[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]


_______________________________________________________________________

Part IV





Department of Health and Human Services





_______________________________________________________________________



National Institutes of Health



_______________________________________________________________________




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 LD50 
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 LD50 100 micrograms per kilograms of body weight. 
Experiments involving genes coding for toxin molecules with an 
LD50 of <100 micrograms per kilograms and >100 nanograms per 
kilograms body weight require Institutional Biosafety Committee 
approval and registration with NIH/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 LD50 of >1 microgram per 
kilogram and <100 microgram per kilogram body weight may proceed under 
BL1 + EK1 containment conditions (e.g., Staphylococcus aureus alpha 
toxin, Staphylococcus aureus beta toxin, ricin, Pseudomonas aeruginosa 
exotoxin A, Bordetella pertussis toxin, the lethal factor of Bacillus 
anthracis, the Pasteurella pestis murine toxins, the oxygen-labile 
hemolysins such as streptolysin O, and certain neurotoxins present in 
snake venoms and other venoms).
    Appendix F-II-C. Some enterotoxins are substantially more toxic 
when administered enterally than parenterally. The following 
enterotoxins shall be subject to BL1 + EK1 containment conditions: 
cholera toxin, the heat labile toxins of Escherichia coli, Klebsiella, 
and other related proteins that may be identified by neutralization 
with an antiserum monospecific for cholera toxin, and the heat stable 
toxins of Escherichia coli and of Yersinia enterocolitica.

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

    Requests involving the cloning of genes coding for molecules toxic 
for vertebrates at an LD50 of <100 nanograms per kilogram body 
weight in host-vector systems other than Escherichia coli K-12 will be 
evaluated by NIH/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 LD50 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 <1/108 under specified conditions. Specific Host-Vector 
2 systems are:
    Appendix I-I-B-1. For Escherichia coli K-12 Host-Vector 2 systems 
(EK2) in which the vector is a plasmid, no more than 1/108 host 
cells shall perpetuate a cloned DNA fragment under the specified non-
permissive laboratory conditions designed to represent the natural 
environment, either by survival of the original host or as a 
consequence of transmission of the cloned DNA fragment.
    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