94-16200. Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines); Notice DEPARTMENT OF HEALTH AND HUMAN SERVICES  

  • [Federal Register Volume 59, Number 127 (Tuesday, July 5, 1994)]
    [Unknown Section]
    [Page 0]
    From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
    [FR Doc No: 94-16200]
    
    
    [[Page Unknown]]
    
    [Federal Register: July 5, 1994]
    
    
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    Part IV
    
    
    
    
    
    Department of Health and Human Services
    
    
    
    
    
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    National Institutes of Health
    
    
    
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    Guidelines for Research Involving Recombinant DNA Molecules (NIH 
    Guidelines); Notice
    DEPARTMENT OF HEALTH AND HUMAN SERVICES
    
    National Institutes of Health
    
     
    Guidelines for Research Involving Recombinant DNA Molecules (NIH 
    Guidelines)
    
    June 1994.
        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
    Section I-A. Purpose
    Section I-B. Definition of Recombinant DNA Molecules
    Section I-C. General Applicability
    Section I-D. General Definitions
    Section II. Containment
    Section III. Experiments Covered by the NIH Guidelines
    Section III-A. Experiments that Require Institutional Biosafety 
    Committee Approval, RAC Review, and NIH Approval Before Initiation
    Section III-B. Experiments that Require NIH/ORDA and Institutional 
    Biosafety Committee Approval Before Initiation
    Section III-B-1. Experiments Involving the Cloning of Toxin 
    Molecules with LD50 of Less than 100 Nanograms Per Kilogram 
    Body Weight
    Section III-B-2. Accelerated Review of Human Gene Transfer 
    Experiments
    Section III-B-3. Minor Modifications to Human Gene Transfer 
    Experiments
    Section III-C. Experiments that Require Institutional Biosafety 
    Committee Approval Before Initiation
    Section III-C-1. Experiments Using Human or Animal Pathogens (Class 
    2, Class 3, Class 4, or Class 5) Agents as Host-Vector Systems
    Section III-C-2. Experiments in which DNA from Human or Animal 
    Pathogens (Class 2, Class 3, Class 4, or Class 5) Agents is Cloned 
    into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector 
    Systems
    Section III-C-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
    Section III-C-4. Experiments Involving Whole Animals
    Section III-C-5. Experiments Involving Whole Plants
    Section III-C-6. Experiments Involving More than 10 Liters of 
    Culture
    Section III-C-7. Human Gene Transfer Experiments Not Covered by 
    Section III-A-2, III-B-2, III-B-3, and Not Considered Exempt under 
    Section V-U
    Section III-D. Experiments that Require Institutional Biosafety 
    Committee Notice Simultaneous with Initiation
    Section III-D-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-2. Experiments Involving Whole Plants
    Section III-E. Exempt Experiments
    Section IV. Roles and Responsibilities
    Section IV-A. Policy
    Section IV-B. Responsibilities of the Institution
    Section IV-B-1. General Information
    Section IV-B-2. Institutional Biosafety Committee (IBC)
    Section IV-B-3. Biological Safety Officer (BSO)
    Section IV-B-4. Principal Investigator (PI)
    Section IV-C. Responsibilities of the National Institutes of Health 
    (NIH)
    Section IV-C-1. NIH Director
    Section IV-C-1-a. General Responsibilities
    Section IV-C-1-b. Specific Responsibilities
    Section IV-C-1-b-(1). Major Actions
    Section IV-C-1-b-(2). Minor Actions
    Section IV-C-2. Recombinant DNA Advisory Committee (RAC)
    Section IV-C-3. Office of Recombinant DNA Activities (ORDA)
    Section IV-C-4. Other NIH Components
    Section IV-D. Compliance with the NIH Guidelines
    Section IV-E. Voluntary Compliance
    Section V. Footnotes and References of Sections I-IV
    Appendix A. Exemptions under Section III-E-5--Sublists of Natural 
    Exchangers
    Appendix B. Classification of Etiologic Agents and Oncogenic Viruses 
    on the Basis of Hazard
    Appendix B-I. Class 1 Agents
    Appendix B-II. Class 2 Agents
    Appendix B-III. Class 3 Agents
    Appendix B-IV. Class 4 Agents
    Appendix B-V. Class 5 Agents
    Appendix B-VI. Footnotes and References of Appendix B
    Appendix C. Exemptions under Section III-E-6
    Appendix C-I. Recombinant DNA in Tissue Culture
    Appendix C-II. Escherichia coli K-12 Host-Vector Systems
    Appendix C-III. Saccharomyces Host-Vector Systems
    Appendix C-IV. Bacillus subtilis or Bacillus licheniformis Host-
    Vector Systems
    Appendix C-V. Extrachromosomal Elements of Gram Positive Organisms
    Appendix C-VI. Footnotes and References of Appendix C
    Appendix D. Major Actions Taken under the NIH Guidelines
    Appendix E. Certified Host-Vector Systems
    Appendix E-I. Bacillus subtilis
    Appendix E-II. Saccharomyces cerevisiae
    Appendix E-III. Escherichia coli
    Appendix E-IV. Neurospora crassa
    Appendix E-V. Streptomyces
    Appendix E-VI. Pseudomonas putida
    Appendix F. Containment Conditions for Cloning of Genes Coding for 
    the Biosynthesis of Molecules Toxic for Vertebrates
    Appendix F-I. General Information
    Appendix F-II. Cloning of Toxin Molecule Genes in Escherichia coli 
    K-12
    Appendix F-III. Cloning of Toxic Molecule Genes in Organisms other 
    than Escherichia coli K-12
    Appendix F-IV. Specific Approvals
    Appendix G. Physical Containment
    Appendix G-I. Standard Practices and Training
    Appendix G-II. Physical Containment Levels
    Appendix G-II-A. Biosafety Level 1 (BL1)
    Appendix G-II-B. Biosafety Level 2 (BL2)
    Appendix G-II-C. Biosafety Level 3 (BL3)
    Appendix G-II-D. Biosafety Level 4 (BL4)
    Appendix G-III. Footnotes and References of Appendix G
    Appendix H. Shipment
    Appendix I. Biological Containment
    Appendix I-I. Levels of Biological Containment
    Appendix I-I-A. Host-Vector 1 Systems
    Appendix I-I-B. Host-Vector 2 Systems
    Appendix I-II. Certification of Host-Vector Systems
    Appendix I-III. Footnotes and References of Appendix I
    Appendix J. Biotechnology Research Subcommittee
    Appendix K. Physical Containment for Large Scale Uses of Organisms 
    Containing Recombinant DNA Molecules
    Appendix K-I. Selection of Physical Containment Levels
    Appendix K-II. Good Large Scale Practices (GLSP)
    Appendix K-III. Biosafety Level 1 (BL1)--Large Scale
    Appendix K-IV. Biosafety Level 2 (BL2)--Large Scale
    Appendix K-V. Biosafety Level 3 (BL3)--Large Scale
    Appendix K-VI. Footnotes of Appendix K
    Appendix K-VII. Definitions to Accompany Containment Grid and 
    Appendix K
    Appendix L. Release into the Environment of Certain Plants
    Appendix M. Points to Consider in the Design and Submission of 
    Protocols for the Transfer of Recombinant DNA Molecules into the 
    Genome of One or More Human Subjects
    Appendix M-I. Description of Proposal
    Appendix M-I-A. Objectives and Rationale of the Proposed Research
    Appendix M-I-B. Research Design, Anticipated Risks and Benefits
    Appendix M-I-C. Selection of the Patients
    Appendix M-I-D. Informed Consent
    Appendix M-I-E. Privacy and Confidentiality
    Appendix M-II. Special Issues
    Appendix M-III. Guidelines for the Submission of Human Gene Transfer 
    Protocols
    Appendix M-III-A. Principal Investigator-Submitted Material
    Appendix M-III-B. Time Frame for Submissions
    Appendix M-III-C. Oral Responses to the RAC
    Appendix M-III-D. Primary Reviewers' Responses
    Appendix M-IV. Reporting Requirements
    Appendix M-V. Procedures to be Followed for Accelerated Review of 
    Human Gene Transfer Experiments by NIH/ORDA under Section III-B-2
    Appendix M-VI. Procedures to be Followed for Expedited Review of 
    Single Patient Human Gene Transfer Experiments by the NIH Director 
    Under Section III-A-2
    Appendix M-VII. Footnotes of Appendix M
    Appendix P. Physical and Biological Containment for Recombinant DNA 
    Research Involving Plants
    Appendix P-I. General Plant Biosafety Levels
    Appendix P-II. Physical Containment Levels
    Appendix P-II-A. Biosafety Level 1--Plants (BL1-P)
    Appendix P-II-B. Biosafety Level 2--Plants (BL2-P)
    Appendix P-II-C. Biosafety Level 3--Plants (BL3-P)
    Appendix P-II-D. Biosafety Level 4--Plants (BL4-P)
    Appendix P-III. Biological Containment Practices
    Appendix P-III-A. Biological Containment Practices (Plants)
    Appendix P-III-B. Biological Containment Practices (Microorganisms)
    Appendix P-III-C. Biological Containment Practices (Macroorganisms)
    Appendix Q. Physical and Biological Containment for Recombinant DNA 
    Research Involving Animals
    Appendix Q-I. General Considerations
    Appendix Q-I-A. Containment Levels
    Appendix Q-I-B. Disposal of Animals (BL1-N through BL4-N)
    Appendix Q-II. Physical and Biological Containment Levels
    Appendix Q-II-A. Biosafety Level 1--Animals (BL1-N)
    Appendix Q-II-B. Biosafety Level 2--Animals (BL2-N)
    Appendix Q-II-C. Biosafety Level 3--Animals (BL3-N)
    Appendix Q-II-D. Biosafety Level 4--Animals (BL4-N)
    Appendix Q-III. Footnotes and References for Appendix Q
    
    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 the NIH, must be submitted to 
    the NIH or to another Federal agency that has jurisdiction for review 
    and approval. Once approval, or other applicable clearances, has been 
    obtained from a Federal agency other than the NIH (whether the 
    experiment is referred to that agency by the NIH or sent directly there 
    by the submitter), the experiment may proceed without the necessity for 
    NIH review or approval (see exceptions in Sections I-A-2 and I-A-3).
        Section I-A-2. Certain experiments that involve the deliberate 
    transfer of recombinant DNA or DNA or RNA derived from recombinant DNA 
    into one or more human subjects (see Section V-U) shall be considered 
    Major Actions (see Section IV-C-1-b-(1)), and shall require RAC review 
    and NIH Director approval, if determined by NIH/ORDA in consultation 
    with the RAC Chair and/or one or more RAC members, as necessary, to: 
    (i) Represent novel characteristics (e.g., target disease or vector), 
    (ii) represent an uncertain degree of risk to human health or the 
    environment, or (iii) contain information determined to require further 
    public review (see Section III-A-2).
        Section I-A-3. Experiments involving the transfer of recombinant 
    DNA to one or more human subjects that are not considered under Section 
    III-A-2 may qualify for Accelerated Review (see Section III-B-2 and 
    Appendix M-V) and will be considered as Minor Actions (see Section IV-
    C-1-b-(2)-(a)). Actions that qualify for Accelerated Review will be 
    reviewed and approved by NIH/ORDA in consultation with the RAC Chair 
    and/or one or more RAC members, as necessary.
        Certain experiments involving the transfer of recombinant DNA or 
    DNA or RNA derived from recombinant DNA into one or more human subjects 
    (see Section V-U) may be considered exempt from RAC and/or NIH/ORDA 
    review and/or NIH Director approval and only require registration with 
    NIH/ORDA (see Section III-C-7).
    
    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 conducted at or sponsored by 
    an institution that receives any support for recombinant DNA research 
    from the NIH, including research performed directly by the 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-b. All recombinant DNA research performed abroad: 
    Specifically:
        Section I-C-1-b-(1). Research supported by NIH funds.
        Section I-C-1-b-(2). If they involve testing in humans of materials 
    containing recombinant DNA developed with NIH funds and 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.
    
    Section I-D. General Definitions
    
        The following terms, which are used throughout the NIH Guidelines, 
    are defined as follows:
        Section I-D-1. An ``institution'' is any public or private entity 
    (including Federal, state, and local government agencies).
        Section I-D-2. An ``Institutional Biosafety Committee'' is a 
    committee that: (i) Meets the requirements for membership specified in 
    Section IV-B-2, and (ii) reviews, approves, and oversees projects in 
    accordance with the responsibilities defined in Section IV-B-2.
        Section I-D-3. The ``Office of Recombinant DNA Activities (ORDA)'' 
    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.
        Section I-D-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.
        Section I-D-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-D-6. ``Deliberate release'' is defined as a planned 
    introduction of recombinant DNA-containing microorganisms, plants, or 
    animals into the environment.
    
    Section II. 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-A). 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. 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).
        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, 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 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. 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. 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.
        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 five categories of experiments involving 
    recombinant DNA: (i) Those that require RAC review and NIH and 
    Institutional Biosafety Committee approval before initiation (see 
    section III-A), (ii) those that require NIH/ORDA and Institutional 
    Biosafety Committee approval before initiation (see section III-B); 
    (iii) those that require Institutional Biosafety Committee approval 
    before initiation (see section III-C), (iv) those that require 
    Institutional Biosafety Committee notification simultaneous with 
    initiation (see section III-D), and (v) those that are exempt from the 
    NIH Guidelines (see section III-E).
    
        Note: If an experiment falls into either section III-A or 
    section III-B and one of the other categories, the rules pertaining 
    to section III-A or section III-B shall be followed. If an 
    experiment falls into section III-E and into either sections III-C 
    or III-D categories as well, the experiment is considered exempt 
    from the NIH Guidelines.
        Any change in containment level, which is different from those 
    specified in the NIH Guidelines, may not be initiated without the 
    express approval of NIH/ORDA (see Minor Actions, section IV-C-1-b-(2) 
    and its subsections).
    
    Section III-A. Experiments that Require Institutional Biosafety 
    Committee Approval, RAC Review, and NIH Approval Before Initiation
    
        Experiments in this category are considered Major Actions (see 
    section IV-C-1-b-(1)) and cannot be initiated without submission of 
    relevant information on the proposed experiment to the Office of 
    Recombinant DNA Activities, National Institutes of Health, Building 31, 
    room 4B11, Bethesda, Maryland 20892, (301) 496-9838, the publication of 
    the proposal in the Federal Register for 15 days of comment, reviewed 
    by the RAC, and specific approval by the NIH (not applicable for 
    Expedited Review single patient human gene transfer experiments 
    considered under Appendix M-VI). The containment conditions for such 
    experiments will be recommended by the RAC and set by the NIH at the 
    time of approval. Such experiments require Institutional Biosafety 
    Committee approval before initiation. Specific experiments already 
    approved are included in Appendix D which may be obtained from the 
    Office of Recombinant DNA Activities, National Institutes of Health, 
    Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-9838.
        Section III-A-1. Deliberate transfer of a drug resistance trait to 
    microorganisms that are not known to acquire the trait naturally (see 
    section V-B), if such acquisition could compromise the use of the drug 
    to control disease agents in humans, veterinary medicine, or 
    agriculture.
        Section III-A-2. Certain experiments involving the deliberate 
    transfer of recombinant DNA or DNA or RNA derived from recombinant DNA 
    into one or more human subjects (see section V-U) shall be considered 
    Major Actions (see section IV-C-1-b-(1) and Appendix M-III), and shall 
    require RAC review and NIH Director approval, if determined by NIH/
    ORDA, in consultation with the RAC Chair and one or more RAC members, 
    as necessary, to: (i) Represent novel characteristics (e.g., target 
    disease or vector), (ii) represent an uncertain degree of risk to human 
    health or the environment, or (iii) contain information determined to 
    require further public review. The requirement for RAC review shall not 
    be considered to preempt any other required review or approval of 
    experiments with one or more human subjects. Relevant Institutional 
    Biosafety Committee and Institutional Review Board reviews and 
    approvals of the proposal should be completed before submission to NIH. 
    Certain experiments involving deliberate transfer of recombinant DNA or 
    DNA or RNA derived from recombinant DNA into one or more human subjects 
    may qualify for the Accelerated Review process (see section III-B-2). 
    Certain categories of experiments involving the deliberate transfer of 
    recombinant DNA or DNA or RNA derived from recombinant DNA into one or 
    more human subjects and that are not covered by section V-U, may be 
    considered exempt from RAC and/or NIH/ORDA review and/or NIH Director 
    approval and only require registration with NIH/ORDA (see section III-
    C-7).
    
    Section III-B. Experiments That Require NIH/ORDA and Institutional 
    Biosafety Committee Approval Before Initiation
    
    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 Recombinant DNA Activities, National Institutes of 
    Health, Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-
    9838.
        Section III-B-1-(a). Experiments in this category cannot be 
    initiated without submission of relevant information on the proposed 
    experiment to NIH/ORDA. The containment conditions for such experiments 
    will be determined by NIH/ORDA in consultation with ad hoc experts. 
    Such experiments require Institutional Biosafety Committee approval 
    before initiation (see section IV-B-2-b-(1)).
    
    Section III-B-2. Accelerated Review of Human Gene Transfer Experiments
    
        As determined by NIH/ORDA, in consultation with the RAC Chair and 
    one or more RAC members, as necessary, certain categories of human gene 
    transfer experiments may be considered as Minor Actions and qualify for 
    Accelerated Review and approval (see section IV-C-1-b-(2)-(a), Appendix 
    M-III-A, and Appendix M-V). The RAC Chair will present a report of all 
    NIH/ORDA approved human gene transfer protocols at the next regularly 
    scheduled RAC meeting. If NIH/ORDA determines that an experiment does 
    not qualify for the Accelerated Review process, the Principal 
    Investigator must submit the proposal for full RAC review  8 
    weeks prior to the next scheduled RAC meeting (See section III-A-2).
    Section III-B-3. Minor Modifications to Human Gene Transfer Experiments
        A minor modification in a human gene transfer protocol is a 
    modification that does not significantly alter the basic design of the 
    protocol and that does not increase risk to human subjects or the 
    environment. After approval has been obtained by the relevant 
    Institutional Biosafety Committee and Institutional Review Board, NIH/
    ORDA will consider the change in consultation with the RAC Chair and 
    one or more RAC members, as necessary. Submit minor modifications to 
    the Office of Recombinant DNA Activities, National Institutes of 
    Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
    9838. The RAC Chair will provide a report on any such approvals at the 
    next regularly scheduled RAC meeting.
    Section III-C. 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)).
    Section III-C-1. Experiments Using Human or Animal Pathogens (Class 2, 
    Class 3, Class 4, or Class 5 Agents (see Section V-A) as Host-Vector 
    Systems
        Section III-C-1-a. Experiments involving the introduction of 
    recombinant DNA into Class 2 agents shall be conducted at Biosafety 
    Level (BL) 2 containment. Experiments with such agents shall be 
    conducted with whole animals at BL2 or BL2-N (Animals) containment.
        Section III-C-1-b. Experiments involving the introduction of 
    recombinant DNA into Class 3 agents shall be conducted at BL3 
    containment. Experiments with such agents shall be conducted with whole 
    animals at BL3 or BL3-N containment.
        Section III-C-1-c. Experiments involving the introduction of 
    recombinant DNA into Class 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-C-1-d. Containment conditions for experiments involving 
    the introduction of recombinant DNA into Class 5 agents shall be set on 
    a case-by-case basis following NIH/ORDA review. A U.S. Department of 
    Agriculture permit is required for work with Class 5 agents (see 
    Sections V-R and V-T). Experiments with such agents shall be conducted 
    with whole animals at BL4 or BL4-N containment.
    Section III-C-2. Experiments in Which DNA From Human or Animal 
    Pathogens (Class 2, Class 3, Class 4, or Class 5 Agents (see Section V-
    A) is Cloned Into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-
    Vector Systems
        Section III-C-2-a. Experiments in which DNA from Class 2 or Class 3 
    agents (see Section V-A) is transferred into nonpathogenic prokaryotes 
    or lower eukaryotes may be performed under BL2 containment. Experiments 
    in which DNA from Class 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-E). 
    Experiments involving the formation of recombinant DNA for certain 
    genes coding for molecules toxic for vertebrates require NIH/ORDA 
    approval (see Section III-B-1) or shall be conducted under NIH 
    specified conditions as described in Appendix F.
        Section III-C-2-b. Containment conditions for experiments in which 
    DNA from Class 5 agents is transferred into nonpathogenic prokaryotes 
    or lower eukaryotes shall be determined by NIH/ORDA following a case-
    by-case review. A U.S. Department of Agriculture permit is required for 
    work with Class 5 agents (see Sections V-R and V-T).
    Section III-C-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
        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-Q) being considered 
    identical (see Section V-S), are considered defective and may be 
    used in the absence of helper under the conditions specified in 
    Section III-D-1.
    
        Section III-C-3-a. Experiments involving the use of infectious or 
    defective Class 2 animal viruses (see Section V-A, Appendix B-II, and 
    Appendix B-II-E) in the presence of helper virus may be conducted at 
    BL2.
        Section III-C-3-b. Experiments involving the use of infectious or 
    defective Class 3 animal viruses (see Section V-A and Appendix B-III-D) 
    in the presence of helper virus may be conducted at BL3.
        Section III-C-3-c. Experiments involving the use of infectious or 
    defective Class 4 animal viruses (see Section V-A and Appendix B-IV-D) 
    in the presence of helper virus may be conducted at BL4.
        Section III-C-3-d. Experiments involving the use of infectious or 
    defective Class 5 viruses (see Section V-A and Appendix B-V) in the 
    presence of helper virus shall be determined on a case-by-case basis 
    following NIH/ORDA review. A U.S. Department of Agriculture permit is 
    required for work with Class 5 agents (see Sections V-R and V-T).
        Section III-C-3-e. Experiments involving the use of infectious or 
    defective animal or plant viruses in the presence of helper virus are 
    not covered in Sections III-C-3-a through III-C-3-d and may be 
    conducted at BL1.
    
    Section III-C-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-C-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). 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-C-
    4-b. For experiments involving recombinant DNA-modified Class 2, 3, 4, 
    or 5 organisms, see Section V-A. 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 Class 5 agents (see Section V-R and V-
    T).
        Section III-C-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-C-1 or III-C-4-a, 
    the appropriate containment shall be determined by the Institutional 
    Biosafety Committee.
    
    Section III-C-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-C-5-a through III-C-5-e. If experiments involving whole plants are 
    not described in Section III-C-5 and do not fall under Sections III-A, 
    III-B, or III-E, they are included in Section III-D.
    
        Note: For recombinant DNA experiments falling under Sections 
    III-C-5-a through III-C-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-C-5-a. BL3-P (Plants) or BL2-P + biological containment 
    is recommended for experiments involving most exotic (see Section V-W) 
    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-C-5-b. BL3-P or BL2-P + biological containment is 
    recommended for experiments involving plants containing cloned genomes 
    of readily transmissible exotic (see Section V-W) 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-C-5-c. BL4-P containment is recommended for experiments 
    with a small number of readily transmissible exotic (see Section V-W) 
    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-C-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="" and="" require="" nih/orda="" and="" institutional="" biosafety="" committee="" approval="" before="" initiation.="" section="" iii-c-5-e.="" bl3-p="" or="" bl2-p="" +="" biological="" containment="" is="" recommended="" for="" experiments="" with="" microbial="" pathogens="" of="" insects="" or="" small="" animals="" associated="" with="" plants="" if="" the="" recombinant="" dna-modified="" organism="" has="" a="" recognized="" potential="" for="" serious="" detrimental="" impact="" on="" managed="" or="" natural="" ecosystems.="" section="" iii-c-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-c-7.="" human="" gene="" transfer="" experiments="" not="" covered="" by="" sections="" iii-a-2,="" iii-b-2,="" iii-b-3,="" and="" not="" considered="" exempt="" under="" section="" v-u="" certain="" experiments="" involving="" the="" transfer="" of="" recombinant="" dna="" or="" dna="" or="" rna="" derived="" from="" recombinant="" dna="" into="" one="" or="" more="" human="" subjects="" that="" are="" not="" covered="" by="" sections="" iii-a-2,="" iii-b-2,="" iii-b-3,="" and="" that="" are="" not="" considered="" exempt="" under="" section="" v-u="" must="" be="" registered="" with="" nih/orda.="" the="" relevant="" institutional="" biosafety="" committee="" and="" institutional="" review="" board="" must="" review="" and="" approve="" all="" experiments="" in="" this="" category="" prior="" to="" their="" initiation.="" section="" iii-d.="" experiments="" that="" require="" institutional="" biosafety="" committee="" notice="" simultaneous="" with="" initiation="" experiments="" not="" included="" in="" sections="" iii-a,="" iii-b,="" iii-c,="" iii-e,="" and="" their="" subsections="" are="" considered="" in="" section="" iii-d.="" all="" such="" experiments="" may="" be="" conducted="" at="" bl1="" containment.="" for="" experiments="" in="" this="" category,="" a="" registration="" document="" (see="" section="" iii-c)="" 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).="" for="" example,="" experiments="" in="" which="" all="" components="" derived="" from="" non-pathogenic="" prokaryotes="" and="" non-pathogenic="" lower="" eukaryotes="" fall="" under="" section="" iii-d="" and="" may="" be="" conducted="" at="" bl1="" containment.="" section="" iii-d-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="" (see="" section="" v-q)="" being="" considered="" identical="" (see="" section="" v-s))="" 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-c-3="" 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-d-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-c,="" or="" iii-e.="" it="" should="" be="" emphasized="" that="" knowledge="" of="" the="" organisms="" and="" judgment="" based="" on="" accepted="" scientific="" practices="" should="" be="" used="" in="" all="" cases="" in="" selecting="" the="" appropriate="" level="" of="" containment.="" for="" example,="" if="" the="" genetic="" modification="" has="" the="" objective="" of="" increasing="" pathogenicity="" or="" converting="" a="" non-pathogenic="" organism="" into="" a="" pathogen,="" then="" a="" higher="" level="" of="" containment="" may="" be="" appropriate="" depending="" on="" the="" organism,="" its="" mode="" of="" dissemination,="" and="" its="" target="" organisms.="" by="" contrast,="" a="" lower="" level="" of="" containment="" may="" be="" appropriate="" for="" small="" animals="" associated="" with="" many="" types="" of="" recombinant="" dna-modified="" plants.="" section="" iii-d-2-a.="" bl1-p="" is="" recommended="" for="" all="" experiments="" with="" recombinant="" dna-containing="" plants="" and="" plant-associated="" microorganisms="" not="" covered="" in="" section="" iii-d-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-w)="" 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-d-2-b.="" bl2-p="" or="" bl1-p="" +="" biological="" containment="" is="" recommended="" for="" the="" following="" experiments:="" section="" iii-d-2-b-(1).="" plants="" modified="" by="" recombinant="" dna="" that="" are="" noxious="" weeds="" or="" can="" interbreed="" with="" noxious="" weeds="" in="" the="" immediate="" geographic="" area.="" section="" iii-d-2-b-(2).="" plants="" in="" which="" the="" introduced="" dna="" represents="" the="" complete="" genome="" of="" a="" non-exotic="" infectious="" agent="" (see="" section="" v-w).="" section="" iii-d-2-b-(3).="" plants="" associated="" with="" recombinant="" dna-="" modified="" non-exotic="" microorganisms="" that="" have="" a="" recognized="" potential="" for="" serious="" detrimental="" impact="" on="" managed="" or="" natural="" ecosystems="" (see="" section="" v-w).="" section="" iii-d-2-b-(4).="" plants="" associated="" with="" recombinant="" dna-="" modified="" exotic="" microorganisms="" that="" have="" no="" recognized="" potential="" for="" serious="" natural="" ecosystems="" (see="" section="" v-w).="" section="" iii-d-2-b-(5).="" experiments="" with="" recombinant="" dna-modified="" arthropods="" or="" small="" animals="" associated="" with="" plants,="" or="" with="" arthropods="" or="" small="" animals="" with="" recombinant="" dna-modified="" microorganisms="" associated="" with="" them="" if="" the="" recombinant="" dna-modified="" microorganisms="" have="" no="" recognized="" potential="" for="" serious="" detrimental="" impact="" on="" managed="" or="" natural="" ecosystems="" (see="" section="" v-w).="" section="" iii-e.="" 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-e-1.="" those="" that="" are="" not="" in="" organisms="" or="" viruses.="" section="" iii-e-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-e-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-e-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-e-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)).="" see="" appendices="" a-i="" through="" a-vi="" for="" a="" list="" of="" natural="" exchangers="" that="" are="" exempt="" from="" the="" nih="" guidelines.="" section="" iii-e-6.="" those="" that="" do="" not="" present="" a="" significant="" risk="" to="" health="" or="" the="" environment="" (see="" section="" iv-c-1-b-(1)-(c)),="" as="" determined="" by="" the="" nih="" director,="" with="" the="" advice="" of="" the="" rac,="" and="" following="" appropriate="" notice="" and="" opportunity="" for="" public="" comment.="" see="" appendix="" c="" for="" other="" classes="" of="" experiments="" which="" are="" exempt="" from="" the="" nih="" guidelines.="" 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="" 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="" recombinant="" dna="" activities="" comply="" 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="" the="" 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.="" assist="" and="" ensure="" compliance="" with="" the="" nih="" guidelines="" by="" principal="" investigators="" conducting="" research="" at="" the="" institution="" as="" specified="" in="" section="" iv-b-4.="" section="" iv-b-1-e.="" ensure="" appropriate="" training="" for="" the="" institutional="" biosafety="" committee="" chair="" and="" members,="" biological="" safety="" officer="" (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-f.="" 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="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838.="" section="" iv-b-1-g.="" report="" any="" significant="" problems,="" violations="" of="" the="" nih="" guidelines,="" or="" any="" significant="" research-related="" accidents="" and="" illnesses="" to="" nih/orda="" 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="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-="" 9838.="" 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="" 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="" require="" institutional="" biosafety="" committee="" prior="" approval.="" when="" the="" institution="" conducts="" recombinant="" dna="" research="" at="" bl3="" or="" bl4,="" a="" biological="" safety="" officer="" is="" mandatory="" and="" shall="" be="" a="" member="" of="" the="" institutional="" biosafety="" committee="" (see="" section="" iv-b-3).="" 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="" a="" report="" with="" nih/="" orda="" which="" includes="" the="" names="" and="" biographical="" sketches="" of="" all="" institutional="" biosafety="" committee="" members,="" including="" community="" members,="" in="" such="" form="" and="" at="" such="" times="" as="" required="" by="" nih/orda.="" 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="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838.="" section="" iv-b-2-b.="" functions="" on="" behalf="" of="" the="" institution,="" the="" institutional="" biosafety="" committee="" is="" responsible="" for:="" section="" iv-b-2-b-(1).="" reviewing="" recombinant="" dna="" research="" conducted="" at="" or="" sponsored="" by="" the="" institution="" for="" compliance="" with="" the="" nih="" guidelines="" as="" specified="" in="" section="" iii="" 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,="" and="" (ii)="" assessment="" of="" the="" facilities,="" procedures,="" practices,="" and="" training="" and="" expertise="" of="" personnel="" involved="" in="" recombinant="" dna="" research.="" 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-c-2-a.="" section="" iv-b-2-b-(4).="" setting="" containment="" levels="" as="" specified="" in="" sections="" iii-c-4-b="" and="" iii-c-5.="" 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/orda="" within="" 30="" days,="" unless="" the="" institutional="" biosafety="" committee="" determines="" that="" a="" report="" has="" already="" been="" filed="" by="" the="" principal="" investigator.="" reports="" to="" nih/orda="" shall="" be="" sent="" to="" the="" office="" of="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838.="" 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.="" 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.="" 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.="" 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.="" section="" iv-b-4-a.="" general="" responsibilities="" as="" part="" of="" this="" general="" responsibility,="" the="" principal="" investigator="" shall:="" section="" iv-b-4-a-(1).="" initiate="" or="" modify="" no="" recombinant="" dna="" research="" which="" requires="" institutional="" biosafety="" committee="" approval="" prior="" to="" initiation="" (see="" sections="" iii-a,="" iii-b,="" and="" iii-c)="" 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-4-a-(2).="" determine="" whether="" experiments="" are="" covered="" by="" section="" iii-d="" and="" that="" the="" appropriate="" procedures="" are="" followed;="" section="" iv-b-4-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/orda,="" and="" other="" appropriate="" authorities="" (if="" applicable)="" within="" 30="" days="" (reports="" to="" nih/orda="" shall="" be="" sent="" to="" the="" office="" of="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838);="" section="" iv-b-4-a-(4).="" report="" any="" new="" information="" bearing="" on="" the="" nih="" guidelines="" to="" the="" institutional="" biosafety="" committee="" and="" to="" nih/orda="" (reports="" to="" nih/orda="" shall="" be="" sent="" to="" the="" office="" of="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838);="" section="" iv-b-4-a-(5).="" be="" adequately="" trained="" in="" good="" microbiological="" techniques;="" section="" iv-b-4-a-(6).="" adhere="" to="" institutional="" biosafety="" committee-="" approved="" emergency="" plans="" for="" handling="" accidental="" spills="" and="" personnel="" contamination;="" and="" section="" iv-b-4-a-(7).="" comply="" with="" shipping="" requirements="" for="" recombinant="" dna="" molecules="" (see="" appendix="" h="" for="" shipping="" requirements="" and="" the="" laboratory="" safety="" monograph="" for="" technical="" recommendations).="" section="" iv-b-4-b.="" submissions="" by="" the="" principal="" investigator="" to="" the="" nih/="" orda="" the="" principal="" investigator="" shall:="" section="" iv-b-4-b-(1).="" submit="" information="" to="" nih/orda="" for="" certification="" of="" new="" host-vector="" systems;="" section="" iv-b-4-b-(2).="" petition="" nih/orda,="" with="" notice="" to="" the="" institutional="" biosafety="" committee,="" for="" proposed="" exemptions="" to="" the="" nih="" guidelines;="" section="" iv-b-4-b-(3).="" petition="" nih/orda,="" with="" concurrence="" of="" the="" institutional="" biosafety="" committee,="" for="" approval="" to="" conduct="" experiments="" specified="" in="" sections="" iii-a="" and="" iii-b="" of="" the="" nih="" guidelines;="" section="" iv-b-4-b-(4).="" petition="" nih/orda="" for="" determination="" of="" containment="" for="" experiments="" requiring="" case-by-case="" review;="" and="" section="" iv-b-4-b-(5).="" petition="" nih/orda="" for="" determination="" of="" containment="" for="" experiments="" not="" covered="" by="" the="" nih="" guidelines.="" section="" iv-b-4-c.="" submissions="" by="" the="" principal="" investigator="" to="" the="" institutional="" biosafety="" committee="" the="" principal="" investigator="" shall:="" section="" iv-b-4-c-(1).="" make="" an="" initial="" determination="" of="" the="" required="" levels="" of="" physical="" and="" biological="" containment="" in="" accordance="" with="" the="" nih="" guidelines;="" section="" iv-b-4-c-(2).="" select="" appropriate="" microbiological="" practices="" and="" laboratory="" techniques="" to="" be="" used="" for="" the="" research;="" section="" iv-b-4-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,="" or="" iii-d,="" to="" the="" institutional="" biosafety="" committee="" for="" review="" and="" approval="" or="" disapproval;="" and="" section="" iv-b-4-c-(4).="" remain="" in="" communication="" with="" the="" institutional="" biosafety="" committee="" throughout="" the="" conduct="" of="" the="" project.="" section="" iv-b-4-d.="" responsibilities="" of="" the="" principal="" investigator="" prior="" to="" initiating="" research="" the="" principal="" investigator="" shall:="" section="" iv-b-4-d-(1).="" make="" available="" to="" all="" laboratory="" staff="" the="" protocols="" that="" describe="" the="" potential="" biohazards="" and="" the="" precautions="" to="" be="" taken;="" section="" iv-b-4-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-4-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-4-e.="" responsibilities="" of="" the="" principal="" investigator="" during="" the="" conduct="" of="" the="" research="" the="" principal="" investigator="" shall:="" section="" iv-b-4-e-(1).="" supervise="" the="" safety="" performance="" of="" the="" laboratory="" staff="" to="" ensure="" that="" the="" required="" safety="" practices="" and="" techniques="" are="" employed;="" section="" iv-b-4-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),="" the="" institutional="" biosafety="" committee,="" nih/orda,="" and="" other="" appropriate="" authorities="" (if="" applicable)="" (reports="" to="" the="" nih/orda="" shall="" be="" sent="" to="" the="" office="" of="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838);="" section="" iv-b-4-e-(3).="" correct="" work="" errors="" and="" conditions="" that="" may="" result="" in="" the="" release="" of="" recombinant="" dna="" materials;="" and="" section="" iv-b-4-e-(4).="" ensure="" the="" integrity="" of="" the="" physical="" containment="" (e.g.,="" biological="" safety="" cabinets)="" and="" the="" biological="" containment="" (e.g.,="" purity="" and="" genotypic="" and="" phenotypic="" characteristics).="" section="" iv-c.="" responsibilities="" of="" the="" national="" institutes="" of="" health="" (nih)="" section="" iv-c-1.="" nih="" director="" the="" nih="" director="" is="" responsible="" for:="" (i)="" establishing="" the="" nih="" guidelines,="" (ii)="" overseeing="" their="" implementation,="" and="" (iii)="" their="" final="" interpretation.="" the="" nih="" director="" has="" responsibilities="" under="" the="" nih="" guidelines="" that="" involve="" orda="" and="" the="" rac.="" orda's="" responsibilities="" under="" the="" nih="" guidelines="" are="" administrative.="" advice="" from="" the="" 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="" the="" rac="" to="" carry="" out="" the="" responsibilities="" set="" forth="" in="" section="" iv-c-2="" (rac="" membership="" is="" specified="" in="" its="" charter="" and="" in="" section="" iv-c-2);="" and="" section="" iv-c-1-a-(3).="" establishing="" and="" maintaining="" orda="" to="" carry="" out="" the="" responsibilities="" defined="" in="" section="" iv-c-3.="" 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="" the="" rac="" and="" provide="" an="" opportunity="" for="" public="" and="" federal="" agency="" comment.="" specifically,="" the="" notice="" of="" meeting="" and="" proposed="" actions="" to="" the="" nih="" guidelines="" shall="" be="" published="" in="" the="" federal="" register="" at="" least="" 15="" days="" before="" the="" rac="" meeting="" (not="" applicable="" for="" expedited="" review="" single="" patient="" human="" gene="" transfer="" experiments="" considered="" under="" appendix="" m-vi).="" the="" nih="" director's="" decision,="" 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,="" 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;="" 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;="" 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).="" 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/orda="" shall="" carry="" out="" certain="" functions="" as="" delegated="" to="" it="" by="" the="" nih="" director="" (see="" section="" iv-c-3).="" minor="" actions="" (as="" determined="" by="" nih/orda="" in="" consultation="" with="" the="" rac="" chair="" and="" one="" or="" more="" rac="" members,="" as="" necessary)="" will="" be="" transmitted="" to="" the="" rac="" and="" institutional="" biosafety="" committee="" chairs:="" section="" iv-c-1-b-(2)-(a).="" reviewing="" and="" approving="" certain="" experiments="" involving="" the="" deliberate="" transfer="" of="" recombinant="" dna="" or="" dna="" or="" rna="" derived="" from="" recombinant="" dna="" into="" one="" or="" more="" human="" subjects="" that="" qualify="" for="" the="" accelerated="" review="" process="" (see="" section="" iii-b-2);="" section="" iv-c-1-b-(2)-(b).="" reviewing="" and="" approving="" minor="" changes="" to="" human="" gene="" transfer="" protocols="" under="" section="" iii-a-2="" and="" iii-b-2;="" section="" iv-c-1-b-(2)-(c).="" changing="" containment="" levels="" for="" experiments="" that="" are="" specified="" in="" section="" iii;="" section="" iv-c-1-b-(2)-(d).="" assigning="" containment="" levels="" for="" experiments="" not="" explicitly="" considered="" in="" the="" nih="" guidelines;="" and="" section="" iv-c-1-b-(2)-(e).="" revising="" the="" classification="" of="" etiologic="" agents="" for="" the="" purpose="" of="" these="" nih="" guidelines="" (see="" section="" v-a).="" section="" iv-c-1-b-(2)-(f).="" interpreting="" the="" nih="" guidelines="" for="" experiments="" to="" which="" the="" nih="" guidelines="" do="" not="" specifically="" assign="" containment="" levels;="" section="" iv-c-1-b-(2)-(g).="" setting="" containment="" under="" sections="" iii-c-="" 1-d="" and="" iii-c-2-="" b;="" section="" iv-c-1-b-(2)-(h).="" approving="" minor="" modifications="" of="" already="" certified="" host-vector="" systems="" (the="" standards="" and="" procedures="" for="" such="" modifications="" are="" described="" in="" appendix="" i-ii);="" section="" iv-c-1-b-(2)-(i).="" decertifying="" already="" certified="" host-="" vector="" systems;="" section="" iv-c-1-b-(2)-(j).="" adding="" new="" entries="" to="" the="" list="" of="" molecules="" toxic="" for="" vertebrates="" (see="" appendix="" f);="" and="" section="" iv-c-1-b-(2)-(k).="" determining="" appropriate="" containment="" conditions="" for="" experiments="" according="" to="" case="" precedents="" developed="" under="" section="" iv-c-1-b-(2)-(c).="" section="" iv-c-1-b-(3).="" the="" nih="" director="" shall="" conduct,="" support,="" and="" assist="" training="" programs="" in="" laboratory="" safety="" for="" institutional="" biosafety="" committee="" members,="" biological="" safety="" officers,="" principal="" investigators,="" and="" laboratory="" staff.="" section="" iv-c-2.="" recombinant="" dna="" advisory="" committee="" (rac)="" the="" rac="" is="" responsible="" for="" carrying="" out="" specified="" functions="" cited="" below="" as="" well="" as="" others="" assigned="" under="" its="" charter="" or="" by="" the="" dhhs="" secretary,="" the="" dhhs="" assistant="" secretary="" for="" health,="" and="" the="" nih="" director.="" the="" rac="" consists="" of="" 25="" members="" including="" the="" chair,="" appointed="" by="" the="" dhhs="" secretary="" or="" his/her="" designee,="" at="" least="" fourteen="" of="" whom="" are="" selected="" from="" authorities="" knowledgeable="" in="" the="" fields="" of="" molecular="" genetics,="" molecular="" biology,="" recombinant="" dna="" research,="" or="" other="" scientific="" fields.="" at="" least="" six="" members="" of="" the="" rac="" shall="" be="" persons="" knowledgeable="" in="" applicable="" law,="" standards="" of="" professional="" conduct="" and="" practice,="" public="" attitudes,="" the="" environment,="" public="" health,="" occupational="" health,="" or="" related="" fields.="" representatives="" from="" federal="" agencies="" shall="" serve="" as="" non-voting="" members.="" nominations="" for="" the="" rac="" may="" be="" submitted="" to="" the="" office="" of="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838.="" 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.="" the="" rac="" shall="" be="" responsible="" for="" advising="" the="" nih="" director="" on="" the="" actions="" listed="" in="" sections="" iv-c-1-b-(1).="" section="" iv-c-3.="" office="" of="" recombinant="" dna="" activities="" (orda)="" orda="" 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.="" orda="" shall="" carry="" out="" such="" other="" functions="" as="" may="" be="" delegated="" to="" it="" by="" the="" nih="" director,="" including="" those="" authorities="" described="" in="" section="" iv-c-1-b-(2).="" orda's="" responsibilities="" include,="" but="" are="" not="" limited="" to="" the="" following:="" section="" iv-c-3-a.="" 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="">50 
    100 nanograms per kilogram body weight in organisms other 
    than Escherichia coli K-12 (see Section III-B-1 and Appendices F-I and 
    F-II);
        Section IV-C-3-b. Reviewing and approving certain experiments 
    involving the deliberate transfer of recombinant DNA or DNA or RNA 
    derived from recombinant DNA into one or more human subjects, in 
    consultation with the RAC Chair and one or more RAC members, as 
    necessary, that qualify for the Accelerated Review process (see Section 
    III-B-2);
        Section IV-C-3-c. Reviewing and approving minor changes to human 
    gene transfer protocols approved under Sections III-A-2 and III-B-2, in 
    consultation with the RAC Chair and one or more RAC members, as 
    necessary;
        Section IV-C-3-d. 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 will give its approval to the Institutional Biosafety 
    Committee membership;
        Section IV-C-3-e. Publishing in the Federal Register:
        Section IV-C-3-e-(1). Announcements of RAC meetings and agendas at 
    least 15 days in advance (NOTE--If the agenda for a RAC meeting is 
    modified, ORDA shall make the revised agenda available to anyone upon 
    request at least 72 hours in advance of the meeting);
        Section IV-C-3-e-(2). Proposed Major Actions to the NIH Guidelines 
    (see Section IV-C-1-b-(1)) at least 15 days prior to the RAC meeting;
        Section IV-C-3-f. Serve as the focal point for data management of 
    NIH-approved human gene transfer protocols approved under Sections III-
    A-2 and III-B-2 and registered with NIH/ORDA as required under Section 
    III-C-7;
        Section IV-C-3-g. Serve as the executive secretary of the RAC; and
        Section IV-C-3-h. Maintain a list of Major and Minor Actions 
    approved under Section III-A-2 and III-B-3 and a list of experiments 
    registered with NIH/ORDA as described in Section III-C-7.
        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.
    Section IV-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. The policies on noncompliance are as follows:
        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 such 
    projects 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.
        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.
        Information concerning noncompliance with the NIH Guidelines may be 
    brought forward by any person. It should be delivered to both NIH/ORDA 
    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 Recombinant DNA 
    Activities, National Institutes of Health, Building 31, Room 4B11, 
    Bethesda, Maryland 20892, (301) 496-9838.
        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.
    Section IV-E. Voluntary Compliance
    Section IV-E-1. Basic Policy
        Individuals, corporations, and institutions not otherwise covered 
    by the NIH Guidelines are encouraged to do so by following 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-E-2 through IV-E-5-b in 
    order to address these concerns.
    Section IV-E-2. Institutional Biosafety Committee Approval
        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) is modified to that extent 
    for purposes of these institutions.
    Section IV-E-3. Certification of Host-Vector Systems
        A host-vector system may be proposed for certification by the NIH 
    Director in accordance with the procedures set forth in Appendix I-II. 
    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-E-5-a will be issued only after 
    consultation with the institution as to the content of the notice.
    Section IV-E-4. Requests for Exemptions and Approvals
        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-E-5-a will be issued only after consultation with the 
    institution as to the content of the notice.
    Section IV-E-5. Protection of Proprietary Data
    Section IV-E-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 the RAC are ``special Government employees.''
        In submitting to NIH for purposes of voluntary compliance with the 
    NIH Guidelines, an institution may designate those items of information 
    which the institution believes constitute trade secrets, privileged, 
    confidential, commercial, or financial information. If NIH receives a 
    request under the Freedom 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 the 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 not be 
    made until the expiration of 15 days after the institution is so 
    advised except to the extent that earlier release in the judgment of 
    the NIH Director is necessary to protect against an imminent hazard to 
    the public or the environment.
    Section IV-E-5-b. Presubmission Review
        Any institution not otherwise covered by the NIH Guidelines, which 
    is considering submission of data or information voluntarily to NIH, 
    may request presubmission 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 presubmission review should be submitted to NIH/ORDA 
    along with the records involved to the Office of Recombinant DNA 
    Activities, National Institutes of Health, Building 31, Room 4B11, 
    Bethesda, Maryland 20892, (301) 496-9838. 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 of 
    Information Act. NIH/ORDA will seek a determination from the 
    responsible official under DHHS regulations (45 Code of Federal 
    Regulations, 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/ORDA files, will be considered records 
    of the institution and not NIH/ORDA, and will not be received as part 
    of NIH/ORDA files. No copies will be made of such records.
        NIH/ORDA will inform the institution of the DHHS 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/ORDA files. 
    If the institution instructs NIH/ORDA to return the records, no copies 
    or summaries of the records will be made or retained by DHHS, NIH, or 
    ORDA. The DHHS 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 of Information Act if at the time of the 
    determination the records were in NIH/ORDA 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 original reference to organisms as Class 1, 2, 3, 
    4, or 5 refers to the classification in the publication Classification 
    of Etiologic Agents on the Basis of Hazard, 4th Edition, July 1974, 
    U.S. Department of Health, Education, and Welfare, Public Health 
    Services, Centers for Disease Control and Prevention, Office of 
    Biosafety, Atlanta, Georgia 30333. 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)). The revised list of 
    organisms in each class is reprinted in Appendix B.
        Section V-B. Section III 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-4-c-(1)). For cases falling under 
    Sections III-A through III-D, 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)). The Institutional Biosafety Committee may refer 
    specific cases to NIH/ORDA as part of NIH/ORDA's functions to ``provide 
    advice to all within and outside NIH'' (see Section IV-C-3). NIH/ORDA 
    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)).
        Section V-C. Laboratory Safety at the Centers for Disease Control, 
    September 1974, U.S. Department of Health, Education, and Welfare 
    Publication No. CDC 75-8118.
        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. National Cancer Institute Safety Standards for 
    Research Involving Oncogenic Viruses, October 1974, U.S. Department of 
    Health, Education, and Welfare, Publication No. (NIH) 75-790.
        Section V-F. National Institutes of Health Biohazards Safety Guide, 
    1974, U.S. Department of Health, Education, and Welfare, Public Health 
    Service, NIH, U.S. Government Printing Office, Stock No. 1740-00383.
        Section V-G. A. Hellman, M. N. Oxman, and R. Pollack (eds.), 1973, 
    Biohazards in Biological Research, Cold Spring Harbor Laboratory, Cold 
    Spring Harbor, NY.
        Section V-H. Furr, A. K., Handbook of Laboratory Safety, 2nd ed, 
    The Chemical Rubber Co., Boca Raton, Florida, 1990.
        Section V-I. American Public Health Association, Bodily, J. L., 
    General Administration of the Laboratory, 6th ed., ``Diagnostic 
    Procedures for Bacterial, Mycotic, and Parasitic Infections,'' New 
    York, 1981.
        Section V-J. H. M. Darlow, Safety in the Microbiological 
    Laboratory, in J. R. Norris and D. W. Robbins (eds.), Methods in 
    Microbiology, Academic Press, Inc, New York, New York, 1969, pp. 169-
    204.
        Section V-K. C. M. Collins, E. G. Hartley, and R. Pilsworth, The 
    Prevention of Laboratory Acquired Infection, Public Health Laboratory 
    Service, Monograph Series No. 6, 1974.
        Section V-L. 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.
        Section V-M.  Design Criteria for Viral Oncology Research 
    Facilities, U.S. Department of Health, Education, and Welfare, Public 
    Health Service, NIH, DHEW Publication No. (NIH) 75-891, 1975.
        Section V-N. Kuehne, R. W., Biological Containment Facility for 
    Studying Infectious Disease, Appl. Microbiol. 26:239-243, 1973.
        Section V-O. Runkle, R. B., and G. B. Phillips, Microbial 
    Containment Control Facilities, Van Nostrand Reinhold, New York, 1969.
        Section V-P. Chatigny, M. A., and D. I. Clinger, ``Contamination 
    Control in Aerobiology,'' in R. L. Dimmick and A. B. Akers (eds.), An 
    Introduction to Experimental Aerobiology, John Wiley & Sons, New York, 
    1969, pp. 194-263.
        Section V-Q. 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.
        Section V-R. A U.S. Department of Agriculture permit is required 
    for the importation, interstate movement, and release into the 
    environment of certain organisms that are plant or animal pathogens, 
    whether genetically engineered or not. Permits are required for 
    veterinary biologics and for certain plants or microorganisms derived 
    through genetic engineering using genetic sequences from plant pests 
    (pathogens). Specific information about regulated organisms and 
    procedures for obtaining a permit for regulated organisms may be 
    obtained from the Director, Biotechnology, Biologics, and Environmental 
    Protection, Animal and Plant Health Inspection Service, U.S. Department 
    of Agriculture, 6505 Belcrest Road, Room 850, Hyattsville, Maryland 
    20782, (301) 436-7601.
        Section V-S. i.e., the total of all genomes within a family shall 
    not exceed two-thirds of the genome.
        Section V-T. 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-U. Human studies in which the induction or enhancement of 
    an immune response to a vector-encoded 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 not covered under Sections III-A-2, III-B-2, or III-B-3. Such 
    studies may be initiated without RAC review and NIH approval if 
    approved by another Federal agency.
        Section V-V. For recombinant DNA experiments in which the intent is 
    to modify stably the genome of cells of one or more human subjects (see 
    Sections III-A-2, III-B-2, and III-B-3).
        Section V-W. 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-R). 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.
    
    Appendix A. Exemptions Under Section III-E-5--Sublists of Natural 
    Exchangers
    
        Certain specified recombinant DNA molecules that ``consist entirely 
    of DNA segments from different species that exchange DNA by known 
    physiological processes, though one or more of the segments may be a 
    synthetic equivalent are exempt from these NIH Guidelines (see Section 
    III-E-5). 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)). See Appendices A-I through A-VI 
    for a list of natural exchangers that are exempt from the NIH 
    Guidelines.'' Section III-E-5 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, 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
    Appendix A-VI. Sublist F
    Streptococcus sanguis
    Streptococcus pneumoniae
    Streptococcus faecalis
    Streptococcus pyogenes
    Streptococcus mutans
    Appendix B. Classification of Etiologic Agents and Oncogenic Viruses on 
    the Basis of Hazard (See Appendix B-VI-A).
    Appendix B-I. Class 1 Agents
        All bacterial, parasitic, fungal, viral, rickettsial, and 
    chlamydial agents not included in higher classes shall be considered 
    Class 1 agents.
    Appendix B-II. Class 2 Agents
    Appendix B-II-A. Class 2 Bacterial Agents
    Acinetobacter calcoaceticus
    Actinobacillus--all species
    Aeromonas hydrophila
    Amycolata autotrophica
    Arizona hinshawii--all serotypes
    Bacillus anthracis
    Bordetella--all species
    Borrelia recurrentis, B. vincenti
    Campylobacter fetus
    Campylobacter jejuni
    Chlamydia psittaci
    Chlamydia trachomatis
    Clostridium botulinum, Cl. chauvoei, Cl. haemolyticum, Cl. 
    histolyticum, Cl. novyi, Cl.
    septicum, Cl. tetani
    Corynebacterium diphtheriae, C. equi, C. haemolyticum, C. 
    pseudotuberculosis, C.
    pyogenes, C. renale
    Dermatophilus congolensis
    Edwardsiella tarda
    Erysipelothrix insidiosa
    Escherichia coli--all enteropathogenic, enterotoxigenic, enteroinvasive 
    and strains
    bearing K1 antigen
    Haemophilus ducreyi, H. influenzae
    Klebsiella--all species except oxytoca
    Legionella pneumophila
    Leptospira interrogans--all serotypes
    Listeria--all species
    Moraxella--all species
    Mycobacteria--all species except those listed in Class 3
    Mycobacterium avium
    Mycoplasma--all species except Mycoplasma mycoides and Mycoplasma 
    agalactiae, which are in Class 5
    Neisseria gonorrhoea, N. meningitides
    Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. 
    transvalensis
    Pasteurella--all species except those listed in Class 3
    Rhodococcus equi
    Salmonella--all species and all serotypes
    Shigella-all species and all serotypes
    Sphaerophorus necrophorus
    Staphylococcus aureus
    Streptobacillus moniliformis
    Streptococcus pneumoniae, S. pyogenes
    Treponema carateum, T. pallidum, and T. pertenue
    Vibrio cholerae, V. parahemolyticus
    Yersinia enterocolitica
    
    Appendix B-II-B. Class 2 Fungal Agents
    
    Blastomyces dermatitidis
    Cryptococcus neoformans
    Paracoccidioides braziliensis
    
    Appendix B-II-C. Class 2 Parasitic Agents
    
    Endamoeba histolytica
    Leishmania sp.
    Naegleria gruberi
    Schistosoma mansoni
    Toxocara canis
    Toxoplasma gondii
    Trichinella spiralis
    Trypanosoma cruzi
    
    Appendix B-II-D. Class 2 Viral, Rickettsial, and Chlamydial Agents
    
    Adenoviruses--human--all types
    Cache Valley virus
    Coronaviruses
    Coxsackie A and B viruses
    Cytomegaloviruses
    Echoviruses--all types
    Encephalomyocarditis virus (EMC)
    Flanders virus
    Hart Park virus
    Hepatitis viruses--associated antigen material
    Herpesviruses--except Herpesvirus simiae (Monkey B virus) which is in 
    Class 4
    Influenza viruses--all types except A/PR8/34, which is in Class 1
    Langat virus
    Lymphogranuloma venereum agent
    Measles virus
    Mumps virus
    Parainfluenza virus--all types except Parainfluenza virus 3, SF4 
    strain, which is in Class 1
    Polioviruses --all types, wild and attenuated
    Poxviruses--all types except Alastrim, Smallpox, and Whitepox which are 
    Class 5 and Monkey pox which depending on experiments is in Class 3 or 
    Class 4
    Rabies virus--all strains except Rabies street virus which should be 
    classified in Class 3
    Reoviruses--all types
    Respiratory syncytial virus
    Rhinoviruses--all types
    Rubella virus
    Simian viruses--all types except Herpesvirus simiae (Monkey B virus) 
    and Marburg virus which are in Class 4
    Sindbis virus
    Tensaw virus
    Turlock virus
    Vaccinia virus
    Varicella virus
    Vesicular stomatitis virus (see Appendix B-VI-B)
    Vole rickettsia
    Yellow fever virus, 17D vaccine strain
    
    Appendix B-II-E. Class 2 Oncogenic Viruses (See Appendix B-VI-C)
    
    Appendix B-II-E-1. Low-Risk Oncogenic Viruses
    
    Adenovirus 7-Simian virus 40 (Ad7-SV40)
    Adenovirus
    Avian leukosis virus
    Bovine leukemia virus
    Bovine papilloma virus
    Chick-embryo-lethal orphan (CELO) virus or fowl adenovirus 1
    Dog sarcoma virus
    Guinea pig herpes virus
    Lucke (Frog) virus
    Hamster leukemia virus
    Marek's disease virus
    Mason-Pfizer monkey virus
    Mouse mammary tumor virus
    Murine leukemia virus
    Murine sarcoma virus
    Polyoma virus
    Rat leukemia virus
    Rous sarcoma virus
    Shope fibroma virus
    Shope papilloma virus
    Simian virus 40 (SV-40)
    
    Appendix B-II-E-2. Moderate-Risk Oncogenic Viruses
    
    Adenovirus 2-Simian virus 40 (Ad2-SV40)
    Epstein-Barr virus (EBV)
    Feline leukemia virus (FeLV)
    Feline sarcoma virus (FeSV)
    Gibbon leukemia virus (GaLV)
    Herpesvirus (HV) ateles
    Herpesvirus (HV) saimiri
    Simian sarcoma virus (SSV)-1
    Yaba
    
    Appendix B-III. Class 3 Agents
    
    Appendix B-III-A. Class 3 Bacterial Agents
    
    Bartonella--all species
    Brucella--all species
    Francisella tularensis
    Mycobacterium bovis, M. tuberculosis
    Pasteurella multocide type--``buffalo'' and other foreign virulent 
    strains (see Appendix B-VI-B)
    Pseudomonas mallei (see Appendix B-VI-B)
    Pseudomonas pseudomallei (see Appendix B-VI-B)
    Yersinia pestis
    
    Appendix B-III-B. Class 3 Fungal Agents
    
    Coccidioides immitis
    Histoplasma capsulatum
    Histoplasma capsulatum var. duboisii
    
    Appendix B-III-C. Class 3 Parasitic Agents
    
    None
    
    Appendix B-III-D. Class 3 Viral, Rickettsial, and Chlamydial Agents
    
    Monkey pox virus--when used in vitro (see Appendix B-VI-D)
    Arboviruses--all strains except those in Class 2 and 4. (Arboviruses 
    indigenous to the United States are in Class 3 except those listed in 
    Class 2. West Nile and Semliki Forest viruses may be classified up or 
    down depending on the conditions of use and geographical location of 
    the laboratory).
    Dengue virus--when used for transmission or animal inoculation 
    experiments
    Lymphocytic choriomeningitis virus (LCM)
    Rickettsia--all species except Vole rickettsia when used for 
    transmission or animal inoculation experiments
    Yellow fever virus--wild, when used in vitro
    
    Appendix B-IV. Class 4 Agents
    
    Appendix B-IV-A. Class 4 Bacterial Agents
    
    None
    
    Appendix B-IV-B. Class 4 Fungal Agents
    
    None
    
    Appendix B-IV-C. Class 4 Parasitic Agents
    
    None
    
    Appendix B-IV-D. Class 4 Viral, Rickettsial, and Chlamydial Agents
    
    Ebola fever virus
    Monkey pox virus--when used for transmission or animal inoculation 
    experiments (see Appendix B-VI-D)
    Hemorrhagic fever agents--including Crimean hemorrhagic fever, (Congo), 
    Junin, and Machupo viruses, and others as yet undefined
    Herpesvirus simiae (Monkey B virus)
    Lassa virus
    Marburg virus
    Tick-borne encephalitis virus complex--including Russian spring-summer 
    encephalitis, Kyasanur forest disease, Omsk hemorrhagic fever, and 
    Central European encephalitis viruses
    Venezuelan equine encephalitis virus, epidemic strains--when used for 
    transmission or animal inoculation experiments
    Yellow fever virus-wild--when used for transmission or animal 
    inoculation experiments
    
    Appendix B-V. Class 5 Agents (see Appendix B-VI-E)
    
    Appendix B-V-A. Animal Disease Organisms which are Forbidden Entry into 
    the United States by Law
    
    Foot and mouth disease virus
    
    Appendix B-V-B. Animal Disease Organisms and Vectors which are 
    Forbidden Entry into the United States by U.S. Department of 
    Agriculture Policy
    
    African horse sickness virus
    African swine fever virus
    Besnoitia besnoiti
    Borna disease virus
    Bovine infectious petechial fever
    Camel pox virus
    Ephemeral fever virus
    Fowl plague virus
    Goat pox virus
    Hog cholera virus
    Louping ill virus
    Lumpy skin disease virus
    Mycoplasma mycoides--contagious bovine pleuropneumonia
    Mycoplasma agalactiae--contagious agalactia of sheep
    Nairobi sheep disease virus
    Newcastle disease virus--Asiatic strains
    Rhinderpest virus
    Rickettsia ruminatium--heart water
    Rift valley fever virus
    Sheep pox virus
    Swine vesicular disease virus
    Teschen disease virus
    Theileria annulata
    Theileria bovis
    Theileria hirci
    Theileria lawrencei
    Theileria parva--East Coast fever
    Trypanosoma evansi
    Trypanosoma vivax--Nagana
    Vesicular exanthema virus
    Wesselsbron disease virus
    Zyonema
    
    Appendix B-V-C. Organisms which may not be Studied in the United States 
    Except at Specified Facilities
    
    Alastrim (see Appendix B-VI-D)
    Small pox (see Appendix B-VI-D)
    White pox (see Appendix B-VI-D)
    
    Appendix B-VI. Footnotes and References of Appendix B
    
        Appendix B-VI-A. The original reference for this classification was 
    the publication Classification of Etiologic Agents on the Basis of 
    Hazard, 4th edition, July 1974, U.S. DHHS, Public Health Service, 
    Centers for Disease Control and Prevention, Office of Biosafety, 
    Atlanta, Georgia 30333. For the purposes of these NIH Guidelines, this 
    list has been revised by the NIH.
        Appendix B-VI-B. A U.S. Department of Agriculture permit, required 
    for import and interstate transport of pathogens, may be obtained from 
    the U.S. Department of Agriculture, ATTN: Animal and Plant Health 
    Inspection Service, Import-Export Products Office, Room 756, Federal 
    Building, 6505 Belcrest Road, Hyattsville, Maryland 20782.
        Appendix B-VI-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 B-VI-D. 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).
        Appendix B-VI-E. U.S. Department of Agriculture, Animal and Plant 
    Health Inspection Service.
    
    Appendix C. Exemptions Under Section III-E-6
    
        Section III-E-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)), as determined by the NIH 
    Director, with the advice of the RAC, and following appropriate notice 
    and opportunity for public comment. See Appendix C for other classes of 
    experiments which are exempt from the NIH Guidelines.'' The following 
    classes of experiments are exempt under Section III-E-6:
    
    Appendix C-I. Recombinant DNA in Tissue Culture
    
        Recombinant DNA molecules containing less than one-half of any 
    eukaryotic viral genome (all viruses from a single family (see Appendix 
    C-VI-D) being considered identical (see Appendix C-VI-E), 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 specific RAC 
    review and NIH and Institutional Biosafety Committee approval before 
    initiation, (ii) experiments described in Section III-B which require 
    NIH/ORDA and Institutional Biosafety Committee approval before 
    initiation, (iii) experiments involving DNA from Class 3, 4, or 5 
    organisms (see Appendix C-VI-A) 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), and (v) whole plants regenerated from 
    plant cells and tissue cultures are covered by the exemption provided 
    they remain axenic cultures even though they differentiate into 
    embryonic tissue and regenerate into plantlets.
    Appendix C-II. Escherichia coli K-12 Host-Vector Systems
        Experiments which use Escherichia coli K-12 host-vector systems, 
    with the exception of those experiments listed in Appendix C-II-A, are 
    exempt from the NIH Guidelines provided that: (i) the Escherichia coli 
    host does not contain conjugation proficient plasmids or generalized 
    transducing phages; or (ii) lambda or lambdoid or Ff bacteriophages or 
    non-conjugative plasmids (see Appendix C-VI-B) shall be used as 
    vectors. However, experiments involving the insertion into Escherichia 
    coli K-12 of DNA from prokaryotes that exchange genetic information 
    (see Appendix C-VI-C) 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 of experiments are not exempt from the NIH 
    Guidelines: (i) experiments described in Section III-A which require 
    Institutional Biosafety Committee approval, RAC review, and NIH 
    approval before initiation, (ii) experiments described in Section III-B 
    which require Institutional Biosafety Committee and NIH/ORDA approval 
    before initiation, (iii) experiments involving DNA from Class 3, 4, or 
    5 organisms (see Appendix C-VI-A) or cells known to be infected with 
    these agents may be conducted under containment conditions specified in 
    Section III-C-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).
    
    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.
    
    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 approval before 
    initiation, (ii) experiments described in Section III-B which require 
    Institutional Biosafety Committee and NIH/ORDA approval before 
    initiation, (iii) experiments involving DNA from Class 3, 4, or 5 
    organisms (see Appendix C-VI-A) or cells known to be infected with 
    these agents may be conducted under containment conditions specified in 
    Section III-C-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).
    
    Appendix C-IV. Bacillus subtilis or Bacillus licheniformis Host-Vector 
    Systems
    
        Any asporogenic Bacillus subtilis or asporogenic Bacillus 
    licheniformis strain which does not revert to a spore-former with a 
    frequency greater than 10-7 may be used for cloning DNA with the 
    exception of those experiments listed in Appendix C-IV-A. 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 approval before 
    initiation, (ii) experiments described in Section III-B which require 
    Institutional Biosafety Committee and NIH/ORDA approval before 
    initiation, (iii) experiments involving DNA from Class 3, 4, or 5 
    organisms (see Appendix C-VI-A) or cells known to be infected with 
    these agents may be conducted under containment conditions specified in 
    Section III-C-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).
    
    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
    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 of experiments are not exempt from the NIH 
    Guidelines: (i) Experiments described in Section III-A which require 
    Institutional Biosafety Committee, specific RAC review, and NIH 
    approval before initiation, (ii) experiments described in Section III-B 
    which require Institutional Biosafety Committee and NIH/ORDA approval 
    before initiation, (iii) experiments involving DNA from Class 3, 4, or 
    5 organisms (see Appendix C-VI-A) or cells known to be infected with 
    these agents may be conducted under containment conditions specified in 
    Section III-C-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).
    
    Appendix C-VI. Footnotes and References of Appendix C
    
        Appendix C-VI-A. The original reference to organisms as Class 1, 2, 
    3, 4, or 5 refers to the classification in the publication 
    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 and Prevention, Office of 
    Biosafety, Atlanta, Georgia 30333.
        Appendix C-VI-A-1. 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)-(d)). The revised list of organisms in each class 
    is reprinted in Appendix B.
        Appendix C-VI-B. A subset of non-conjugative plasmid vectors are 
    poorly mobilizable (e.g., pBR322, pBR313). Where practical, these 
    vectors should be employed.
        Appendix C-VI-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-E-5.
        Appendix C-VI-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-VI-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
    
        Under Section IV-C-1-b-(1), the NIH Director may take certain 
    actions with regard to the NIH Guidelines after the issues have been 
    considered by the RAC. An updated list of these actions are available 
    from the Office of Recombinant DNA Activities, National Institutes of 
    Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
    9838.
    
    Appendix E. Certified Host-Vector Systems (see Appendix I)
    
        While many experiments using Escherichia coli K-12, Saccharomyces 
    cerevisiae, and Bacillus subtilis are currently exempt from the NIH 
    Guidelines under Section III-E, 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.
    
    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                
    ------------------------------------------------------------------------
    gt WESB'.........  DP50supF                           
    gt WESB*.........  DP50supF                           
    gt ZJ virB'......  Escherichia coli K-12              
    gtALOB...  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 (inositolless) 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.
    
    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="" and="" require="" institutional="" biosafety="" committee="" and="" nih/orda="" approval="" before="" initiation.="" no="" specific="" restrictions="" shall="" apply="" to="" the="" cloning="" of="" genes="" if="" the="" protein="" specified="" by="" the="" gene="" has="" an="">50 100 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/ORDA prior to initiating the 
    experiments. A list of toxin molecules classified as to LD50 is 
    available from NIH/ORDA. Testing procedures for determining toxicity of 
    toxin molecules not on the list are available from the Office of 
    Recombinant DNA Activities, National Institutes of Health, Building 31, 
    Room 4B11, Bethesda, Maryland 20892, (301) 496-9838. The results of 
    such tests shall be forwarded to NIH/ORDA, which will consult with ad 
    hoc experts, prior to inclusion of the molecules on the list (see 
    Section IV-C-1-b-(2)-(e)).
    
    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="">50 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="" molecules="" toxic="" for="" vertebrates="" at="" an="">50 of <100 nanograms="" per="" kilogram="" body="" weight="" in="" host-vector="" systems="" other="" than="" escherichia="" coli="" k-12="" will="" be="" evaluated="" by="" nih/orda="" in="" consultation="" with="" ad="" hoc="" toxin="" experts="" (see="" sections="" iii-b-1="" and="" iv-c-1-b-(2)-(e)).="" 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="">50 of <100 nanograms="" per="" kilogram="" body="" weight="" is="" available="" from="" the="" office="" of="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838.="" 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="" supersedes="" appendix="" g.="" appendix="" k="" defines="" good="" large="" scale="" practice="" through="" biosafety="" level="" 3--large="" scale.="" for="" certain="" work="" with="" plants,="" appendix="" p="" supersedes="" appendix="" g.="" appendix="" p="" defines="" biosafety="" levels="" 1="" through="" 4--plants.="" for="" certain="" work="" with="" animals,="" appendix="" q="" 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).="" consequently,="" all="" personnel="" directly="" or="" indirectly="" involved="" in="" experiments="" using="" recombinant="" dna="" shall="" receive="" adequate="" instruction="" (see="" sections="" iv-b-1-e="" and="" iv-b-4-d).="" 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-4-d="" and="" iv-b-4-e).="" 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).="" the="" laboratory="" safety="" monograph="" (see="" appendix="" g-iii-o)="" and="" biosafety="" in="" microbiological="" and="" biomedical="" laboratories="" (see="" appendix="" g-iii-b)="" 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).="" 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).="" 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="" 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="" by="" the="" nih="" director,="" with="" the="" advice="" of="" the="" rac="" to="" other="" combinations="" which="" achieve="" an="" equivalent="" level="" of="" containment="" (see="" section="" iv-c-1-b-(2)-="" (c)).="" appendix="" g-ii-a.="" biosafety="" level="" 1="" (bl1)="" (see="" appendix="" g-iii-m)="" 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)="" 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.="" 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/="" orda.="" reports="" to="" nih/orda="" shall="" be="" sent="" to="" the="" office="" of="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838.="" 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="" (bl="" 2).="" appendix="" g-ii-b-3-="" a.="" biological="" safety="" cabinets="" (class="" i="" or="" ii)="" (see="" appendix="" g-iii-l)="" 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).="" 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="" (bl="" 2).="" 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)="" appendix="" g-ii-c-1.="" standard="" microbiological="" practices="" (bl3).="" appendix="" g-ii-c-1-a.="" work="" surfaces="" are="" decontaminated="" at="" least="" once="" a="" day="" and="" after="" any="" spill="" of="" 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.="" 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),="" 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/orda.="" reports="" to="" nih/orda="" shall="" be="" sent="" to="" the="" office="" of="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838.="" 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.="" 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)="" 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))="" that="" avoids="" any="" interference="" with="" the="" air="" balance="" of="" the="" cabinets="" or="" building="" exhaust="" system.="" 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="" persons="" 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.="" 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/orda.="" reports="" to="" the="" nih/orda="" shall="" be="" sent="" to="" the="" office="" of="" recombinant="" dna="" activities,="" national="" institutes="" of="" health,="" building="" 31,="" room="" 4b11,="" bethesda,="" maryland="" 20892,="" (301)="" 496-9838.="" 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),="" 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.="" 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.="" 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.="" 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))="" 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="" alternative="" combinations="" of="" physical="" and="" biological="" containment="" safeguards="" --------------------------------------------------------------------------------------------------------------------------------------------------------="" alternate="" physical="" containment="" ------------------------------------------------------="" alternate="" biological="" classification="" of="" physical="" &="" biological="" containment="" laboratory="" laboratory="" laboratory="" containment="" facilities="" practices="" equipment="" --------------------------------------------------------------------------------------------------------------------------------------------------------="" bl3/hv2.................................................................="" bl3="" bl3="" bl3="" hv2="" bl3="" bl3="" bl4="" hv1="" bl3/hv1.................................................................="" bl3="" bl3="" bl3="" hv1="" bl3="" bl3="" bl2="" hv2="" bl4/hv1.................................................................="" bl4="" bl4="" bl4="" hv1="" bl4="" bl4="" bl3="" hv2="" --------------------------------------------------------------------------------------------------------------------------------------------------------="" bl--biosafety="" level.="" hv--host-vector="" system.="" appendix="" g-iii.="" footnotes="" and="" references="" of="" appendix="" g="" appendix="" g-iii-a.="" laboratory="" safety="" at="" the="" center="" for="" disease="" control,="" u.s.="" department="" of="" health,="" education,="" and="" welfare="" publication="" no.="" cdc="" 75-8118,="" september="" 1974.="" 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.="" 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).="" 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).="" 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).="" 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.="" 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,="" import-="" export="" products,="" room="" 756,="" federal="" building,="" 6505="" belcrest="" road,="" hyattsville,="" maryland="" 20782;="" for="" animal="" pathogens="" call="" (301)="" 436-7885;="" for="" plant="" pests="" (301)="" 436-6799.="" appendix="" i.="" biological="" containment="" (see="" appendix="" e)="" 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-i-a="" 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)="" and="" variants="" of="" bacteriophage,="" such="" as="" lambda="" (see="" appendices="" i-iii-="" h="" through="" o).="" 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="" non-conjugative="" plasmid="" or="" bacteriophage="" vector.="" appendix="" i-ii="" describes="" the="" data="" to="" be="" considered="" and="" mechanism="" for="" approval="" of="" host-="" vector="" 1="" systems.="" appendix="" i-i-b.="" host-vector="" 2="" systems="" 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>8 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.
        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 1/10\8\ 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 Recombinant DNA Activities, National Institutes of Health, Building 
    31, room 4B11, Bethesda, Maryland 20892, (301) 496-9838. Proposed host-
    vector systems will be reviewed by the RAC (see section IV-C-1-b-(1)-
    (e)). 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)-(h)). Once a host-vector system has 
    been certified by the NIH Director, a notice of certification will be 
    sent by NIH/ORDA 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 Recombinant DNA Activities, National 
    Institutes of Health, Building 31, room 4B11, Bethesda, Maryland 20892, 
    (301) 496-9838. The NIH Director may rescind the certification of a 
    host-vector system (see section IV-C-1-b-(2)-(i)). 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 host-vector 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 Host-Vector 1 system.
        Appendix I-II-B-2. Host-Vector 2 Systems. Investigators planning to 
    request Host-Vector 2 systems certification may obtain instructions 
    from NIH/ORDA concerning data to be submitted (see Appendices I-III-N 
    and O). 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 non-permissive 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 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 Recombinant DNA Activities, National Institutes of 
    Health, Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-
    9838. 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 Recombinant DNA Activities, National Institutes of 
    Health, Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-
    9838.
    
    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 gt WES System, Science, 1977, 196, pp. 
    175-177.
        Appendix I-III-N. Skalka, A., Current Status of Coliphage AEK2 
    Vectors, Gene, 1978, 3, pp. 29-35.
        Appendix I-III-O. Szybalski, W., A. Skalka, S. Gottesman, A. 
    Campbell, and D. Botstein, Standardized Laboratory Tests for EK2 
    Certification, Gene, 1978, 3, pp. 36-38.
    
    Appendix J. Biotechnology Research 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-C-6. 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.
        All provisions shall apply to large scale research or production 
    activities with the following modifications: (i) Appendix K shall 
    supersede Appendix G 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; (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. 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 
    large scale physical containment are referred to as Good Large Scale 
    Practice, BL1-Large Scale, BL2-Large Scale, and BL3-Large Scale. Good 
    Large Scale Practice is recommended for 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 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 
    workplace 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.
        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. 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/ORDA, and other appropriate 
    authorities (if applicable). Reports to NIH/ORDA shall be sent to the 
    Office of Recombinant DNA Activities, National Institutes of Health, 
    Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-9838.
    
    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 Biological Safety Officer, Institutional 
    Biosafety Committee, NIH/ORDA, and other appropriate authorities (if 
    applicable). Reports to NIH/ORDA shall be sent to the Office of 
    Recombinant DNA Activities, National Institutes of Health, Building 31, 
    room 4B11, Bethesda, Maryland 20892, (301) 496-9838. 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 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.
        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. 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) 
    and IV-B-3-c-(3) 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/ORDA, and other appropriate authorities (if 
    applicable). Reports to NIH/ORDA shall be sent to the Office of 
    Recombinant DNA Activities, National Institutes of Health, Building 31, 
    Room 4B11, Bethesda, Maryland 20892, (301) 496-9838. Medical 
    evaluation, surveillance, and treatment are provided as appropriate and 
    written records are maintained.
        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 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.
        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. 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.
        Appendix K-IV-L. Emergency plans required by Sections IV-B-2-b-(6) 
    and IV-B-3-c-(3) 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/ORDA, and other appropriate authorities (if 
    applicable). Reports to NIH/ORDA shall be sent to the Office of 
    Recombinant DNA Activities, National Institutes of Health, Building 31, 
    Room 4B11, Bethesda, Maryland 20892, (301) 496-9838. 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 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.
        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. 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.
        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) 
    and IV-B-3-c-(3) 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:
        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-0-11. 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.
    
    Appendix K--Table 1. Comparison of Good Large Scale Practice (GLSP) 
    and Biosafety Level (BL)--Large Scale (LS) Practice (see Appendix 
    K-VI-A)
    
    ----------------------------------------------------------------------------------------------------------------
     Criterion [See Appendix                                                                                        
             K-VI-B]                  GLSP                 BL1-LS                BL2-LS                BL3-LS       
    ----------------------------------------------------------------------------------------------------------------
    1.Formulate and           K-II-A                G-I                   G-I                   G-I                 
     implement institutional                                                                                        
     codes of practice for                                                                                          
     safety of personnel and                                                                                        
     adequate control of                                                                                            
     hygiene and safety                                                                                             
     measures.                                                                                                      
    2.Provide adequate        K-II-B                G-I 1                G-I 1                G-I 1              
     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.                                                                                       
    3.Provide changing and    K-II-C                G-II-A-1-h            G-II-B-2-f            G-II-C-2-i          
     hand washing facilities                                                                                        
     as well as protective                                                                                          
     clothing, appropriate                                                                                          
     to the risk, to be worn                                                                                        
     during work.                                                                                                   
    4.Prohibit eating,        K-II-C                G-II-A-1-d            G-II-B-1-d            G-II-C-1-c          
     drinking, smoking,                             G-II-A-1-e            G-II-B-1-e            G-II-C-1-d          
     mouth pipetting, and                                                                                           
     applying cosmetics in                                                                                          
     the work place.                                                                                                
    5.Internal accident       K-II-G                K-III-A               K-IV-A                K-IV-A              
     reporting.                                                                                                     
    6.Medical surveillance..  NR                    NR                    K-IV-A                K-V-A               
    7.Viable organisms        NR                    K-III-B               K-IV-B                K-V-B               
     should be handled in a                                                                                         
     system that physically                                                                                         
     separates the process                                                                                          
     from the external                                                                                              
     environment (closed                                                                                            
     system or other primary                                                                                        
     containment).                                                                                                  
    8.Culture fluids not      NR                    K-III-C               K-IV-C                K-V-C               
     removed from a system                                                                                          
     until organisms are                                                                                            
     inactivated.                                                                                                   
    9.Inactivation of waste   K-II-E                K-III-C               K-V-C                 K-V-C               
     solutions and materials                                                                                        
     with respect to their                                                                                          
     biohazard potential.                                                                                           
    10.Control of aerosols    Minimize              Minimize              Prevent               Prevent             
     by engineering or        Procedure             Engineer              Engineer              Engineer            
     procedural controls to   K-II-F                K-III-B               K-IV-B                K-V-B               
     prevent or minimize                            K-III-D               K-IV-D                K-V-D               
     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.                                                                                        
    11.Treatment of exhaust   NR                    Minimize              Prevent               Prevent             
     gases from a closed                            K-III-E               K-IV-E                K-V-E               
     system to minimize or                                                                                          
     prevent release of                                                                                             
     viable organisms.                                                                                              
    12.Closed system that     NR                    K-III-F               K-IV-F                K-V-F               
     has contained viable                                                                                           
     organisms not to be                                                                                            
     opened until sterilized                                                                                        
     by a validated                                                                                                 
     procedure.                                                                                                     
    13.Closed system to be    NR                    NR                    NR                    K-V-G               
     maintained at as a low                                                                                         
     pressure as possible to                                                                                        
     maintain integrity of                                                                                          
     containment features.                                                                                          
    14.Rotating seals and     NR                    NR                    Prevent               Prevent             
     other penetrations into                                              K-IV-G                K-V-H               
     closed system designed                                                                                         
     to prevent or minimize                                                                                         
     leakage.                                                                                                       
    15.Closed system shall    NR                    NR                    K-IV-H                K-V-I               
     incorporate monitoring                                                                                         
     or sensing devices to                                                                                          
     monitor the integrity                                                                                          
     of containment.                                                                                                
    16.Validated integrity    NR                    NR                    K-IV-I                K-V-J               
     testing of closed                                                                                              
     containment system.                                                                                            
    17.Closed system to be    NR                    NR                    K-IV-J                K-V-K               
     permanently identified                                                                                         
     for record keeping                                                                                             
     purposes.                                                                                                      
    18.Universal biosafety    NR                    NR                    K-IV-K                K-V-L               
     sign to be posted on                                                                                           
     each closed system.                                                                                            
    19.Emergency plans        K-II-G                K-III-G               K-IV-L                K-V-M               
     required for handling                                                                                          
     large losses of                                                                                                
     cultures.                                                                                                      
    20.Access to the work     NR                    G-II-A-1-a            G-II-B-1-a            K-V-N               
     place.                                                                                                         
    21.Requirements for       NR                    NR                    NR                    K-V-N&O             
     controlled access area.                                                                                        
    ----------------------------------------------------------------------------------------------------------------
    NR=not required.                                                                                                
    
    Appendix K-VI. Footnotes of Appendix K
    
        Appendix K-VI-A. This table is derived from the text in Appendices 
    G 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.
        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. Release into the Environment of Certain Plants
    
    Appendix L-I. General Information
    
        Appendix L specifies conditions under which certain plants as 
    specified below, may be approved for release into the environment. 
    Experiments in this category cannot be initiated without submission of 
    relevant information on the proposed experiment to NIH, review by the 
    RAC Plant Subcommittee, and specific approval by the NIH Director. Such 
    experiments also require the approval of the Institutional Biosafety 
    Committee before initiation.
    
    Appendix L-II. Criteria Allowing Review by the RAC Plant Subcommittee 
    Without the Requirement for Full RAC Review
    
        In consultation with the RAC Plant Subcommittee and without the 
    requirement for full RAC review (Institutional Biosafety Committee 
    review and approval is necessary), NIH/ORDA may approve the growing of 
    plants containing recombinant DNA in the field under the following 
    conditions: (i) The plant species is a cultivated crop of a genus that 
    has no species known to be a noxious weed; (ii) the introduced DNA 
    consists of well-characterized genes containing no sequences harmful to 
    humans, animals, or plants; (iii) the vector consists of DNA from 
    exempt host-vector systems (see Appendix C), from plants of the same or 
    closely related species, from nonpathogenic prokaryotes or 
    nonpathogenic lower eukaryotic plants, from plants pathogens only if 
    sequences resulting in production of disease symptoms have been 
    deleted, or chimeric vectors constructed from sequences of exempt host-
    vector systems (see Appendix C) or from sequences from plant pathogens 
    in which the disease symptoms have been deleted. The DNA may be 
    introduced by any suitable method. If sequences resulting in production 
    of disease symptoms are retained for purposes of introducing the DNA 
    into the plant, greenhouse-grown plants must be shown to be free of 
    such sequences before such plants, their derivatives, or seed can be 
    used in field tests; (iv) plants are grown in controlled access fields 
    under specified conditions appropriate for the plant under study and 
    the geographical location. Such conditions should include provisions 
    for using good cultural and pest control practices, for physical 
    isolation from plants of the same species outside of the experimental 
    plot in accordance with pollination characteristics of the species, and 
    the prevention of plants containing recombinant DNA from becoming 
    established in the environment. Review by the Institutional Biosafety 
    Committee should include an appraisal by scientists knowledgeable of 
    the crop, its production practices, and the local geographical 
    conditions. Procedures for assessing alterations in and the spread of 
    organisms containing recombinant DNA must be developed. The results of 
    the outlined tests must be submitted for review and approval by the 
    Institutional Biosafety Committee. Copies of such results must be 
    submitted to the Office of Recombinant DNA Activities, National 
    Institutes of Health, Building 31, Room 4B11, Bethesda, Maryland 20892, 
    (301) 496-9838.
    
    Appendix M. Points to Consider in the Design and Submission of 
    Protocols for the Transfer of Recombinant DNA Molecules Into the 
    Genome of One or More Human Subjects
    
        Appendix M applies to research conducted at or sponsored by an 
    institution that receives any support for recombinant DNA research from 
    the NIH. Researchers not covered by the NIH Guidelines are encouraged 
    to use Appendix M. Experiments in which recombinant DNA or DNA or RNA 
    derived from recombinant DNA is introduced into one or more human 
    subjects with the intent of stably modifying his/her genome are covered 
    by Sections III-A-2, III-B-2, and III-B-3 (see Section V-U). 
    Experiments in which recombinant DNA or DNA or RNA derived from 
    recombinant DNA and that are not covered by Sections III-A-2, III-B-2, 
    or III-B-3 and that are not considered exempt under Section V-U, are 
    covered under Section III-C-7.
        This document is intended to provide guidance in preparing 
    proposals for NIH consideration under Sections III-A-2 and III-B-2. 
    Section III-A-2 addresses Major Actions involving the transfer of 
    recombinant DNA or DNA or RNA derived from recombinant DNA into one or 
    more human subjects that have been determined by NIH/ORDA, in 
    consultation with the RAC Chair and one or more RAC members, as 
    necessary, to: (i) Represent novel characteristics (e.g., target 
    disease or vector), (ii) represent an uncertain degree of risk to human 
    health or the environment, or (iii) contain information determined to 
    require further public review. Proposals considered under Section III-
    A-2 will be reviewed by the RAC and approved by the NIH Director. RAC 
    review of experiments considered under Section III-A-2 will follow 
    publication of a precis of the proposal in the Federal Register and an 
    opportunity for public comment. Section III-B-2 addresses Minor Actions 
    involving the transfer of recombinant DNA or DNA or RNA derived from 
    recombinant DNA into one or more human subjects that have been 
    determined by NIH/ORDA, in consultation with the RAC Chair and one or 
    more RAC members, as necessary, to qualify for the Accelerated Review 
    process. Proposals considered under Sections III-A-2 and III-B-2 will 
    be on a case-by-case basis. A list of actions approved under Sections 
    III-A-2 and III-B-2 involving the transfer of recombinant DNA or DNA or 
    RNA derived from recombinant DNA into one or more human subjects is 
    available from the Office of Recombinant DNA Activities, National 
    Institutes of Health, Building 31, Room 4B11, Bethesda, Maryland 20892, 
    (301) 496-9838. The list of actions to the NIH Guidelines involving the 
    transfer of recombinant DNA or DNA or RNA derived from recombinant DNA 
    into one or more human subjects does not include experiments considered 
    to be exempt from RAC and NIH/ORDA review under Section III-C-7.
        Since the recombinant DNA or DNA or RNA derived from recombinant 
    DNA is expected to be confined following transfer to one or more human 
    subjects, no risk to public health or to the environment is expected. 
    Nevertheless, Appendix M-I-B-4-b specifically asks the researchers to 
    address this point.
        This appendix will be considered for revision as experience in 
    evaluating proposals accumulates and as new scientific developments 
    occur. This review will be carried out periodically as needed.
        A proposal involving the transfer of recombinant DNA or DNA or RNA 
    derived from recombinant DNA into one or more human subjects will be 
    considered by the RAC and/or NIH/ORDA only after the protocol has been 
    approved by the local Institutional Biosafety Committee and 
    Institutional Review Board in accordance with DHHS Regulations for the 
    Federal Regulations for the Protection of Human Subjects (45 Code of 
    Federal Regulations, Part 46). If a proposal involves children, special 
    attention should be paid to subpart D of these DHHS regulations. The 
    Institutional Review Board and Institutional Biosafety Committee may, 
    at their discretion, condition their approval on further specific 
    deliberation by the RAC and/or NIH/ORDA. Consideration of human gene 
    transfer proposals by the RAC and/or NIH/ORDA may proceed 
    simultaneously with review by other involved Federal agencies (see 
    Appendix M-VII-A) provided that NIH/ORDA is notified of the 
    simultaneous review. Meetings of the full RAC and its subcommittee will 
    be open to the public except where trade secrets or proprietary 
    information would be disclosed. The committee prefers that proposals 
    submitted for RAC review contain no proprietary information or trade 
    secrets, enabling all aspects of the review to be open to the public. 
    Public review of these protocols will serve to inform the public about 
    the technical aspects of the proposals as well as the meaning and 
    significance of the research.
        The clinical application of recombinant DNA techniques raises two 
    general kinds of questions: (i) the questions usually discussed by 
    Institutional Review Boards in their review of any proposed research 
    involving one or more human subjects; and (ii) broader issues. The 
    first type of question is addressed principally in Appendix M-I of this 
    document. Several broader issues are discussed throughout Appendix M.
        Appendix M-I requests a description of the protocol with special 
    attention to the short-term risks and benefits of the proposed research 
    to the patient and to other people, the selection of patients, informed 
    consent, privacy, and confidentiality. Appendix M-II addresses special 
    issues pertaining to the free flow of information about the clinical 
    trials. These issues lie outside the usual purview of Institutional 
    Review Boards and reflect general public concerns about biomedical 
    research. Appendix M-III summarizes guidelines for submission of human 
    gene transfer protocols for RAC review. Appendix M-IV specifies 
    reporting requirements. Appendix M-V describes the procedures for 
    Accelerated Review of human gene transfer experiments. Appendix M-VI 
    describes the procedures to be followed for Expedited Review of single 
    patient human gene transfer experiments. Appendix M-VII contains the 
    footnotes to Appendix M.
        The RAC will not at present entertain proposals for germ-line 
    alterations but will consider for approval protocols involving somatic 
    cell gene transfer. The purpose of somatic cell gene therapy is to 
    treat an individual patient, e.g., by inserting a properly functioning 
    gene into a patient's somatic cells. In germ-line alterations, a 
    specific attempt is made 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 acceptability of human somatic cell gene therapy 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 deliberations and hearing. 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: civic, 
    religious, scientific, and medical groups have all accepted, in 
    principle, the appropriateness of gene therapy of somatic cells in 
    humans for specific genetic diseases. Somatic cell gene therapy is seen 
    as an extension of present methods of therapy that might be preferable 
    to other technologies. In light of this public support, the RAC is 
    prepared to consider proposals for somatic cell gene therapy.
        In its evaluation of proposals involving the transfer of 
    recombinant DNA or DNA or RNA derived from recombinant DNA into one or 
    more human subjects, the 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 investigations, namely, to protect the health and well-
    being of one or more 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, 
    this document requests information that will enable the RAC and/or NIH/
    ORDA to assess the possibility that the proposed experiments will 
    inadvertently affect reproductive cells or lead to infection of other 
    people (e.g., medical personnel or relatives).
        In recognition of the social concern that surrounds the subject of 
    gene transfer, the RAC and NIH/ORDA will cooperate with other groups in 
    assessing the possible long-term consequences of the transfer of 
    recombinant DNA or DNA or RNA derived from recombinant DNA into one or 
    more human subjects and related laboratory and animal experiments in 
    order to define appropriate human applications of this emerging 
    technology.
        Responses to Appendix M should be provided in the form of either 
    written answers or references to specific sections of the protocol or 
    its appendices. Principal Investigators should indicate points which 
    are not applicable with a brief explanation. Principal Investigators 
    submitting proposals that employ essentially the same vector systems 
    (or with minor variations), and/or that are based on the same 
    preclinical testing as proposals previously reviewed by the RAC, may 
    refer to preceding documents without having to rewrite such material.
    
    Appendix M-I. Description of Proposal
    
    Appendix M-I-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-I-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-I-A-1-a. Why is the disease selected for treatment by 
    means of gene therapy a good candidate for such treatment?
        Appendix M-I-A-1-b. Describe the natural history and range of 
    expression of the disease selected for 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 therapy?
        Appendix M-I-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-I-A-1-d. What alternative therapies exist? In what 
    groups of patients are these therapies effective? What are their 
    relative advantages and disadvantages as compared with the proposed 
    gene therapy?
        Appendix M-I-A-2. Transfer of DNA for Other Purposes. Appendix M-I-
    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-I-A-2-b. What alternative methodologies exist? What are 
    their relative advantages and disadvantages as compared to the use of 
    recombinant DNA?
    Appendix M-I-B. Research Design, Anticipated Risks and Benefits
        Appendix M-I-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-I-B-1-a. What is the structure of the cloned DNA that 
    will be used?
        Appendix M-I-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-I-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.
        Appendix M-I-B-1-a-(3). Describe the steps used to derive the DNA 
    construct.
        Appendix M-I-B-1-b. What is the structure of the material that will 
    be administered to the patient?
        Appendix M-I-B-1-b-(1). Describe the preparation, structure, and 
    composition of the materials that will be given to the patient or used 
    to treat the patient'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 patient 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-I-B-1-b-(1)-(i) through (iii) 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-I-B-1-b-(2). Describe any other material to be used in 
    preparation of the material to be administered to the patient. 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-I-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-I-B-2-a. Delivery System. Appendix M-I-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 patient, 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-I-B-2-a-(2). Is the delivery system efficient? What 
    percentage of the target cells contain the added DNA?
        Appendix M-I-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-I-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-I-B-2-b. Gene Transfer and Expression. Appendix M-I-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-I-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?
        Appendix M-I-B-2-b-(3). Explain in detail all results from animal 
    and cultured cell model experiments which assess the effectiveness of 
    the delivery system (see Appendix M-I-B-2-a) in achieving the minimally 
    required level of gene transfer and expression (see Appendix M-I-B-2-b-
    (2)).
        Appendix M-I-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-I-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-I-B-2-b-(6). Is the gene expressed in cells other than 
    the target cells? If so, to what extent?
        Appendix M-I-B-2-c. Retrovirus Delivery Systems. Appendix M-I-B-2-
    c-(1). What cell types have been infected with the retroviral vector 
    preparation? Which cells, if any, produce infectious particles?
        Appendix M-I-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 of 
    recombination with endogenous or other viral sequences is likely to 
    occur in the patient'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-I-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-I-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 the analyses?
        Appendix M-I-B-2-c-(5). Has a protocol similar to the one proposed 
    for a clinical trial been conducted in non-human 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-I-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-I-B-3. Clinical Procedures, Including Patient 
    Monitoring. Describe the treatment that will be administered to 
    patients and the diagnostic methods that will be used to monitor the 
    success or failure of the 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-I-B-3-a. Will cells (e.g., bone marrow cells) be removed 
    from patients and treated ex vivo? If so, describe the type, number, 
    and intervals at which these cells will be removed.
        Appendix M-I-B-3-b. Will patients be treated to eliminate or reduce 
    the number of cells containing malfunctioning genes (e.g., through 
    radiation or chemotherapy)?
        Appendix M-I-B-3-c. What treated cells (or vector/DNA combination) 
    will be given to patients? How will the treated cells be administered? 
    What volume of cells will be used? Will there be single or multiple 
    treatments? If so, over what period of time?
        Appendix M-I-B-3-d. How will it be determined that new gene 
    sequences have been inserted into the patient'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-I-B-3-e. What studies will be conducted to assess the 
    presence and effects of the contaminants?
        Appendix M-I-B-3-f. What are the clinical endpoints of the study? 
    Are there objections and quantitative measurements to assess the 
    natural history of the disease? Will such measurements be used in 
    patient follow-up? How will patients 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 patient follow-up continue?
        Appendix M-I-B-3-g. What are the major beneficial and adverse 
    effects of 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-I-B-3-h. If a treated patient dies, what special post-
    mortem studies will be performed?
        Appendix M-I-B-4. Public Health Considerations. Describe any 
    potential benefits and hazards of the proposed therapy to persons other 
    than the patients being treated. Specifically:
        Appendix M-I-B-4-a. On what basis are potential public health 
    benefits or hazards postulated?
        Appendix M-I-B-4-b. Is there a significant possibility that the 
    added DNA will spread from the patient to other persons or to the 
    environment?
        Appendix M-I-B-4-c. What precautions will be taken against such 
    spread (e.g., patients sharing a room, health-care workers, or family 
    members)?
        Appendix M-I-B-4-d. What measures will be undertaken to mitigate 
    the risks, if any, to public health?
        Appendix M-I-B-4-e. In light of possible risks to offspring, 
    including vertical transmission, will birth control measures be 
    recommended to patients? Are such concerns applicable to health care 
    personnel?
        Appendix M-I-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-I-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-III-
    E).
        Appendix M-I-B-5-b. At what hospital or clinic will the treatment 
    be given? Which facilities of the hospital or clinic will be especially 
    important for the proposed study? Will patients occupy regular hospital 
    beds or clinical research center beds? Where will patients reside 
    during the follow-up period? What special arrangements will be made for 
    the comfort and consideration of the patients. Will the research 
    institution designate an ombudsman, patient care representative, or 
    other individual to help protect the rights and welfare of the patient?
    Appendix M-I-C. Selection of the Patients
        Estimate the number of patients to be involved in the proposed 
    study. Describe recruitment procedures and patient eligibility 
    requirements, paying particular attention to whether these procedures 
    and requirements are fair and equitable. Specifically:
        Appendix M-I-C-1. How many patients do you plan to involve in the 
    proposed study?
        Appendix M-I-C-2. How many eligible patients do you anticipate 
    being able to identify each year?
        Appendix M-I-C-3. What recruitment procedures do you plan to use?
        Appendix M-I-C-4. What selection criteria do you plan to employ? 
    What are the exclusion and inclusion criteria for the study?
        Appendix M-I-C-5. How will patients be selected if it is not 
    possible to include all who desire to participate?
    Appendix M-I-D. Informed Consent
        Indicate how patients will be informed about the proposed study and 
    how their consent will be solicited. The consent procedure should 
    adhere to the requirements of DHHS regulations for the protection of 
    human subjects (45 Code of Federal Regulations, Part 46). If the study 
    involves pediatric or mentally handicapped patients, describe 
    procedures for seeking the permission of parents or guardians and, 
    where applicable, the assent of each patient. Areas of special concern 
    include potential adverse effects, financial costs, privacy, long-term 
    follow-up and post-mortem examination. When gene transfer is a 
    procedure separate from a clinical protocol, Informed Consent documents 
    shall be submitted for both the gene transfer and clinical protocols.
        Appendix M-I-D-1. How will the major points covered in Appendices 
    M-I-A through M-I-C be disclosed to potential participants in this 
    study and/or parents or guardians in language that is understandable to 
    them?
        Appendix M-I-D-2. How will the innovative character and the 
    theoretically possible adverse effects of the experiment be discussed 
    with patients and/or parents or guardians? How will the potential 
    adverse effects be compared with the consequences of the disease?
        Appendix M-I-D-3. What explanation of the financial costs of the 
    experiment, follow-up care, and any available alternatives will be 
    provided to patients and/or parents or guardians?
        Appendix M-I-D-4. How will patients and/or their parents or 
    guardians be informed that the innovative character of the experiment 
    may lead to great interest by the media in the research and in the 
    treated patients?
        Appendix M-I-D-5. How will the patients and/or their parents or 
    guardians be informed about: (i) the irreversible consequences of some 
    of the procedures performed? (ii) any adverse medical consequences that 
    may occur if the subject(s) withdraws from the study once it has begun? 
    (iii) expectations of willingness to cooperate in long-term follow-up? 
    and (iv) expectations that permission to perform an autopsy will be 
    granted in the event of a patient's death as a precondition for a 
    patient's participation in the study? This stipulation is included 
    because an accurate determination of the precise cause of a patient's 
    death would be of vital importance to all future patients.
    Appendix M-I-E. Privacy and Confidentiality
        Indicate what measure will be taken to protect the privacy of 
    patients and their families as well as to maintain the confidentiality 
    of research data.
        Appendix M-I-E-1. What provisions will be made to honor the wishes 
    of individual patients (and the parents or guardians of pediatric or 
    mentally handicapped patients) as to whether, when, or how the identity 
    of patients is publicly disclosed.
        Appendix M-I-E-2. What provision will be made to maintain the 
    confidentiality of research data, at least in cases where data could be 
    linked to individual patients?
    
    Appendix M-II. Special Issues
    
        Although the following issues are beyond the normal purview of 
    local Institutional Review Boards, the RAC requests that Principal 
    Investigators respond to Appendices M-II-A and M-II-B below:
        Appendix M-II-A. What steps will be taken, consistent with Appendix 
    M-I-E, 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-II-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 Principal Investigators 
    and clinicians concerning research methods and results?
    
    Appendix M-III. Guidelines for the Submission of Human Gene Transfer 
    Protocols
    
        Appendices M-III-A through M-III-D and M-IV apply to human gene 
    transfer protocols considered under Section III-A-2 and III-B-2. 
    Appendices M-III-A, M- IV, and M-V apply to human gene transfer 
    protocols considered under Section III- B-2.
    Appendix M-III-A. Principal Investigator-Submitted Material
        Principal Investigators should submit the following materials to 
    the Office of Recombinant DNA Activities, National Institutes of 
    Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
    9838.
        Appendix M-III-A-1. Written proposals shall be submitted in the 
    following order:
        (1) Scientific abstract--1 page; (2) non-technical abstract--1 
    page; (3) Institutional Biosafety Committee and Institutional Review 
    Board approvals and their deliberations pertaining to your protocol; 
    (4) Response to Points to Consider--5 pages (see Appendix M through M-
    III); (6) protocol--20 pages excluding appendices--approved by the 
    local Institutional Biosafety Committee and Institutional Review 
    Board); (7) Informed Consent document--approved by the Institutional 
    Review Board; (8) appendices including tables, figures, and 
    manuscripts; (9) curricula vitae--2 pages for each key professional 
    person in biographical sketch format; and (10) an indication of other 
    Federal agencies to which the protocol is being submitted for review.
        Appendix M-III-A-2. When a proposal has been submitted previously, 
    there should be a short section ( 200 words) immediately 
    following the abstracts that summarizes the major revisions since the 
    last review.
        Appendix M-III-A-3. Data provided shall include: (i) A description 
    of the elements in the vector, (ii) the source of that information, 
    (iii) the method by which sequence data were compiled, and (iv) three 
    3\1/2\ inch diskettes with the vector sequence in ASCII format.
    Appendix M-III-B. Time Frame for Submissions
        Note: Time frames are applicable only to protocols that are 
    determined by NIH/ORDA to require full RAC review and NIH Director 
    approval. Time frames do not apply to Accelerated Review human gene 
    transfer experiments (see Section III-B-2 or those that only require 
    registration with NIH/ORDA (see Section III-C- 7).
    
        Appendix M-III-B-1. Written material from Principal Investigator 
    shall be submitted  8 weeks before the RAC meeting at which 
    it will be reviewed.
        Appendix M-III-B-2. Written comments from the primary reviewers to 
    the Principal Investigator shall be submitted  4 weeks 
    before the RAC meeting at which it will be reviewed.
        Appendix M-III-B-3. Written responses (including critical data in 
    response to the primary reviewers' comments) shall be submitted by the 
    Principal Investigator to NIH/ORDA  2 weeks before the RAC 
    meeting.
    Appendix M-III-C. Oral Responses to the RAC
        Principal Investigators shall limit their oral responses to the RAC 
    only to those questions that are raised during the meeting. Oral 
    presentations of previously submitted material and/or critical data 
    that was not submitted  2 weeks prior to the RAC meeting are 
    prohibited.
    Appendix M-III-D. Primary Reviewers' Responses
        Appendix M-III-D-1. Primary Reviewers' Written Comments. The 
    primary reviewers' written comments on a proposal should include the 
    following:
        Appendix M-III-D-1-a. Emphasize the issues related to gene marking, 
    gene transfer, or gene therapy.
        Appendix M-III-D-1-b. State explicitly whether the Points to 
    Consider have been addressed satisfactorily.
        Appendix M-III-D-1-c. Examine the scientific rationale, scientific 
    context (relative to other proposals reviewed by the RAC), whether the 
    preliminary in vitro and in vivo data were obtained in appropriate 
    models and are sufficient, and whether questions related to safety, 
    efficacy, and social/ethical context have been resolved.
        Appendix M-III-D-1-d. Whenever possible, criticisms of Informed 
    Consent documents should include written alternatives for suggested 
    revisions for the RAC to consider.
        Appendix M-III-D-1-e. Primary reviews should state whether the 
    proposal is: (i) acceptable as written, (ii) expected to be acceptable 
    with specific revisions or after satisfactory responses to specific 
    questions raised on review, or (iii) unacceptable in its present form.
        Appendix M-III-D-2. Oral Discussions by Primary Reviewers at the 
    RAC Meeting. Appendix M-III-D-2-a. It should be possible for most 
    primary reviewers to present their oral reviews in  5 
    minutes.
    
    Appendix M-IV. Reporting Requirements
    
        Appendix M-IV-A. Serious adverse effects of treatment should be 
    reported immediately to the local Institutional Review Board, the NIH 
    Office for Protection from Research Risks, and NIH/ORDA followed by the 
    submission of a written report filed with each group. Reports submitted 
    to NIH/ORDA shall be sent to the Office of Recombinant DNA Activities, 
    National Institutes of Health, Building 31, Room 4B11, Bethesda, 
    Maryland 20892, (301) 496-9838.
        Appendix M-IV-B. Reports regarding the general progress of patients 
    should be filed with both the local Institutional Review Board and NIH/
    ORDA within six months of the commencement of the experiment and at 
    six-month intervals thereafter. These twice-yearly reports should 
    continue for a sufficient period of time to allow observation of all 
    major effects. In the event of a patient's death, a summary of the 
    special post-mortem studies and statement of the cause of death should 
    be submitted to the Institutional Review Board and NIH/ORDA, if 
    available.
    
    Appendix M-V. Procedures to the Followed for Accelerated Review of 
    Human Gene Transfer Experiments by NIH/ORDA under Section III-B-2
    
        Requests for Accelerated Review should be submitted to the Office 
    of Recombinant DNA Activities, National Institutes of Health, Bethesda, 
    Maryland 20892, (301) 496-9838.
        Appendix M-V-A. Human gene transfer experiments in this category 
    must be in accordance with the provisions of Section III-B-2. If the 
    human gene transfer protocol does not qualify for Accelerated Review 
    (see Section III-B-2) as determined by NIH/ORDA, then the Principal 
    Investigator must submit the experiment for full RAC review and NIH 
    approval in accordance with Section III-A-2.
        Appendix M-V-B. No protocol shall be considered without 
    Institutional Biosafety Committee and Institutional Review Board 
    approval.
        Appendix M-V-C. At this time, all gene transfer protocols must be 
    considered experimental.
        Appendix M-V-D. Principal Investigators requesting Accelerated 
    Review (see Section III-B-2), must submit the relevant documentation in 
    accordance with Appendix M-III. NIH/ORDA will notify the Principal 
    Investigator whether the proposed study qualifies for the Accelerated 
    Review process. If NIH/ORDA determines that an experiment does not 
    qualify for Accelerated Review process, the Principal Investigator must 
    submit the proposal for full RAC review  8 weeks prior to 
    the next scheduled RAC meeting.
        Appendix M-V-E. It is expected that NIH/ORDA will consult with the 
    RAC Chair and one or more RAC members, as necessary, when considering 
    Accelerated Review human gene transfer protocols (see Section III-B-2).
        Appendix M-V-F. The RAC Chair will provide a report on all human 
    gene transfer protocols that have been approved by NIH/ORDA at the next 
    regularly scheduled RAC meeting.
        Appendix M-V-F-1. In accordance with Reporting Requirements (See 
    Appendix M- IV), any adverse effects of the treatment should be 
    reported immediately to the local Institutional Review Board, the NIH 
    Office for Protection from Research Risks, and NIH/ORDA followed by the 
    submission of a written report filed with each group. Reports submitted 
    to NIH/ORDA shall be sent to the Office of Recombinant DNA Activities, 
    National Institutes of Health, Building 31, Room 4B11, Bethesda, 
    Maryland 20892, (301) 496-9838.
        Appendix M-V-F-2. In accordance with Reporting Requirements (see 
    Appendix M- IV), reports regarding the general progress of patients 
    should be filed with both the local Institutional Review Board and NIH/
    ORDA within six months of the commencement of the experiment and at 
    six-month intervals thereafter. In the event of a patient's death, a 
    summary of the special post-mortem studies and statement of the cause 
    of death should be submitted to the Institutional Review Board and NIH/
    ORDA, if available.
        Appendix M-VI. Procedures to be Followed for Expedited Review of 
    Single Patient Human Gene Transfer Experiments by NIH Director Under 
    Section III-A-2 Requests for Expedited Review should be submitted to 
    the Office of Recombinant DNA Activities, National Insitutes of Health, 
    Bethesda, Maryland 20892, (301) 496-9838.
        Appendix M-VI-A. A Principal Investigator submitting a request to 
    the NIH/ORDA for Expedited Review of a single patient gene transfer 
    protocol shall provide detailed information regarding the necessity of 
    Expedited Review.
        Appendix M-VI-B. No protocol shall be considered without relevant 
    Institutional Biosafety Committee and Institutional Review Board 
    approvals.
        Appendix M-VI-C. At this time, all gene transfer protocols are 
    considered experimental.
        Appendix M-VI-D. Regardless of the method of review, the Points to 
    Consider is the standard of review for all gene transfer protocols.
        Appendix M-VI-E. Review of such protocols may include intramural 
    NIH experts but must include extramural experts.
        Appendix M-VI-F. The reviewers shall consider similarity of the new 
    protocol to previously approved protocols.
        Appendix M-VI-G. The NIH/ORDA shall report to the RAC following 
    Expedited Review and include all of the materials on which the decision 
    was based. The RAC shall formally review the protocol at its next 
    scheduled meeting. Patient privacy shall be maintained.
        Appendix M-VI-H. Protocols that are deferred or not approved by the 
    RAC in its normal review process are not eligible for Expedited Review. 
    No protocol shall have more than one patient approved under Expedited 
    Review.
        Appendix M-VI-I. As requested in the context of non-expedited 
    review, none of the costs of the experimental protocol shall be borne 
    by the patient or the patient's family.
        Appendix M-VI-J. Data on all patients undergoing gene transfer 
    shall be provided to the RAC within six months of the procedure.
    
    Appendix M-VII. Footnotes of Appendix M
    
        Appendix M-VII-A. The Food and Drug Administration has jurisdiction 
    over products intended for use in human gene transfer clinical trials. 
    For general information on the Food and Drug Administration's policies 
    and regulatory requirements, see the Federal Register, Volume 51, pages 
    23309-23313, 1986.
        Appendix M-VII-B. The term ``patient'' and its variants are used in 
    the text as a shorthand designation for ``patient-subject.''
    
    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 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 
    Section II. The selection of containment levels required for research 
    involving recombinant DNA molecules in plants or associated with plants 
    is specified in Section III. These biosafety levels are described in 
    Appendix P-II. This appendix describes greenhouse practices and special 
    greenhouse facilities for physical containment.
        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, 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. 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), 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)
        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/ORDA and other appropriate authorities immediately (if 
    applicable). Reports to the NIH/ORDA shall be sent to the Office of 
    Recombinant DNA Activities, National Institutes of Health, Building 31, 
    Room 4B11, Bethesda, Maryland 20892, (301) 496-9838. 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.
        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).
    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/ORDA, and other appropriate 
    authorities immediately (if applicable). Reports to the NIH/ORDA shall 
    be sent to the Office of Recombinant DNA Activities, National 
    Institutes of Health, Building 31, Room 4B11, Bethesda, Maryland 20892, 
    (301) 496-9838. 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.
        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.
    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.
    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/ORDA, and other appropriate 
    authorities immediately (if applicable). Reports to the NIH/ORDA shall 
    be sent to the Office of Recombinant DNA Activities, National 
    Institutes of Health, Building 31, Room 4B11, Bethesda, Maryland 20892, 
    (301) 496-9838. Documentation of any such accident shall be prepared 
    and maintained.
    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 non-breakable, 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-g-(2). Supplies and materials shall be brought into 
    the greenhouse facility through a double-door 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.
    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.
    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.
    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 non-motile or sterile strains of small animals; (iii) conduct 
    experiments at a time of year that precludes the survival of escaping 
    organisms; (iv) use animals that have an obligate association with a 
    plant that is not present within the dispersal range of the organism; 
    or (v) prevent the escape of organisms present in run-off water by 
    chemical treatment or evaporation of run-off water.
    
    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 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). 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. 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 
    sections of Appendix Q. The descriptions include: (i) standard 
    practices for physical and biological containment, and (ii) animal 
    facilities.
    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)
    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.
        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/ORDA, and other appropriate 
    authorities (if applicable). Reports to the NIH/ORDA shall be sent to 
    the Office of Recombinant DNA Activities, National Institutes of 
    Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
    9838. 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.
    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 syringe-needle units (i.e., needle is integral to the 
    syringe) shall be used for the injection or aspiration of fluids 
    containing organisms that contain recombinant 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.
    Appendix Q-II-C. Biosafety Level 3--Animals (BL3-N) (see Appendix Q-
    III-B)
    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.
        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/ORDA, and other 
    appropriate authorities (if applicable). Reports to the NIH/ORDA shall 
    be sent to the Office of Recombinant DNA Activities, National 
    Institutes of Health, Building 31, Room 4B11, Bethesda, Maryland 20892, 
    (301) 496-9838. 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 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 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.
        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 syringe-needle units (i.e., needle is integral to the 
    syringe) shall be used for the injection or aspiration of fluids 
    containing organisms that contain recombinant 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.
        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)
    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.
        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 Agency-
    approved germicide. The efficacy of the chemical treatment process 
    shall be validated with an indicator organism. Chemical disinfectants 
    shall be neutralized or diluted before release into general effluent 
    waste systems.
    Appendix 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/ORDA, and other appropriate 
    authorities (if applicable). Reports to the NIH/ORDA shall be sent to 
    the Office of Recombinant DNA Activities, National Institutes of 
    Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
    9838. 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.
        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 syringe-needle units (i.e., needle is integral to the 
    syringe) shall be used for the injection or aspiration of fluids 
    containing organisms that contain recombinant 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-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 double-door 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 Agency-
    approved germicide. The efficacy of the chemical treatment process 
    shall be validated with an indicator organism. Chemical disinfectants 
    shall be neutralized or diluted before release into general effluent 
    waste systems.
        Appendix 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.
        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 1 x 10-3 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 1 x 10\8\ 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. Classes 4 and 5 microorganisms pose a high level 
    of individual risk for acquiring life-threatening diseases to personnel 
    and/or animals. To import Class 5 agents, special approval must be 
    obtained from U.S. Department of Agriculture, Animal and Plant Health 
    Inspection Service, Import-Export Products, Room 756, Federal Building, 
    6505 Belcrest Road, Hyattsville, Maryland 20782.
        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.
        OMB's ``Mandatory Information Requirements for Federal Assistance 
    Program Announcements'' (45 FR 39592) requires a statement concerning 
    the official government programs contained in the Catalog of Federal 
    Domestic Assistance. Normally NIH lists in its announcements the number 
    and title of affected individual programs for the guidance of the 
    public. Because the guidance in this notice covers not only virtually 
    every NIH program but also essentially every Federal research program 
    in which DNA recombinant molecule techniques could be used, it has been 
    determined to be not cost effective or in the public interest to 
    attempt to list these programs. Such a list would likely require 
    several additional pages. In addition, NIH could not be certain that 
    every Federal program would be included as many Federal agencies, as 
    well as private organizations, both national and international, have 
    elected to follow the NIH Guidelines. In lieu of the individual program 
    listing, NIH invites readers to direct questions to the information 
    address above about whether individual programs listed in the Catalog 
    of Federal Domestic Assistance are affected.
    
        Effective Date: June 24, 1994.
    Harold Varmus,
    Director, National Institutes of Health.
    [FR Doc. 94-16200 Filed 7-1-94; 8:45 am]
    BILLING CODE 4140-01-P
    
    
    

Document Information

Published:
07/05/1994
Entry Type:
Uncategorized Document
Document Number:
94-16200
Dates:
June 24, 1994. Harold Varmus, Director, National Institutes of Health. [FR Doc. 94-16200 Filed 7-1-94; 8:45 am] BILLING CODE 4140-01-P
Pages:
0-0 (1 pages)
Docket Numbers:
Federal Register: July 5, 1994