications: 

Appendix Q 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 Q-III-D, Footnotes and References for Appendix Q). The animals 

covered in Appendix Q 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 Q require Institutional Biosafety Committee prior approval. 

The institution shall establish and maintain a health surveillance program for personnel engaged in animal 

research involving viable recombinant DNA-containing microorganisms that require Biosafety Level (BL) 3 or 

greater containment in the laboratory. 

Appendix Q-I. General Considerations 

Appendix Q-I-A. Containment Levels 

The containment levels required for research involving recombinant DNA 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 Q. The descriptions include: (i) 

standard practices for physical and biological containment, and (ii) animal facilities. 
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Appendix Q-I-B. Disposal of Animals (BL1-N through BL4-N) 

Appendix Q-I-B-1. When an animal covered by Appendix Q containing recombinant DNA or a recombinant 

DNA-derived organism is euthanized or dies, the carcass shall be disposed of to avoid its use as food for human 

beings or animals unless food use is specifically authorized by an appropriate Federal agency. 

Appendix Q-I-B-2. A permanent record shall be maintained of the experimental use and disposal of each 

animal or group of animals. 

Appendix Q-II. Physical and Biological Containment Levels 

Appendix Q-II-A. Biosafety Level 1 - Animals (BL1-N) 

Appendix Q-II-A-1. Standard Practices (BL1-N) 

Appendix Q-II-A-1-a. Animal Facility Access (BL1-N) 

Appendix Q-II-A-1-a-(1). The containment area shall be locked. 

Appendix Q-II-A-1-a-(2). Access to the containment area shall be limited or restricted when experimental 

animals are being held. 

Appendix Q-II-A-1-a-(3). The containment area shall be patrolled or monitored at frequent intervals. 

Appendix Q-II-A-1-b. Other (BL1-N) 

Appendix Q-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 be marked. In 

addition, transgenic animals should contain distinct and biochemically assayable DNA sequences that allow 

identification of transgenic animals from among non-transgenic animals. 

Appendix Q-II-A-1-b-(2) A double barrier shall be provided to separate male and female animals unless 

reproductive studies are part of the experiment or other measures are taken to avoid reproductive transmission. 

Reproductive incapacitation may be used. 

Appendix Q-II-A-1-b-(3). The containment area shall be in accordance with state and Federal laws and animal 

care requirements. 

Appendix Q-II-A-2. Animal Facilities (BL1-N) 

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

rooms) to minimize the possibility of theft or unintentional release. 

Appendix Q-II-B. Biosafety Level 2 - Animals (BL2-N) (See Appendix Q-III-A, Footnotes and References 

for Appendix Q)

Appendix Q-II-B-1. Standard Practices (BL2-N) 

Appendix Q-II-B-1-a. Animal Facility Access (BL2-N) 

Appendix Q-II-B-1-a-(1). The containment area shall be locked. 

Appendix Q-II-B-1-a-(2). The containment area shall be patrolled or monitored at frequent intervals. 

Appendix Q-II-B-1-a-(3). The containment building shall be controlled and have a locking access. 

Appendix Q-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. 
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Appendix Q-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 Q-II-B-1-b. Decontamination and Inactivation (BL2-N) 

Appendix Q-II-B-1-b-(1). Contaminated materials that are decontaminated at a site away from the laboratory 

shall be placed in a closed durable leak-proof container prior to removal from the laboratory. 

Appendix Q-II-B-1-b-(2). Needles and syringes shall be promptly placed in a puncture-resistant container and 

decontaminated, preferably by autoclaving, before discard or reuse. 

Appendix Q-II-B-1-c. Signs (BL2-N) 

Appendix Q-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 access doors to the animal work 

area. The sign shall indicate: (i) the agent, (ii) the animal species, (iii) the name and telephone number of the 

Animal Facility Director or other responsible individual, and (iv) any special requirements for entering the 

laboratory. 

Appendix Q-II-B-1-d. Protective Clothing (BL2-N) 

Appendix Q-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 Q-II-B-1-d-(2). Special care shall be taken to avoid skin contamination with microorganisms 

containing recombinant DNA. Impervious and/or protective gloves shall be worn when handling experimental 

animals and when skin contact with an infectious agent is unavoidable. 

Appendix Q-II-B-1-e. Records (BL2-N) 

Appendix Q-II-B-1-e-(1). Any incident involving spills and accidents that result in environmental release or 

exposures of animals or laboratory workers to organisms containing recombinant DNA molecules shall be 

reported immediately to the Animal Facility Director, Institutional Biosafety Committee, NIH/OBA, and other 

appropriate authorities (if applicable). Reports to the NIH/OBA shall be sent to the Office of Biotechnology 

Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 

20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). Medical evaluation, surveillance, 

and treatment shall be provided as appropriate and written records maintained. If necessary, the area shall be 

appropriately decontaminated. 

Appendix Q-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 the animal facility. 

Appendix Q-II-B-1-f. Transfer of Materials (BL2-N) 

Appendix Q-II-B-1-f-(1). Biological materials removed from the animal containment area in a viable or intact 

state shall be transferred to a non-breakable sealed primary container and then enclosed in a 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 from the Animal Facility Director. 

Packages containing viable agents may only be opened in a facility having an equivalent or higher level of 

physical containment unless the agent is biologically inactivated or incapable of reproduction. 
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Appendix Q-II-B-1-g. Other (BL2-N) 

Appendix Q-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 be marked. In 

addition, transgenic animals should contain distinct and biochemically assayable DNA sequences that allow 

identification of transgenic animals from among non-transgenic animals. 

Appendix Q-II-B-1-g-(2). Needles and syringes shall be used only for parenteral injection and aspiration of 

fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringeneedle units (i.e., needle is integral to the syringe) shall be used for the injection or aspiration of fluids containing 

organisms that contain recombinant DNA. Extreme caution shall be used when handling needles and syringes 

to avoid autoinoculation and the generation 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. Needles and 

syringes shall be promptly placed in a puncture-resistant container and decontaminated, preferably by 

autoclaving, before discard or reuse. 

Appendix Q-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 particular 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 horizontal transmission (e.g., arthropods, 

contaminated bedding, or animal waste, etc.) should be prevented. 

Appendix Q-II-B-1-g-(4). Eating, drinking, smoking, and applying cosmetics shall not be permitted in the work 

area. 

Appendix Q-II-B-1-g-(5). Individuals who handle materials and animals containing recombinant DNA 

molecules shall be required to wash their hands before exiting the containment area. 

Appendix Q-II-B-1-g-(6). A double barrier shall be provided to separate male and female animals unless 

reproductive studies are part of the experiment or other measures are taken to avoid reproductive transmission. 

Reproductive incapacitation may be used. 

Appendix Q-II-B-1-g-(7). The containment area shall be in accordance with state and Federal laws and animal 

care requirements. 

Appendix Q-II-B-1-g-(8). A biosafety manual shall be prepared or adopted. Personnel shall be advised of 

special hazards and required to read and follow instructions on practices and procedures. 

Appendix Q-II-B-2. Animal Facilities (BL2-N) 

Appendix Q-II-B-2-a. Animals shall be contained within an enclosed structure (animal room or equivalent) 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 the agent in use is not known to 

be transmitted by arthropods. 

Appendix Q-II-B-2-b. Surfaces shall be impervious to water and resistant to acids, alkalis, organic solvents, 

and moderate heat. 

Appendix Q-II-B-2-c. The animal containment area shall be designed so that it can be easily cleaned. 

Appendix Q-II-B-2-d. Windows that open shall be fitted with fly screens. 

Appendix Q-II-B-2-e. An autoclave shall be available for decontamination of laboratory wastes. 

Appendix Q-II-B-2-f. If arthropods are used in the experiment or the agent under study can be transmitted 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. 
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Appendix Q-II-C. Biosafety Level 3 - Animals (BL3-N) (See Appendix Q-III-B, Footnotes and References 

for Appendix Q) 

Appendix Q-II-C-1. Standard Practices (BL3-N) 

Appendix Q-II-C-1-a. Animal Facility Access (BL3-N) 

Appendix Q-II-C-1-a-(1). The containment area shall be locked. 

Appendix Q-II-C-1-a-(2). The containment area shall be patrolled or monitored at frequent intervals. 

Appendix Q-II-C-1-a-(3). The containment building shall be controlled and have a locking access. 

Appendix Q-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 Q-II-C-1-a-(5). Animal room doors, gates, or other closures shall be kept closed when experiments 

are in progress. 

Appendix Q-II-C-1-b. Decontamination and Inactivation (BL3-N) 

Appendix Q-II-C-1-b-(1). The work surfaces of containment equipment shall be decontaminated when work 

with organisms containing recombinant DNA molecules is finished. Where feasible, plastic-backed paper 

toweling shall be used on nonporous work surfaces to facilitate clean-up. 

Appendix Q-II-C-1-b-(2). All animals shall be euthanized at the end of their experimental usefulness and the 

carcasses decontaminated before disposal in an approved manner. 

Appendix Q-II-C-1-b-(3). Needles and syringes shall be promptly placed in a puncture-resistant container and 

decontaminated, preferably by autoclaving, before discard or reuse. 

Appendix Q-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 facility 

to a facility with a lower containment classification. 

Appendix Q-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 every 30 days with an indicator organism. 

Appendix Q-II-C-1-c. Signs (BL3-N) 

Appendix Q-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 access doors to the animal work 

area. The sign shall indicate: (i) the agent, (ii) the animal species, (iii) the name and telephone number of the 

Animal Facility Director or other responsible individual, and (iv) any special requirements for entering the 

laboratory. 

Appendix Q-II-C-1-d. Protective Clothing (BL3-N) 

Appendix Q-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 containment area 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. 
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Appendix Q-II-C-1-d-(2). Special care shall be taken to avoid skin contamination with microorganisms 

containing recombinant DNA. Impervious and/or protective gloves shall be worn when handling experimental 

animals and when skin contact with an infectious agent is unavoidable. 

Appendix Q-II-C-1-d-(3). Appropriate respiratory protection shall be worn in rooms containing experimental 

animals. 

Appendix Q-II-C-1-e. Records (BL3-N) 

Appendix Q-II-C-1-e-(1). Documents regarding experimental animal use and disposal shall be maintained in a 

permanent record book. 

Appendix Q-II-C-1-e-(2). Any incident involving spills and accidents that result in environmental release or 

exposure of animals or laboratory workers to organisms containing recombinant DNA shall be reported 

immediately to the Biological Safety Office, Animal Facility Director, Institutional Biosafety Committee, NIH/OBA, 

and other appropriate authorities (if applicable). Reports to the NIH/OBA shall be sent to the Office of 

Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, 

MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). Medical evaluation, 

surveillance, and treatment shall be provided as appropriate and written records maintained. If necessary, the 

area shall be appropriately decontaminated. 

Appendix Q-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 Q-II-C-1-f. Transfer of Materials (BL3-N) 

Appendix Q-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 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 the 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 or incapable of reproduction. 

Appendix Q-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 Q-II-C-1-g. Other (BL3-N) 

Appendix Q-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 be marked. In 

addition, transgenic animals should contain distinct and biochemically assayable DNA sequences that allow 

identification of transgenic animals from among non-transgenic animals. 

Appendix Q-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 a particular 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 horizontal transmission (e.g., arthropods, 

contaminated bedding, or animal waste) should be prevented. 

Appendix Q-II-C-1-g-(3). Eating, drinking, smoking, and applying cosmetics shall not be permitted in the work 

area. 

Appendix Q-II-C-1-g-(4). Individuals who handle materials and animals containing recombinant DNA 

molecules shall be required to wash their hands before exiting the containment area. 
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Appendix Q-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 Q-II-C-1-g-(6). Animal holding areas shall be cleaned at least once a day and decontaminated 

immediately following any spill of viable materials. 

Appendix Q-II-C-1-g-(7). All procedures shall be performed carefully to minimize the creation of aerosols. 

Appendix Q-II-C-1-g-(8). A double barrier shall be provided to separate male and female animals unless 

reproductive studies are part of the experiment or other measures are taken to avoid reproductive transmission. 

Reproductive incapacitation may be used. 

Appendix Q-II-C-1-g-(9). The containment area shall be in accordance with state and Federal laws and animal 

care requirements. 

Appendix Q-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 Q-II-C-1-g-(11). Personnel shall be required to shower before exiting the BL3-N area and wearing 

personal clothing. 

Appendix Q-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. 

Appendix Q-II-C-1-g-(13). Needles and syringes shall be used only for parenteral injection and aspiration of 

fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringeneedle units (i.e., needle is integral to the syringe) shall be used for the injection or aspiration of fluids containing 

organisms that contain recombinant DNA. Extreme caution shall be used when handling needles and syringes 

to avoid autoinoculation and the generation 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 be promptly placed in a puncture-resistant container and decontaminated, preferably by 

autoclaving, before discard or reuse. 

Appendix Q-II-C-1-g-(14). A biosafety manual shall be prepared or adopted. Personnel shall be advised of 

special hazards and required to read and follow instructions on practices and procedures. 

Appendix Q-II-C-2. Animal Facilities (BL3-N) 

Appendix Q-II-C-2-a. Animals shall be contained within an enclosed structure (animal room or equivalent) 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 the agent in use is not known to 

be transmitted by arthropods. 

Appendix Q-II-C-2-b. The interior walls, floors, and ceilings shall be impervious to water and resistant to acids, 

alkalis, organic solvents, and moderate heat, to facilitate cleaning. Penetrations in these structures and 

surfaces (e.g., plumbing and utilities) shall be sealed. 

Appendix Q-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 be considered 

when constructing or renovating the animal facility. 

Appendix Q-II-C-2-d. An autoclave, incinerator, or other effective means to decontaminate animals and waste 

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. 
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Appendix Q-II-C-2-e. If arthropods are used in the experiment or the agent under study can be transmitted 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 to minimize arthropod entry and 

propagation, including appropriate screening, or the equivalent of access doors. 

Appendix Q-II-C-2-f. Access doors to the containment area shall be self-closing. 

Appendix Q-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 other contiguous 

areas. The animal containment area shall be physically separated from access corridors and other laboratories 

or areas by a double-door clothes change room, equipped with integral showers and airlock. 

Appendix Q-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 every 30 days with an indicator organism. 

Appendix Q-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 exhaust from 

primary containment units, may be used for this purpose if the exhaust air is discharged to the outside and shall 

be dispersed away from occupied areas and air intakes. Personnel shall verify that the direction of the airflow 

(into the animal room) is proper. 

Appendix Q-II-C-2-j. If the agent is transmitted by aerosol, then the exhaust air shall pass through a high 

efficiency particulate air/HEPA filter. 

Appendix Q-II-C-2-k. Vacuum lines shall be protected with high efficiency particulate air/HEPA filters and liquid 

disinfectant traps. 

Appendix Q-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 other special 

containment primary barriers). Prudent judgment must be exercised to implement this ventilation system (e.g., 

animal species) and its discharge location. 

Appendix Q-II-C-2-m. Each animal area shall contain a foot, elbow, or automatically operated sink for hand 

washing. The sink shall be located near the exit door. 

Appendix Q-II-C-2-n. Restraining devices for animals may be required to avoid damage to the integrity of the 

animal containment facility. 

Appendix Q-II-D. Biosafety Level 4 - Animals (BL4-N) (See Appendix Q-III-C, Footnotes and References 

for Appendix Q)

Appendix Q-II-D-1. Standard Practices (BL4-N) 

Appendix Q-II-D-1-a. Animal Facility Access (BL4-N) 

Appendix Q-II-D-1-a-(1). Individuals under 16 years of age shall not be permitted to enter the animal area. 

Appendix Q-II-D-1-a-(2). The containment area shall be locked. 

Appendix Q-II-D-1-a-(3). The containment area shall be patrolled or monitored at frequent intervals. 

Appendix Q-II-D-1-a-(4). The containment building shall be controlled and have a locking access. 
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Appendix Q-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 Q-II-D-1-a-(6). Individuals shall enter and exit the animal facility only through the clothing change 

and shower rooms. 

Appendix Q-II-D-1-a-(7). Personnel shall use the airlocks to enter or exit the laboratory only in an emergency. 

Appendix Q-II-D-1-a-(8). Animal room doors, gates, and other closures shall be kept closed when experiments 

are in progress. 

Appendix Q-II-D-1-b. Decontamination and Inactivation (BL4-N) 

Appendix Q-II-D-1-b-(1). All contaminated liquid or solid wastes shall be decontaminated before disposal. 

Appendix Q-II-D-1-b-(2). The work surfaces and containment equipment shall be decontaminated when work 

with organisms containing recombinant DNA molecules is finished. Where feasible, plastic-backed paper 

toweling shall be used on nonporous work surfaces to facilitate clean-up. 

Appendix Q-II-D-1-b-(3). All wastes from animal rooms and laboratories shall be appropriately decontaminated 

before disposal in an approved manner. 

Appendix Q-II-D-1-b-(4). No materials, except for biological materials that are to remain in a viable or intact 

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 this purpose. 

Appendix Q-II-D-1-b-(5). When ventilated suits are required, the animal personnel shower entrance/exit area 

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 be through an airlock fitted with airtight 

doors. 

Appendix Q-II-D-1-b-(6). Needles and syringes shall be promptly placed in a puncture-resistant container and 

decontaminated, preferably by autoclaving, before discard or reuse. 

Appendix Q-II-D-1-b-(7). Supplies and materials needed in the animal facility shall be brought in by way of the 

double-door autoclave, fumigation chamber, or airlock that shall be appropriately decontaminated between each 

use. 

Appendix Q-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 Q-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. Liquid wastes from shower rooms and toilets shall be decontaminated with 

chemical disinfectants or 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 every 30 days with an indicator organism. Liquid 

wastes from the shower shall be chemically decontaminated using an Environmental Protection Agencyapproved 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. 
Page 127 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Appendix Q-II-D-1-c. Signs (BL4-N) 

Appendix Q-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 access doors to the animal work 

area. The sign shall indicate: (i) the agent, (ii) the animal species, (iii) the name and telephone number of the 

Animal Facility Director, or other responsible individual, and (iv) any special requirements for entering the 

laboratory. 

Appendix Q-II-D-1-d. Protective Clothing (BL4-N) 

Appendix Q-II-D-1-d-(1). Individuals shall enter and exit the animal facility only through the clothing change 

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, and shoes shall be 

provided for all personnel entering the animal facility. When exiting the BL4-N area and before proceeding into 

the shower area, personnel shall remove their laboratory clothing in the inner change room. All laboratory 

clothing shall be autoclaved before laundering. Personnel shall shower each time they exit the animal facility. 

Appendix Q-II-D-1-d-(2). A ventilated head-hood or a one-piece positive pressure suit, which is ventilated by a 

life-support system, shall be worn by all personnel entering rooms that contain experimental 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 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 be through an airlock fitted with airtight doors. 

Appendix Q-II-D-1-d-(3). Appropriate respiratory protection shall be worn in rooms containing experimental 

animals. 

Appendix Q-II-D-1-e. Records (BL4-N) 

Appendix Q-II-D-1-e-(1). Documents regarding experimental animal use and disposal shall be maintained in a 

permanent record book. 

Appendix Q-II-D-1-e-(2). A system shall be established for: (i) reporting laboratory accidents and exposures 

that are a result of overt exposures to organisms containing recombinant DNA, (ii) employee absenteeism, and 

(iii) medical surveillance of potential laboratory-associated 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 to organisms containing recombinant DNA molecules shall be reported 

immediately to the Biological Safety Officer, Animal Facility Director, Institutional Biosafety Committee, 

NIH/OBA, and other appropriate authorities (if applicable). Reports to the NIH/OBA shall be sent to the Office of 

Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, 

MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax). Medical evaluation, 

surveillance, and treatment shall be provided as appropriate and written records maintained. If necessary, the 

area shall be appropriately decontaminated. 

Appendix Q-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 Q-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 Q-II-D-1-f. Transfer of Materials (BL4-N) 

Appendix Q-II-D-1-f-(1). No materials, except for biological materials that are to remain in a viable or intact 

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 this purpose. 
Page 128 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Appendix Q-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 then enclosed 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. Advance approval 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 biologically inactivated or incapable of reproduction. 

Special safety testing, decontamination procedures, and Institutional Biosafety Committee approval shall be 

required to transfer agents or tissue/organ specimens from a BL4-N animal facility to one with a lower 

containment classification. 

Appendix Q-II-D-1-f-(3). Supplies and materials needed in the animal facility shall be brought in by way of the 

double-door autoclave, fumigation chamber, or airlock that shall be appropriately decontaminated between each 

use. After securing the outer doors, personnel within the animal facility retrieve the materials 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 Q-II-D-1-g. Other (BL4-N) 

Appendix Q-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 be marked. In 

addition, transgenic animals should contain distinct and biochemically assayable DNA sequences that allow 

identification of transgenic animals from among non-transgenic animals. 

Appendix Q-II-D-1-g-(2). Eating, drinking, smoking, and applying cosmetics shall not be permitted in the work 

area. 

Appendix Q-II-D-1-g-(3). Individuals who handle materials and animals containing recombinant DNA 

molecules shall be required to wash their hands before exiting the containment area. 

Appendix Q-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 Q-II-D-1-g-(5). Animal holding areas shall be cleaned at least once a day and decontaminated 

immediately following any spill of viable materials. 

Appendix Q-II-D-1-g-(6). All procedures shall be performed carefully to minimize the creation of aerosols. 

Appendix Q-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 Q-II-D-1-g-(8). The containment area shall be in accordance with state and Federal laws and animal 

care requirements. 

Appendix Q-II-D-1-g-(9). The life support system for the ventilated suit or head hood is equipped with alarms 

and emergency back-up air tanks. The exhaust air from the suit area shall be filtered by two sets of 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 waste materials to be removed from the suit 

area. 

Appendix Q-II-D-1-g-(10). Needles and syringes shall be used only for parenteral injection and aspiration of 

fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringeneedle units (i.e., needle is integral to the syringe) shall be used for the injection or aspiration of fluids containing 

organisms that contain recombinant DNA. Extreme caution shall be used when handling needles and syringes 

to avoid autoinoculation and the generation of aerosols during use and disposal. Following use, needles shall 
Page 129 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

not be bent, sheared, replaced in the needle sheath or guard, or removed from the syringe. The needles and 

syringes shall be promptly placed in a puncture-resistant container and decontaminated, preferably by 

autoclaving, before discard or reuse. 

Appendix Q-II-D-1-g-(11). An essential adjunct to the reporting-surveillance system is the availability of a 

facility for quarantine, isolation, and medical care of personnel with potential or known laboratory-associated 

illnesses. 

Appendix Q-II-D-1-g-(12). A biosafety manual shall be prepared or adopted. Personnel shall be advised of 

special hazards and required to read and follow instructions on practices and procedures. 

Appendix Q-II-D-1-g-(13). Vacuum lines shall be protected with high efficiency particulate air/HEPA filters and 

liquid disinfectant traps. 

Appendix Q-II-D-2. Animal Facilities (BL4-N) 

Appendix Q-II-D-2-a. Animals shall be contained within an enclosed structure (animal room or equivalent) to 

minimize the possibility of theft or unintentional release and avoid arthropod access. 

Appendix Q-II-D-2-b. The interior walls, floors, and ceilings shall be impervious to water and resistant to acids, 

alkalis, organic solvents, and moderate heat, to facilitate cleaning. Penetrations in these structures and 

surfaces (e.g., plumbing and utilities) shall be sealed. 

Appendix Q-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 Q-II-D-2-d. 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 Q-II-D-2-e. Access doors to the containment area shall be self-closing. 

Appendix Q-II-D-2-f. All perimeter joints and openings shall be sealed to form an arthropod-proof structure. 

Appendix Q-II-D-2-g. The BL4-N laboratory provides a double barrier to prevent the release of recombinant 

DNA containing microorganisms into the environment. Design of 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 access corridors or other contiguous areas. Physical separation of the 

animal containment area from access corridors or other laboratories or activities shall be provided by a doubledoor clothes change room equipped with integral showers and airlock. 

Appendix Q-II-D-2-h. A necropsy room shall be provided within the BL4-N containment area. 

Appendix Q-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. Liquid wastes from shower rooms and toilets shall be decontaminated with 

chemical disinfectants or 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 every 30 days with an indicator organism. Liquid 

wastes from the shower shall be chemically decontaminated using an Environmental Protection Agencyapproved 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 Q-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 the occupied areas and air 

intakes. Personnel shall verify that the direction of the airflow (into the animal room) is proper. 
Page 130 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

Appendix Q-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 Q-II-D-2-l. All high efficiency particulate air/HEPA filters' frames and housings shall be certified to 

have no detectable smoke [dioctylphthalate] leaks when the exit face (direction of flow) of the filter 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 dioctylphthalate particles 

and a challenge concentration of 80-120 micrograms per liter. The air sampling rate should be at least 1 cfm 

(28.3 liters per minute). 

Appendix Q-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 is capable of killing spores, then assurance 

is provided that all other known agents are inactivated by the parameters established to operate the equipment. 

Test spores meeting this criterion are Bacillus subtilis var. niger or Bacillus stearothermophilis. The operating 

temperature of the incinerator shall be continuously monitored and recorded during use. 

Appendix Q-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 Q-II-D-2-o. Each animal area shall contain a foot, elbow, or automatically operated sink for hand 

washing. The sink shall be located near the exit door. 

Appendix Q-II-D-2-p. Restraining devices for animals may be required to avoid damage to the integrity of the 

containment animal facility. 

Appendix Q-II-D-2-q. The supply water distribution system shall be fitted with a back-flow preventer or break 

tank. 

Appendix Q-II-D-2-r. All utilities, liquid and gas services, shall be protected with devices that avoid back-flow. 

Appendix Q-II-D-2-s. Sewer and other atmospheric ventilation lines shall be equipped minimally with a single 

high efficiency particulate/HEPA filter. Condensate drains from these type housings shall be appropriately 

connected to a contaminated or sanitary drain system. The drain position in the housing dictates the 

appropriate system to be used. 

Appendix Q-III. Footnotes and References for Appendix Q 

Appendix Q-III-A. If recombinant DNA 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 Q-III-B. Personnel who handle pathogenic and potentially lethal agents shall be required to have 

specific training and be supervised by knowledgeable scientists who are experienced in working with these 

agents. BL3-N containment also minimizes escape of recombinant DNA-containing organisms from exhaust air 

or waste material from the containment area. 

Appendix Q-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 of Sections I 

through IV) pose a high level of individual risk for acquiring life-threatening diseases to personnel 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. 
Page 131 - NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) 

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 are trained 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 animal rooms 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 Q-III-D. Other research with non-laboratory animals, which may not appropriately be conducted 

under conditions described in Appendix Q, 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 by 

utilizing 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 that neither 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, the construction 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 non-laboratory 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 P. 

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