[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
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Guidelines for Research Involving Recombinant DNA Molecules (NIH
Guidelines); Notice
DEPARTMENT OF HEALTH AND HUMAN SERVICES
National Institutes of Health
Guidelines for Research Involving Recombinant DNA Molecules (NIH
Guidelines)
June 1994.
These NIH Guidelines supersede all earlier versions and shall be in
effect until further notice.
Table of Contents
Section I. Scope of the NIH Guidelines
Section I-A. Purpose
Section I-B. Definition of Recombinant DNA Molecules
Section I-C. General Applicability
Section I-D. General Definitions
Section II. Containment
Section III. Experiments Covered by the NIH Guidelines
Section III-A. Experiments that Require Institutional Biosafety
Committee Approval, RAC Review, and NIH Approval Before Initiation
Section III-B. Experiments that Require NIH/ORDA and Institutional
Biosafety Committee Approval Before Initiation
Section III-B-1. Experiments Involving the Cloning of Toxin
Molecules with LD50 of Less than 100 Nanograms Per Kilogram
Body Weight
Section III-B-2. Accelerated Review of Human Gene Transfer
Experiments
Section III-B-3. Minor Modifications to Human Gene Transfer
Experiments
Section III-C. Experiments that Require Institutional Biosafety
Committee Approval Before Initiation
Section III-C-1. Experiments Using Human or Animal Pathogens (Class
2, Class 3, Class 4, or Class 5) Agents as Host-Vector Systems
Section III-C-2. Experiments in which DNA from Human or Animal
Pathogens (Class 2, Class 3, Class 4, or Class 5) Agents is Cloned
into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector
Systems
Section III-C-3. Experiments Involving the Use of Infectious Animal
or Plant DNA or RNA Viruses or Defective Animal or Plant DNA or RNA
Viruses in the Presence of Helper Virus in Tissue Culture Systems
Section III-C-4. Experiments Involving Whole Animals
Section III-C-5. Experiments Involving Whole Plants
Section III-C-6. Experiments Involving More than 10 Liters of
Culture
Section III-C-7. Human Gene Transfer Experiments Not Covered by
Section III-A-2, III-B-2, III-B-3, and Not Considered Exempt under
Section V-U
Section III-D. Experiments that Require Institutional Biosafety
Committee Notice Simultaneous with Initiation
Section III-D-1. Experiments Involving the Formation of Recombinant
DNA Molecules Containing No More than Two-Thirds of the Genome of
any Eukaryotic Virus
Section III-D-2. Experiments Involving Whole Plants
Section III-E. Exempt Experiments
Section IV. Roles and Responsibilities
Section IV-A. Policy
Section IV-B. Responsibilities of the Institution
Section IV-B-1. General Information
Section IV-B-2. Institutional Biosafety Committee (IBC)
Section IV-B-3. Biological Safety Officer (BSO)
Section IV-B-4. Principal Investigator (PI)
Section IV-C. Responsibilities of the National Institutes of Health
(NIH)
Section IV-C-1. NIH Director
Section IV-C-1-a. General Responsibilities
Section IV-C-1-b. Specific Responsibilities
Section IV-C-1-b-(1). Major Actions
Section IV-C-1-b-(2). Minor Actions
Section IV-C-2. Recombinant DNA Advisory Committee (RAC)
Section IV-C-3. Office of Recombinant DNA Activities (ORDA)
Section IV-C-4. Other NIH Components
Section IV-D. Compliance with the NIH Guidelines
Section IV-E. Voluntary Compliance
Section V. Footnotes and References of Sections I-IV
Appendix A. Exemptions under Section III-E-5--Sublists of Natural
Exchangers
Appendix B. Classification of Etiologic Agents and Oncogenic Viruses
on the Basis of Hazard
Appendix B-I. Class 1 Agents
Appendix B-II. Class 2 Agents
Appendix B-III. Class 3 Agents
Appendix B-IV. Class 4 Agents
Appendix B-V. Class 5 Agents
Appendix B-VI. Footnotes and References of Appendix B
Appendix C. Exemptions under Section III-E-6
Appendix C-I. Recombinant DNA in Tissue Culture
Appendix C-II. Escherichia coli K-12 Host-Vector Systems
Appendix C-III. Saccharomyces Host-Vector Systems
Appendix C-IV. Bacillus subtilis or Bacillus licheniformis Host-
Vector Systems
Appendix C-V. Extrachromosomal Elements of Gram Positive Organisms
Appendix C-VI. Footnotes and References of Appendix C
Appendix D. Major Actions Taken under the NIH Guidelines
Appendix E. Certified Host-Vector Systems
Appendix E-I. Bacillus subtilis
Appendix E-II. Saccharomyces cerevisiae
Appendix E-III. Escherichia coli
Appendix E-IV. Neurospora crassa
Appendix E-V. Streptomyces
Appendix E-VI. Pseudomonas putida
Appendix F. Containment Conditions for Cloning of Genes Coding for
the Biosynthesis of Molecules Toxic for Vertebrates
Appendix F-I. General Information
Appendix F-II. Cloning of Toxin Molecule Genes in Escherichia coli
K-12
Appendix F-III. Cloning of Toxic Molecule Genes in Organisms other
than Escherichia coli K-12
Appendix F-IV. Specific Approvals
Appendix G. Physical Containment
Appendix G-I. Standard Practices and Training
Appendix G-II. Physical Containment Levels
Appendix G-II-A. Biosafety Level 1 (BL1)
Appendix G-II-B. Biosafety Level 2 (BL2)
Appendix G-II-C. Biosafety Level 3 (BL3)
Appendix G-II-D. Biosafety Level 4 (BL4)
Appendix G-III. Footnotes and References of Appendix G
Appendix H. Shipment
Appendix I. Biological Containment
Appendix I-I. Levels of Biological Containment
Appendix I-I-A. Host-Vector 1 Systems
Appendix I-I-B. Host-Vector 2 Systems
Appendix I-II. Certification of Host-Vector Systems
Appendix I-III. Footnotes and References of Appendix I
Appendix J. Biotechnology Research Subcommittee
Appendix K. Physical Containment for Large Scale Uses of Organisms
Containing Recombinant DNA Molecules
Appendix K-I. Selection of Physical Containment Levels
Appendix K-II. Good Large Scale Practices (GLSP)
Appendix K-III. Biosafety Level 1 (BL1)--Large Scale
Appendix K-IV. Biosafety Level 2 (BL2)--Large Scale
Appendix K-V. Biosafety Level 3 (BL3)--Large Scale
Appendix K-VI. Footnotes of Appendix K
Appendix K-VII. Definitions to Accompany Containment Grid and
Appendix K
Appendix L. Release into the Environment of Certain Plants
Appendix M. Points to Consider in the Design and Submission of
Protocols for the Transfer of Recombinant DNA Molecules into the
Genome of One or More Human Subjects
Appendix M-I. Description of Proposal
Appendix M-I-A. Objectives and Rationale of the Proposed Research
Appendix M-I-B. Research Design, Anticipated Risks and Benefits
Appendix M-I-C. Selection of the Patients
Appendix M-I-D. Informed Consent
Appendix M-I-E. Privacy and Confidentiality
Appendix M-II. Special Issues
Appendix M-III. Guidelines for the Submission of Human Gene Transfer
Protocols
Appendix M-III-A. Principal Investigator-Submitted Material
Appendix M-III-B. Time Frame for Submissions
Appendix M-III-C. Oral Responses to the RAC
Appendix M-III-D. Primary Reviewers' Responses
Appendix M-IV. Reporting Requirements
Appendix M-V. Procedures to be Followed for Accelerated Review of
Human Gene Transfer Experiments by NIH/ORDA under Section III-B-2
Appendix M-VI. Procedures to be Followed for Expedited Review of
Single Patient Human Gene Transfer Experiments by the NIH Director
Under Section III-A-2
Appendix M-VII. Footnotes of Appendix M
Appendix P. Physical and Biological Containment for Recombinant DNA
Research Involving Plants
Appendix P-I. General Plant Biosafety Levels
Appendix P-II. Physical Containment Levels
Appendix P-II-A. Biosafety Level 1--Plants (BL1-P)
Appendix P-II-B. Biosafety Level 2--Plants (BL2-P)
Appendix P-II-C. Biosafety Level 3--Plants (BL3-P)
Appendix P-II-D. Biosafety Level 4--Plants (BL4-P)
Appendix P-III. Biological Containment Practices
Appendix P-III-A. Biological Containment Practices (Plants)
Appendix P-III-B. Biological Containment Practices (Microorganisms)
Appendix P-III-C. Biological Containment Practices (Macroorganisms)
Appendix Q. Physical and Biological Containment for Recombinant DNA
Research Involving Animals
Appendix Q-I. General Considerations
Appendix Q-I-A. Containment Levels
Appendix Q-I-B. Disposal of Animals (BL1-N through BL4-N)
Appendix Q-II. Physical and Biological Containment Levels
Appendix Q-II-A. Biosafety Level 1--Animals (BL1-N)
Appendix Q-II-B. Biosafety Level 2--Animals (BL2-N)
Appendix Q-II-C. Biosafety Level 3--Animals (BL3-N)
Appendix Q-II-D. Biosafety Level 4--Animals (BL4-N)
Appendix Q-III. Footnotes and References for Appendix Q
Section I. Scope of the NIH Guidelines
Section I-A. Purpose
The purpose of the NIH Guidelines is to specify practices for
constructing and handling: (i) Recombinant deoxyribonucleic acid (DNA)
molecules, and (ii) organisms and viruses containing recombinant DNA
molecules.
Section I-A-1. Any recombinant DNA experiment, which according to
the NIH Guidelines requires approval by the NIH, must be submitted to
the NIH or to another Federal agency that has jurisdiction for review
and approval. Once approval, or other applicable clearances, has been
obtained from a Federal agency other than the NIH (whether the
experiment is referred to that agency by the NIH or sent directly there
by the submitter), the experiment may proceed without the necessity for
NIH review or approval (see exceptions in Sections I-A-2 and I-A-3).
Section I-A-2. Certain experiments that involve the deliberate
transfer of recombinant DNA or DNA or RNA derived from recombinant DNA
into one or more human subjects (see Section V-U) shall be considered
Major Actions (see Section IV-C-1-b-(1)), and shall require RAC review
and NIH Director approval, if determined by NIH/ORDA in consultation
with the RAC Chair and/or one or more RAC members, as necessary, to:
(i) Represent novel characteristics (e.g., target disease or vector),
(ii) represent an uncertain degree of risk to human health or the
environment, or (iii) contain information determined to require further
public review (see Section III-A-2).
Section I-A-3. Experiments involving the transfer of recombinant
DNA to one or more human subjects that are not considered under Section
III-A-2 may qualify for Accelerated Review (see Section III-B-2 and
Appendix M-V) and will be considered as Minor Actions (see Section IV-
C-1-b-(2)-(a)). Actions that qualify for Accelerated Review will be
reviewed and approved by NIH/ORDA in consultation with the RAC Chair
and/or one or more RAC members, as necessary.
Certain experiments involving the transfer of recombinant DNA or
DNA or RNA derived from recombinant DNA into one or more human subjects
(see Section V-U) may be considered exempt from RAC and/or NIH/ORDA
review and/or NIH Director approval and only require registration with
NIH/ORDA (see Section III-C-7).
Section I-B. Definition of Recombinant DNA Molecules
In the context of the NIH Guidelines, recombinant DNA molecules are
defined as either: (i) Molecules that are constructed outside living
cells by joining natural or synthetic DNA segments to DNA molecules
that can replicate in a living cell, or (ii) molecules that result from
the replication of those described in (i) above.
Synthetic DNA segments which are likely to yield a potentially
harmful polynucleotide or polypeptide (e.g., a toxin or a
pharmacologically active agent) are considered as equivalent to their
natural DNA counterpart. If the synthetic DNA segment is not expressed
in vivo as a biologically active polynucleotide or polypeptide product,
it is exempt from the NIH Guidelines.
Genomic DNA of plants and bacteria that have acquired a
transposable element, even if the latter was donated from a recombinant
vector no longer present, are not subject to the NIH Guidelines unless
the transposon itself contains recombinant DNA.
Section I-C. General Applicability
Section I-C-1. The NIH Guidelines are applicable to:
Section I-C-1-a. All recombinant DNA research within the United
States (U.S.) or its territories that is conducted at or sponsored by
an institution that receives any support for recombinant DNA research
from the NIH, including research performed directly by the NIH. An
individual who receives support for research involving recombinant DNA
must be associated with or sponsored by an institution that assumes the
responsibilities assigned in the NIH Guidelines.
Section I-C-1-b. All recombinant DNA research performed abroad:
Specifically:
Section I-C-1-b-(1). Research supported by NIH funds.
Section I-C-1-b-(2). If they involve testing in humans of materials
containing recombinant DNA developed with NIH funds and if the
institution that developed those materials sponsors or participates in
those projects. Participation includes research collaboration or
contractual agreements, not mere provision of research materials.
Section I-C-1-b-(3). If the host country has established rules for
the conduct of recombinant DNA research, then the research must be in
compliance with those rules. If the host country does not have such
rules, the proposed research must be reviewed and approved by an NIH-
approved Institutional Biosafety Committee or equivalent review body
and accepted in writing by an appropriate national governmental
authority of the host country. The safety practices that are employed
abroad must be reasonably consistent with the NIH Guidelines.
Section I-D. General Definitions
The following terms, which are used throughout the NIH Guidelines,
are defined as follows:
Section I-D-1. An ``institution'' is any public or private entity
(including Federal, state, and local government agencies).
Section I-D-2. An ``Institutional Biosafety Committee'' is a
committee that: (i) Meets the requirements for membership specified in
Section IV-B-2, and (ii) reviews, approves, and oversees projects in
accordance with the responsibilities defined in Section IV-B-2.
Section I-D-3. The ``Office of Recombinant DNA Activities (ORDA)''
is the office within the NIH that is responsible for: (i) Reviewing and
coordinating all activities relating to the NIH Guidelines, and (ii)
performing other duties as defined in Section IV-C-3.
Section I-D-4. The ``Recombinant DNA Advisory Committee'' is the
public advisory committee that advises the Department of Health and
Human Services (DHHS) Secretary, the DHHS Assistant Secretary for
Health, and the NIH Director concerning recombinant DNA research. The
RAC shall be constituted as specified in Section IV-C-2.
Section I-D-5. The ``NIH Director'' is the Director of the National
Institutes of Health, or any other officer or employee of NIH to whom
authority has been delegated.
Section I-D-6. ``Deliberate release'' is defined as a planned
introduction of recombinant DNA-containing microorganisms, plants, or
animals into the environment.
Section II. Containment
Effective biological safety programs have been operative in a
variety of laboratories for many years. Considerable information
already exists about the design of physical containment facilities and
selection of laboratory procedures applicable to organisms carrying
recombinant DNA (see section V-A). The existing programs rely upon
mechanisms that can be divided into two categories: (i) A set of
standard practices that are generally used in microbiological
laboratories; and (ii) special procedures, equipment, and laboratory
installations that provide physical barriers that are applied in
varying degrees according to the estimated biohazard. Four biosafety
levels are described in Appendix G. These biosafety levels consist of
combinations of laboratory practices and techniques, safety equipment,
and laboratory facilities appropriate for the operations performed and
are based on the potential hazards imposed by the agents used and for
the laboratory function and activity. Biosafety Level 4 provides the
most stringent containment conditions, Biosafety Level 1 the least
stringent.
Experiments involving recombinant DNA lend themselves to a third
containment mechanism, namely, the application of highly specific
biological barriers. Natural barriers exist that limit either: (i) The
infectivity of a vector or vehicle (plasmid or virus) for specific
hosts, or (ii) its dissemination and survival in the environment.
Vectors, which provide the means for recombinant DNA and/or host cell
replication, can be genetically designed to decrease, by many orders of
magnitude, the probability of dissemination of recombinant DNA outside
the laboratory (see Appendix I).
Since these three means of containment are complementary, different
levels of containment can be established that apply various
combinations of the physical and biological barriers along with a
constant use of standard practices. Categories of containment are
considered separately in order that such combinations can be
conveniently expressed in the NIH Guidelines.
Physical containment conditions within laboratories, described in
Appendix G, may not always be appropriate for all organisms because of
their physical size, the number of organisms needed for an experiment,
or the particular growth requirements of the organism. Likewise,
biological containment for microorganisms described in Appendix I may
not be appropriate for all organisms, particularly higher eukaryotic
organisms. However, significant information exists about the design of
research facilities and experimental procedures that are applicable to
organisms containing recombinant DNA that is either integrated into the
genome or into microorganisms associated with the higher organism as a
symbiont, pathogen, or other relationship. This information describes
facilities for physical containment of organisms used in non-
traditional laboratory settings and special practices for limiting or
excluding the unwanted establishment, transfer of genetic information,
and dissemination of organisms beyond the intended location, based on
both physical and biological containment principles. Research conducted
in accordance with these conditions effectively confines the organism.
For research involving plants, four biosafety levels (BL1-P through
BL4-P) are described in Appendix P. BL1-P is designed to provide a
moderate level of containment for experiments for which there is
convincing biological evidence that precludes the possibility of
survival, transfer, or dissemination of recombinant DNA into the
environment, or in which there is no recognizable and predictable risk
to the environment in the event of accidental release. BL2-P is
designed to provide a greater level of containment for experiments
involving plants and certain associated organisms in which there is a
recognized possibility of survival, transmission, or dissemination of
recombinant DNA containing organisms, but the consequence of such an
inadvertent release has a predictably minimal biological impact. BL3-P
and BL4-P describe additional containment conditions for research with
plants and certain pathogens and other organisms that require special
containment because of their recognized potential for significant
detrimental impact on managed or natural ecosystems. BL1-P relies upon
accepted scientific practices for conducting research in most ordinary
greenhouse or growth chamber facilities and incorporates accepted
procedures for good pest control and cultural practices. BL1-P
facilities and procedures provide a modified and protected environment
for the propagation of plants and microorganisms associated with the
plants and a degree of containment that adequately controls the
potential for release of biologically viable plants, plant parts, and
microorganisms associated with them. BL2-P and BL3-P rely upon accepted
scientific practices for conducting research in greenhouses with
organisms infecting or infesting plants in a manner that minimizes or
prevents inadvertent contamination of plants within or surrounding the
greenhouse. BL4-P describes facilities and practices known to provide
containment of certain exotic plant pathogens.
For research involving animals, which are of a size or have growth
requirements that preclude the use of conventional primary containment
systems used for small laboratory animals, four biosafety levels (BL1-N
through BL4-N) are described in Appendix Q. BL1-N describes containment
for animals that have been modified by stable introduction of
recombinant DNA, or DNA derived therefrom, into the germ-line
(transgenic animals) and experiments involving viable recombinant DNA-
modified microorganisms and is designed to eliminate the possibility of
sexual transmission of the modified genome or transmission of
recombinant DNA-derived viruses known to be transmitted from animal
parent to offspring only by sexual reproduction. Procedures, practices,
and facilities follow classical methods of avoiding genetic exchange
between animals. BL2-N describes containment which is used for
transgenic animals associated with recombinant DNA-derived organisms
and is designed to eliminate the possibility of vertical or horizontal
transmission. Procedures, practices, and facilities follow classical
methods of avoiding genetic exchange between animals or controlling
arthropod transmission. BL3-N and BL4-N describe higher levels of
containment for research with certain transgenic animals involving
agents which pose recognized hazard.
In constructing the NIH Guidelines, it was necessary to define
boundary conditions for the different levels of physical and biological
containment and for the classes of experiments to which they apply.
These definitions do not take into account all existing and anticipated
information on special procedures that will allow particular
experiments to be conducted under different conditions than indicated
here without affecting risk. Individual investigators and Institutional
Biosafety Committees are urged to devise simple and more effective
containment procedures and to submit recommended changes in the NIH
Guidelines to permit the use of these procedures.
Section III. Experiments Covered by the NIH Guidelines
This section describes five categories of experiments involving
recombinant DNA: (i) Those that require RAC review and NIH and
Institutional Biosafety Committee approval before initiation (see
section III-A), (ii) those that require NIH/ORDA and Institutional
Biosafety Committee approval before initiation (see section III-B);
(iii) those that require Institutional Biosafety Committee approval
before initiation (see section III-C), (iv) those that require
Institutional Biosafety Committee notification simultaneous with
initiation (see section III-D), and (v) those that are exempt from the
NIH Guidelines (see section III-E).
Note: If an experiment falls into either section III-A or
section III-B and one of the other categories, the rules pertaining
to section III-A or section III-B shall be followed. If an
experiment falls into section III-E and into either sections III-C
or III-D categories as well, the experiment is considered exempt
from the NIH Guidelines.
Any change in containment level, which is different from those
specified in the NIH Guidelines, may not be initiated without the
express approval of NIH/ORDA (see Minor Actions, section IV-C-1-b-(2)
and its subsections).
Section III-A. Experiments that Require Institutional Biosafety
Committee Approval, RAC Review, and NIH Approval Before Initiation
Experiments in this category are considered Major Actions (see
section IV-C-1-b-(1)) and cannot be initiated without submission of
relevant information on the proposed experiment to the Office of
Recombinant DNA Activities, National Institutes of Health, Building 31,
room 4B11, Bethesda, Maryland 20892, (301) 496-9838, the publication of
the proposal in the Federal Register for 15 days of comment, reviewed
by the RAC, and specific approval by the NIH (not applicable for
Expedited Review single patient human gene transfer experiments
considered under Appendix M-VI). The containment conditions for such
experiments will be recommended by the RAC and set by the NIH at the
time of approval. Such experiments require Institutional Biosafety
Committee approval before initiation. Specific experiments already
approved are included in Appendix D which may be obtained from the
Office of Recombinant DNA Activities, National Institutes of Health,
Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-9838.
Section III-A-1. Deliberate transfer of a drug resistance trait to
microorganisms that are not known to acquire the trait naturally (see
section V-B), if such acquisition could compromise the use of the drug
to control disease agents in humans, veterinary medicine, or
agriculture.
Section III-A-2. Certain experiments involving the deliberate
transfer of recombinant DNA or DNA or RNA derived from recombinant DNA
into one or more human subjects (see section V-U) shall be considered
Major Actions (see section IV-C-1-b-(1) and Appendix M-III), and shall
require RAC review and NIH Director approval, if determined by NIH/
ORDA, in consultation with the RAC Chair and one or more RAC members,
as necessary, to: (i) Represent novel characteristics (e.g., target
disease or vector), (ii) represent an uncertain degree of risk to human
health or the environment, or (iii) contain information determined to
require further public review. The requirement for RAC review shall not
be considered to preempt any other required review or approval of
experiments with one or more human subjects. Relevant Institutional
Biosafety Committee and Institutional Review Board reviews and
approvals of the proposal should be completed before submission to NIH.
Certain experiments involving deliberate transfer of recombinant DNA or
DNA or RNA derived from recombinant DNA into one or more human subjects
may qualify for the Accelerated Review process (see section III-B-2).
Certain categories of experiments involving the deliberate transfer of
recombinant DNA or DNA or RNA derived from recombinant DNA into one or
more human subjects and that are not covered by section V-U, may be
considered exempt from RAC and/or NIH/ORDA review and/or NIH Director
approval and only require registration with NIH/ORDA (see section III-
C-7).
Section III-B. Experiments That Require NIH/ORDA and Institutional
Biosafety Committee Approval Before Initiation
Section III-B-1. Experiments Involving the Cloning of Toxin Molecules
with LD50 of Less than 100 Nanograms per Kilogram Body Weight
Deliberate formation of recombinant DNA containing genes for the
biosynthesis of toxin molecules lethal for vertebrates at an LD50
of less than 100 nanograms per kilogram body weight (e.g., microbial
toxins such as the botulinum toxins, tetanus toxin, diphtheria toxin,
and Shigella dysenteriae neurotoxin). Specific approval has been given
for the cloning in Escherichia coli K-12 of DNA containing genes coding
for the biosynthesis of toxic molecules which are lethal to vertebrates
at 100 nanograms to 100 micrograms per kilogram body weight. Specific
experiments already approved under this section may be obtained from
the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-
9838.
Section III-B-1-(a). Experiments in this category cannot be
initiated without submission of relevant information on the proposed
experiment to NIH/ORDA. The containment conditions for such experiments
will be determined by NIH/ORDA in consultation with ad hoc experts.
Such experiments require Institutional Biosafety Committee approval
before initiation (see section IV-B-2-b-(1)).
Section III-B-2. Accelerated Review of Human Gene Transfer Experiments
As determined by NIH/ORDA, in consultation with the RAC Chair and
one or more RAC members, as necessary, certain categories of human gene
transfer experiments may be considered as Minor Actions and qualify for
Accelerated Review and approval (see section IV-C-1-b-(2)-(a), Appendix
M-III-A, and Appendix M-V). The RAC Chair will present a report of all
NIH/ORDA approved human gene transfer protocols at the next regularly
scheduled RAC meeting. If NIH/ORDA determines that an experiment does
not qualify for the Accelerated Review process, the Principal
Investigator must submit the proposal for full RAC review 8
weeks prior to the next scheduled RAC meeting (See section III-A-2).
Section III-B-3. Minor Modifications to Human Gene Transfer Experiments
A minor modification in a human gene transfer protocol is a
modification that does not significantly alter the basic design of the
protocol and that does not increase risk to human subjects or the
environment. After approval has been obtained by the relevant
Institutional Biosafety Committee and Institutional Review Board, NIH/
ORDA will consider the change in consultation with the RAC Chair and
one or more RAC members, as necessary. Submit minor modifications to
the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
9838. The RAC Chair will provide a report on any such approvals at the
next regularly scheduled RAC meeting.
Section III-C. Experiments that Require Institutional Biosafety
Committee Approval Before Initiation
Prior to the initiation of an experiment that falls into this
category, the Principal Investigator must submit a registration
document to the Institutional Biosafety Committee which contains the
following information: (i) The source(s) of DNA; (ii) the nature of the
inserted DNA sequences; (iii) the host(s) and vector(s) to be used;
(iv) if an attempt will be made to obtain expression of a foreign gene,
and if so, indicate the protein that will be produced; and (v) the
containment conditions that will be implemented as specified in the NIH
Guidelines. For experiments in this category, the registration document
shall be dated, signed by the Principal Investigator, and filed with
the Institutional Biosafety Committee. The Institutional Biosafety
Committee shall review and approve all experiments in this category
prior to their initiation. Requests to decrease the level of
containment specified for experiments in this category will be
considered by NIH (see Section IV-C-1-b-(2)-(c)).
Section III-C-1. Experiments Using Human or Animal Pathogens (Class 2,
Class 3, Class 4, or Class 5 Agents (see Section V-A) as Host-Vector
Systems
Section III-C-1-a. Experiments involving the introduction of
recombinant DNA into Class 2 agents shall be conducted at Biosafety
Level (BL) 2 containment. Experiments with such agents shall be
conducted with whole animals at BL2 or BL2-N (Animals) containment.
Section III-C-1-b. Experiments involving the introduction of
recombinant DNA into Class 3 agents shall be conducted at BL3
containment. Experiments with such agents shall be conducted with whole
animals at BL3 or BL3-N containment.
Section III-C-1-c. Experiments involving the introduction of
recombinant DNA into Class 4 agents shall be conducted at BL4
containment. Experiments with such agents shall be conducted with whole
animals at BL4 or BL4-N containment.
Section III-C-1-d. Containment conditions for experiments involving
the introduction of recombinant DNA into Class 5 agents shall be set on
a case-by-case basis following NIH/ORDA review. A U.S. Department of
Agriculture permit is required for work with Class 5 agents (see
Sections V-R and V-T). Experiments with such agents shall be conducted
with whole animals at BL4 or BL4-N containment.
Section III-C-2. Experiments in Which DNA From Human or Animal
Pathogens (Class 2, Class 3, Class 4, or Class 5 Agents (see Section V-
A) is Cloned Into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-
Vector Systems
Section III-C-2-a. Experiments in which DNA from Class 2 or Class 3
agents (see Section V-A) is transferred into nonpathogenic prokaryotes
or lower eukaryotes may be performed under BL2 containment. Experiments
in which DNA from Class 4 agents is transferred into nonpathogenic
prokaryotes or lower eukaryotes may be performed under BL2 containment
after demonstration that only a totally and irreversibly defective
fraction of the agent's genome is present in a given recombinant. In
the absence of such a demonstration, BL4 containment shall be used. The
Institutional Biosafety Committee may approve the specific lowering of
containment for particular experiments to BL1. Many experiments in this
category are exempt from the NIH Guidelines (see Section III-E).
Experiments involving the formation of recombinant DNA for certain
genes coding for molecules toxic for vertebrates require NIH/ORDA
approval (see Section III-B-1) or shall be conducted under NIH
specified conditions as described in Appendix F.
Section III-C-2-b. Containment conditions for experiments in which
DNA from Class 5 agents is transferred into nonpathogenic prokaryotes
or lower eukaryotes shall be determined by NIH/ORDA following a case-
by-case review. A U.S. Department of Agriculture permit is required for
work with Class 5 agents (see Sections V-R and V-T).
Section III-C-3. Experiments Involving the Use of Infectious Animal or
Plant DNA or RNA Viruses or Defective Animal or Plant DNA or RNA
Viruses in the Presence of Helper Virus in Tissue Culture Systems
Caution: Special care should be used in the evaluation of
containment levels for experiments which are likely to either enhance
the pathogenicity (e.g., insertion of a host oncogene) or to extend the
host range (e.g., introduction of novel control elements) of viral
vectors under conditions that permit a productive infection. In such
cases, serious consideration should be given to increasing physical
containment by at least one level.
Note: Recombinant DNA or RNA molecules derived therefrom, which
contain less than two-thirds of the genome of any eukaryotic virus
(all viruses from a single Family (see Section V-Q) being considered
identical (see Section V-S), are considered defective and may be
used in the absence of helper under the conditions specified in
Section III-D-1.
Section III-C-3-a. Experiments involving the use of infectious or
defective Class 2 animal viruses (see Section V-A, Appendix B-II, and
Appendix B-II-E) in the presence of helper virus may be conducted at
BL2.
Section III-C-3-b. Experiments involving the use of infectious or
defective Class 3 animal viruses (see Section V-A and Appendix B-III-D)
in the presence of helper virus may be conducted at BL3.
Section III-C-3-c. Experiments involving the use of infectious or
defective Class 4 animal viruses (see Section V-A and Appendix B-IV-D)
in the presence of helper virus may be conducted at BL4.
Section III-C-3-d. Experiments involving the use of infectious or
defective Class 5 viruses (see Section V-A and Appendix B-V) in the
presence of helper virus shall be determined on a case-by-case basis
following NIH/ORDA review. A U.S. Department of Agriculture permit is
required for work with Class 5 agents (see Sections V-R and V-T).
Section III-C-3-e. Experiments involving the use of infectious or
defective animal or plant viruses in the presence of helper virus are
not covered in Sections III-C-3-a through III-C-3-d and may be
conducted at BL1.
Section III-C-4. Experiments Involving Whole Animals
This section covers experiments involving whole animals in which
the animal's genome has been altered by stable introduction of
recombinant DNA, or DNA derived therefrom, into the germ-line
(transgenic animals) and experiments involving viable recombinant DNA-
modified microorganisms tested on whole animals. For the latter, other
than viruses which are only vertically transmitted, the experiments may
not be conducted at BL1-N containment. A minimum containment of BL2 or
BL2-N is required.
Caution--Special care should be used in the evaluation of
containment conditions for some experiments with transgenic animals.
For example, such experiments might lead to the creation of novel
mechanisms or increased transmission of a recombinant pathogen or
production of undesirable traits in the host animal. In such cases,
serious consideration should be given to increasing the containment
conditions.
Section III-C-4-a. Recombinant DNA, or DNA or RNA molecules derived
therefrom, from any source except for greater than two-thirds of
eukaryotic viral genome may be transferred to any non-human vertebrate
or any invertebrate organism and propagated under conditions of
physical containment comparable to BL1 or BL1-N and appropriate to the
organism under study (see Section V-B). Animals that contain sequences
from viral vectors, which do not lead to transmissible infection either
directly or indirectly as a result of complementation or recombination
in animals, may be propagated under conditions of physical containment
comparable to BL1 or BL1-N and appropriate to the organism under study.
Experiments involving the introduction of other sequences from
eukaryotic viral genomes into animals are covered under Section III-C-
4-b. For experiments involving recombinant DNA-modified Class 2, 3, 4,
or 5 organisms, see Section V-A. It is important that the investigator
demonstrate that the fraction of the viral genome being utilized does
not lead to productive infection. A U.S. Department of Agriculture
permit is required for work with Class 5 agents (see Section V-R and V-
T).
Section III-C-4-b. For experiments involving recombinant DNA, or
DNA or RNA derived therefrom, involving whole animals, including
transgenic animals, and not covered by Sections III-C-1 or III-C-4-a,
the appropriate containment shall be determined by the Institutional
Biosafety Committee.
Section III-C-5. Experiments Involving Whole Plants
Experiments to genetically engineer plants by recombinant DNA
methods, to use such plants for other experimental purposes (e.g.,
response to stress), to propagate such plants, or to use plants
together with microorganisms or insects containing recombinant DNA, may
be conducted under the containment conditions described in Sections
III-C-5-a through III-C-5-e. If experiments involving whole plants are
not described in Section III-C-5 and do not fall under Sections III-A,
III-B, or III-E, they are included in Section III-D.
Note: For recombinant DNA experiments falling under Sections
III-C-5-a through III-C-5-d, physical containment requirements may
be reduced to the next lower level by appropriate biological
containment practices, such as conducting experiments on a virus
with an obligate insect vector in the absence of that vector or
using a genetically attenuated strain.
Section III-C-5-a. BL3-P (Plants) or BL2-P + biological containment
is recommended for experiments involving most exotic (see Section V-W)
infectious agents with recognized potential for serious detrimental
impact on managed or natural ecosystems when recombinant DNA techniques
are associated with whole plants.
Section III-C-5-b. BL3-P or BL2-P + biological containment is
recommended for experiments involving plants containing cloned genomes
of readily transmissible exotic (see Section V-W) infectious agents
with recognized potential for serious detrimental effects on managed or
natural ecosystems in which there exists the possibility of
reconstituting the complete and functional genome of the infectious
agent by genomic complementation in planta.
Section III-C-5-c. BL4-P containment is recommended for experiments
with a small number of readily transmissible exotic (see Section V-W)
infectious agents, such as the soybean rust fungus (Phakospora
pachyrhizi) and maize streak or other viruses in the presence of their
specific arthropod vectors, that have the potential of being serious
pathogens of major U.S. crops.
Section III-C-5-d. BL3-P containment is recommended for experiments
involving sequences encoding potent vertebrate toxins introduced into
plants or associated organisms. Recombinant DNA containing genes for
the biosynthesis of toxin molecules lethal for vertebrates at an
LD50 of <100 nanograms per kilogram body weight fall under Section
III-B-1 and require NIH/ORDA and Institutional Biosafety Committee
approval before initiation.
Section III-C-5-e. BL3-P or BL2-P + biological containment is
recommended for experiments with microbial pathogens of insects or
small animals associated with plants if the recombinant DNA-modified
organism has a recognized potential for serious detrimental impact on
managed or natural ecosystems.
Section III-C-6. Experiments Involving More than 10 Liters of Culture
The appropriate containment will be decided by the Institutional
Biosafety Committee. Where appropriate, Appendix K, Physical
Containment for Large Scale Uses of Organisms Containing Recombinant
DNA Molecules, shall be used. Appendix K describes containment
conditions Good Large Scale Practice through BL3-Large Scale.
Section III-C-7. Human Gene Transfer Experiments Not Covered by
Sections III-A-2, III-B-2, III-B-3, and Not Considered Exempt Under
Section V-U
Certain experiments involving the transfer of recombinant DNA or
DNA or RNA derived from recombinant DNA into one or more human subjects
that are not covered by Sections III-A-2, III-B-2, III-B-3, and that
are not considered exempt under Section V-U must be registered with
NIH/ORDA. The relevant Institutional Biosafety Committee and
Institutional Review Board must review and approve all experiments in
this category prior to their initiation.
Section III-D. Experiments that Require Institutional Biosafety
Committee Notice Simultaneous With Initiation
Experiments not included in Sections III-A, III-B, III-C, III-E,
and their subsections are considered in Section III-D. All such
experiments may be conducted at BL1 containment. For experiments in
this category, a registration document (see Section III-C) shall be
dated and signed by the investigator and filed with the local
Institutional Biosafety Committee at the time the experiment is
initiated. The Institutional Biosafety Committee reviews and approves
all such proposals, but Institutional Biosafety Committee review and
approval prior to initiation of the experiment is not required (see
Section IV-A). For example, experiments in which all components derived
from non-pathogenic prokaryotes and non-pathogenic lower eukaryotes
fall under Section III-D and may be conducted at BL1 containment.
Section III-D-1. Experiments Involving the Formation of Recombinant DNA
Molecules Containing No More than Two-Thirds of the Genome of Any
Eukaryotic Virus
Recombinant DNA molecules containing no more than two-thirds of the
genome of any eukaryotic virus (all viruses from a single Family (see
Section V-Q) being considered identical (see Section V-S)) may be
propagated and maintained in cells in tissue culture using BL1
containment. For such experiments, it must be demonstrated that the
cells lack helper virus for the specific Families of defective viruses
being used. If helper virus is present, procedures specified under
Section III-C-3 should be used. The DNA may contain fragments of the
genome of viruses from more than one Family but each fragment shall be
less than two-thirds of a genome.
Section III-D-2. Experiments Involving Whole Plants
This section covers experiments involving recombinant DNA-modified
whole plants, and/or experiments involving recombinant DNA-modified
organisms associated with whole plants, except those that fall under
Section III-A, III-B, III-C, or III-E. It should be emphasized that
knowledge of the organisms and judgment based on accepted scientific
practices should be used in all cases in selecting the appropriate
level of containment. For example, if the genetic modification has the
objective of increasing pathogenicity or converting a non-pathogenic
organism into a pathogen, then a higher level of containment may be
appropriate depending on the organism, its mode of dissemination, and
its target organisms. By contrast, a lower level of containment may be
appropriate for small animals associated with many types of recombinant
DNA-modified plants.
Section III-D-2-a. BL1-P is recommended for all experiments with
recombinant DNA-containing plants and plant-associated microorganisms
not covered in Section III-D-2-b or other sections of the NIH
Guidelines. Examples of such experiments are those involving
recombinant DNA-modified plants that are not noxious weeds or that
cannot interbreed with noxious weeds in the immediate geographic area,
and experiments involving whole plants and recombinant DNA-modified
non-exotic (see Section V-W) microorganisms that have no recognized
potential for rapid and widespread dissemination or for serious
detrimental impact on managed or natural ecosystems (e.g., Rhizobium
spp. and Agrobacterium spp.).
Section III-D-2-b. BL2-P or BL1-P + biological containment is
recommended for the following experiments:
Section III-D-2-b-(1). Plants modified by recombinant DNA that are
noxious weeds or can interbreed with noxious weeds in the immediate
geographic area.
Section III-D-2-b-(2). Plants in which the introduced DNA
represents the complete genome of a non-exotic infectious agent (see
Section V-W).
Section III-D-2-b-(3). Plants associated with recombinant DNA-
modified non-exotic microorganisms that have a recognized potential for
serious detrimental impact on managed or natural ecosystems (see
Section V-W).
Section III-D-2-b-(4). Plants associated with recombinant DNA-
modified exotic microorganisms that have no recognized potential for
serious natural ecosystems (see Section V-W).
Section III-D-2-b-(5). Experiments with recombinant DNA-modified
arthropods or small animals associated with plants, or with arthropods
or small animals with recombinant DNA-modified microorganisms
associated with them if the recombinant DNA-modified microorganisms
have no recognized potential for serious detrimental impact on managed
or natural ecosystems (see Section V-W).
Section III-E. Exempt Experiments
The following recombinant DNA molecules are exempt from the NIH
Guidelines and registration with the Institutional Biosafety Committee
is not required:
Section III-E-1. Those that are not in organisms or viruses.
Section III-E-2. Those that consist entirely of DNA segments from a
single nonchromosomal or viral DNA source, though one or more of the
segments may be a synthetic equivalent.
Section III-E-3. Those that consist entirely of DNA from a
prokaryotic host including its indigenous plasmids or viruses when
propagated only in that host (or a closely related strain of the same
species), or when transferred to another host by well established
physiological means.
Section III-E-4. Those that consist entirely of DNA from an
eukaryotic host including its chloroplasts, mitochondria, or plasmids
(but excluding viruses) when propagated only in that host (or a closely
related strain of the same species).
Section III-E-5. Those that consist entirely of DNA segments from
different species that exchange DNA by known physiological processes,
though one or more of the segments may be a synthetic equivalent. A
list of such exchangers will be prepared and periodically revised by
the NIH Director with advice of the RAC after appropriate notice and
opportunity for public comment (see Section IV-C-1-b-(1)-(c)). See
Appendices A-I through A-VI for a list of natural exchangers that are
exempt from the NIH Guidelines.
Section III-E-6. Those that do not present a significant risk to
health or the environment (see Section IV-C-1-b-(1)-(c)), as determined
by the NIH Director, with the advice of the RAC, and following
appropriate notice and opportunity for public comment. See Appendix C
for other classes of experiments which are exempt from the NIH
Guidelines.
Section IV, Roles and Responsibilities
Section IV-A. Policy
The safe conduct of experiments involving recombinant DNA depends
on the individual conducting such activities. The NIH Guidelines cannot
anticipate every possible situation. Motivation and good judgment are
the key essentials to protection of health and the environment. The NIH
Guidelines are intended to assist the institution, Institutional
Biosafety Committee, Biological Safety Officer, and Principal
Investigator in determining safeguards that should be implemented. The
NIH Guidelines will never be complete or final since all conceivable
experiments involving recombinant DNA cannot be foreseen. Therefore, it
is the responsibility of the institution and those associated with it
to adhere to the intent of the NIH Guidelines as well as to their
specifics. Each institution (and the Institutional Biosafety Committee
acting on its behalf) is responsible for ensuring that recombinant DNA
activities comply with the NIH Guidelines. General recognition of
institutional authority and responsibility properly establishes
accountability for safe conduct of the research at the local level. The
following roles and responsibilities constitute an administrative
framework in which safety is an essential and integral part of research
involving recombinant DNA molecules. Further clarifications and
interpretations of roles and responsibilities will be issued by the NIH
as necessary.
Section IV-B. Responsibilities of the Institution
Section IV-B-1. General Information
Each institution conducting or sponsoring recombinant DNA research
which is covered by the NIH Guidelines is responsible for ensuring that
the research is conducted in full conformity with the provisions of the
NIH Guidelines. In order to fulfill this responsibility, the
institution shall:
Section IV-B-1-a. Establish and implement policies that provide for
the safe conduct of recombinant DNA research and that ensure compliance
with the NIH Guidelines. As part of its general responsibilities for
implementing the NIH Guidelines, the institution may establish
additional procedures, as deemed necessary, to govern the institution
and its components in the discharge of its responsibilities under the
NIH Guidelines. Such procedures may include: (i) Statements formulated
by the institution for the general implementation of the NIH
Guidelines, and (ii) any additional precautionary steps the institution
deems appropriate.
Section IV-B-1-b. Establish an Institutional Biosafety Committee
that meets the requirements set forth in Section IV-B-2-a and carries
out the functions detailed in
Section IV-B-2-b.
Section IV-B-1-c. Appoint a Biological Safety Officer (who is also
a member of the Institutional Biosafety Committee) if the institution:
(i) Conducts recombinant DNA research at Biosafety Level (BL) 3 or BL4,
or (ii) engages in large scale (greater than 10 liters) research. The
Biological Safety Officer carries out the duties specified in Section
IV-B-3.
Section IV-B-1-d. Assist and ensure compliance with the NIH
Guidelines by Principal Investigators conducting research at the
institution as specified in
Section IV-B-4.
Section IV-B-1-e. Ensure appropriate training for the Institutional
Biosafety Committee Chair and members, Biological Safety Officer (when
applicable), Principal Investigators, and laboratory staff regarding
laboratory safety and implementation of the NIH Guidelines. The
Institutional Biosafety Committee Chair is responsible for ensuring
that Institutional Biosafety Committee members are appropriately
trained. The Principal Investigator is responsible for ensuring that
laboratory staff are appropriately trained. The institution is
responsible for ensuring that the Principal Investigator has sufficient
training; however, this responsibility may be delegated to the
Institutional Biosafety Committee.
Section IV-B-1-f. Determine the necessity for health surveillance
of personnel involved in connection with individual recombinant DNA
projects; and if appropriate, conduct a health surveillance program for
such projects. The institution shall establish and maintain a health
surveillance program for personnel engaged in large scale research or
production activities involving viable organisms containing recombinant
DNA molecules which require BL3 containment at the laboratory scale.
The institution shall establish and maintain a health surveillance
program for personnel engaged in animal research involving viable
recombinant DNA-containing microorganisms that require BL3 or greater
containment in the laboratory. The Laboratory Safety Monograph
discusses various components of such a program (e.g., records of agents
handled, active investigation of relevant illnesses, and the
maintenance of serial serum samples for monitoring serologic changes
that may result from the employees' work experience). Certain medical
conditions may place a laboratory worker at increased risk in any
endeavor where infectious agents are handled. Examples cited in the
Laboratory Safety Monograph include gastrointestinal disorders and
treatment with steroids, immunosuppressive drugs, or antibiotics.
Workers with such disorders or treatment should be evaluated to
determine whether they should be engaged in research with potentially
hazardous organisms during their treatment or illness. Copies of the
Laboratory Safety Monograph are available from the Office of
Recombinant DNA Activities, National Institutes of Health, Building 31,
Room 4B11, Bethesda, Maryland 20892, (301) 496-9838.
Section IV-B-1-g. Report any significant problems, violations of
the NIH Guidelines, or any significant research-related accidents and
illnesses to NIH/ORDA within thirty days, unless the institution
determines that a report has already been filed by the Principal
Investigator or Institutional Biosafety Committee. Reports shall be
sent to the Office of Recombinant DNA Activities, National Institutes
of Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
9838.
Section IV-B-2. Institutional Biosafety Committee (IBC)
The institution shall establish an Institutional Biosafety
Committee whose responsibilities need not be restricted to recombinant
DNA. The Institutional Biosafety Committee shall meet the following
requirements:
Section IV-B-2-a. Membership and Procedures
Section IV-B-2-a-(1). The Institutional Biosafety Committee must be
comprised of no fewer than five members so selected that they
collectively have experience and expertise in recombinant DNA
technology and the capability to assess the safety of recombinant DNA
research and to identify any potential risk to public health or the
environment. At least two members shall not be affiliated with the
institution (apart from their membership on the Institutional Biosafety
Committee) and who represent the interest of the surrounding community
with respect to health and protection of the environment (e.g.,
officials of state or local public health or environmental protection
agencies, members of other local governmental bodies, or persons active
in medical, occupational health, or environmental concerns in the
community). The Institutional Biosafety Committee shall include at
least one individual with expertise in plant, plant pathogen, or plant
pest containment principles when experiments utilizing Appendix P
require prior approval by the Institutional Biosafety Committee. The
Institutional Biosafety Committee shall include at least one scientist
with expertise in animal containment principles when experiments
utilizing Appendix Q require Institutional Biosafety Committee prior
approval. When the institution conducts recombinant DNA research at BL3
or BL4, a Biological Safety Officer is mandatory and shall be a member
of the Institutional Biosafety Committee (see Section IV-B-3).
Section IV-B-2-a-(2). In order to ensure the competence necessary
to review and approve recombinant DNA activities, it is recommended
that the Institutional Biosafety Committee: (i) include persons with
expertise in recombinant DNA technology, biological safety, and
physical containment; (ii) include or have available as consultants
persons knowledgeable in institutional commitments and policies,
applicable law, standards of professional conduct and practice,
community attitudes, and the environment, and (iii) include at least
one member representing the laboratory technical staff.
Section IV-B-2-a-(3). The institution shall file a report with NIH/
ORDA which includes the names and biographical sketches of all
Institutional Biosafety Committee members, including community members,
in such form and at such times as required by NIH/ORDA.
Section IV-B-2-a-(4). No member of an Institutional Biosafety
Committee may be involved (except to provide information requested by
the Institutional Biosafety Committee) in the review or approval of a
project in which he/she has been or expects to be engaged or has a
direct financial interest.
Section IV-B-2-a-(5). The institution, that is ultimately
responsible for the effectiveness of the Institutional Biosafety
Committee, may establish procedures that the Institutional Biosafety
Committee shall follow in its initial and continuing review and
approval of applications, proposals, and activities.
Section IV-B-2-a-(6). When possible and consistent with protection
of privacy and proprietary interests, the institution is encouraged to
open its Institutional Biosafety Committee meetings to the public.
Section IV-B-2-a-(7). Upon request, the institution shall make
available to the public all Institutional Biosafety Committee meeting
minutes and any documents submitted to or received from funding
agencies which the latter are required to make available to the public.
If public comments are made on Institutional Biosafety Committee
actions, the institution shall forward both the public comments and the
Institutional Biosafety Committee's response to the Office of
Recombinant DNA Activities, National Institutes of Health, Building 31,
Room 4B11, Bethesda, Maryland 20892, (301) 496-9838.
Section IV-B-2-b. Functions
On behalf of the institution, the Institutional Biosafety Committee
is responsible for:
Section IV-B-2-b-(1). Reviewing recombinant DNA research conducted
at or sponsored by the institution for compliance with the NIH
Guidelines as specified in Section III and approving those research
projects that are found to conform with the NIH Guidelines. This review
shall include: (i) independent assessment of the containment levels
required by the NIH Guidelines for the proposed research, and (ii)
assessment of the facilities, procedures, practices, and training and
expertise of personnel involved in recombinant DNA research.
Section IV-B-2-b-(2). Notifying the Principal Investigator of the
results of the Institutional Biosafety Committee's review and approval.
Section IV-B-2-b-(3). Lowering containment levels for certain
experiments as specified in Section III-C-2-a.
Section IV-B-2-b-(4). Setting containment levels as specified in
Sections III-C-4-b and III-C-5.
Section IV-B-2-b-(5). Periodically reviewing recombinant DNA
research conducted at the institution to ensure compliance with the NIH
Guidelines.
Section IV-B-2-b-(6). Adopting emergency plans covering accidental
spills and personnel contamination resulting from recombinant DNA
research.
Note: The Laboratory Safety Monograph describes basic elements
for developing specific procedures dealing with major spills of
potentially hazardous materials in the laboratory, including
information and references about decontamination and emergency
plans. The NIH and the Centers for Disease Control and Prevention
are available to provide consultation and direct assistance, if
necessary, as posted in the Laboratory Safety Monograph. The
institution shall cooperate with the state and local public health
departments by reporting any significant research-related illness or
accident that may be hazardous to the public health.
Section IV-B-2-b-(7). Reporting any significant problems with or
violations of the NIH Guidelines and any significant research-related
accidents or illnesses to the appropriate institutional official and
NIH/ORDA within 30 days, unless the Institutional Biosafety Committee
determines that a report has already been filed by the Principal
Investigator. Reports to NIH/ORDA shall be sent to the Office of
Recombinant DNA Activities, National Institutes of Health, Bethesda,
Maryland 20892, (301) 496-9838.
Section IV-B-2-b-(8). The Institutional Biosafety Committee may not
authorize initiation of experiments which are not explicitly covered by
the NIH Guidelines until NIH (with the advice of the RAC when required)
establishes the containment requirement.
Section IV-B-2-b-(9). Performing such other functions as may be
delegated to the Institutional Biosafety Committee under Section IV-B-
2.
Section IV-B-3. Biological Safety Officer (BSO)
Section IV-B-3-a. The institution shall appoint a Biological Safety
Officer if it engages in large scale research or production activities
involving viable organisms containing recombinant DNA molecules.
Section IV-B-3-b. The institution shall appoint a Biological Safety
Officer if it engages in recombinant DNA research at BL3 or BL4. The
Biological Safety Officer shall be a member of the Institutional
Biosafety Committee.
Section IV-B-3-c. The Biological Safety Officer's duties include,
but are not be limited to:
Section IV-B-3-c-(1). Periodic inspections to ensure that
laboratory standards are rigorously followed;
Section IV-B-3-c-(2). Reporting to the Institutional Biosafety
Committee and the institution any significant problems, violations of
the NIH Guidelines, and any significant research-related accidents or
illnesses of which the Biological Safety Officer becomes aware unless
the Biological Safety Officer determines that a report has already been
filed by the Principal Investigator;
Section IV-B-3-c-(3). Developing emergency plans for handling
accidental spills and personnel contamination and investigating
laboratory accidents involving recombinant DNA research;
Section IV-B-3-c-(4). Providing advice on laboratory security;
Section IV-B-3-c-(5). Providing technical advice to Principal
Investigators and the Institutional Biosafety Committee on research
safety procedures.
Note: See the Laboratory Safety Monograph for additional
information on the duties of the Biological Safety Officer.
Section IV-B-4. Principal Investigator (PI)
On behalf of the institution, the Principal Investigator is
responsible for full compliance with the NIH Guidelines in the conduct
of recombinant DNA research.
Section IV-B-4-a. General Responsibilities
As part of this general responsibility, the Principal Investigator
shall:
Section IV-B-4-a-(1). Initiate or modify no recombinant DNA
research which requires Institutional Biosafety Committee approval
prior to initiation (see Sections III-A, III-B, and III-C) until that
research or the proposed modification thereof has been approved by the
Institutional Biosafety Committee and has met all other requirements of
the NIH Guidelines;
Section IV-B-4-a-(2). Determine whether experiments are covered by
Section III-D and that the appropriate procedures are followed;
Section IV-B-4-a-(3). Report any significant problems, violations
of the NIH Guidelines, or any significant research-related accidents
and illnesses to the Biological Safety Officer (where applicable),
Greenhouse/Animal Facility Director (where applicable), Institutional
Biosafety Committee, NIH/ORDA, and other appropriate authorities (if
applicable) within 30 days (reports to NIH/ORDA shall be sent to the
Office of Recombinant DNA Activities, National Institutes of Health,
Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-9838);
Section IV-B-4-a-(4). Report any new information bearing on the NIH
Guidelines to the Institutional Biosafety Committee and to NIH/ORDA
(reports to NIH/ORDA shall be sent to the Office of Recombinant DNA
Activities, National Institutes of Health, Building 31, Room 4B11,
Bethesda, Maryland 20892, (301) 496-9838);
Section IV-B-4-a-(5). Be adequately trained in good microbiological
techniques;
Section IV-B-4-a-(6). Adhere to Institutional Biosafety Committee-
approved emergency plans for handling accidental spills and personnel
contamination; and
Section IV-B-4-a-(7). Comply with shipping requirements for
recombinant DNA molecules (see Appendix H for shipping requirements and
the Laboratory Safety Monograph for technical recommendations).
Section IV-B-4-b. Submissions by the Principal Investigator to the NIH/
ORDA
The Principal Investigator shall:
Section IV-B-4-b-(1). Submit information to NIH/ORDA for
certification of new host-vector systems;
Section IV-B-4-b-(2). Petition NIH/ORDA, with notice to the
Institutional Biosafety Committee, for proposed exemptions to the NIH
Guidelines;
Section IV-B-4-b-(3). Petition NIH/ORDA, with concurrence of the
Institutional Biosafety Committee, for approval to conduct experiments
specified in Sections III-A and III-B of the NIH Guidelines;
Section IV-B-4-b-(4). Petition NIH/ORDA for determination of
containment for experiments requiring case-by-case review; and
Section IV-B-4-b-(5). Petition NIH/ORDA for determination of
containment for experiments not covered by the NIH Guidelines.
Section IV-B-4-c. Submissions by the Principal Investigator to the
Institutional Biosafety Committee
The Principal Investigator shall:
Section IV-B-4-c-(1). Make an initial determination of the required
levels of physical and biological containment in accordance with the
NIH Guidelines;
Section IV-B-4-c-(2). Select appropriate microbiological practices
and laboratory techniques to be used for the research;
Section IV-B-4-c-(3). Submit the initial research protocol and any
subsequent changes (e.g., changes in the source of DNA or host-vector
system), if covered under
Sections III-A, III-B, III-C, or III-D, to the Institutional Biosafety
Committee for review and approval or disapproval; and
Section IV-B-4-c-(4). Remain in communication with the
Institutional Biosafety Committee throughout the conduct of the
project.
Section IV-B-4-d. Responsibilities of the Principal Investigator Prior
to Initiating Research
The Principal Investigator shall:
Section IV-B-4-d-(1). Make available to all laboratory staff the
protocols that describe the potential biohazards and the precautions to
be taken;
Section IV-B-4-d-(2). Instruct and train laboratory staff in: (i)
the practices and techniques required to ensure safety, and (ii) the
procedures for dealing with accidents; and
Section IV-B-4-d-(3). Inform the laboratory staff of the reasons
and provisions for any precautionary medical practices advised or
requested (e.g., vaccinations or serum collection).
Section IV-B-4-e. Responsibilities of the Principal Investigator During
the Conduct of the Research
The Principal Investigator shall:
Section IV-B-4-e-(1). Supervise the safety performance of the
laboratory staff to ensure that the required safety practices and
techniques are employed;
Section IV-B-4-e-(2). Investigate and report any significant
problems pertaining to the operation and implementation of containment
practices and procedures in writing to the Biological Safety Officer
(where applicable), Greenhouse/Animal Facility Director (where
applicable), the Institutional Biosafety Committee, NIH/ORDA, and other
appropriate authorities (if applicable) (reports to the NIH/ORDA shall
be sent to the Office of Recombinant DNA Activities, National
Institutes of Health, Building 31, Room 4B11, Bethesda, Maryland 20892,
(301) 496-9838);
Section IV-B-4-e-(3). Correct work errors and conditions that may
result in the release of recombinant DNA materials; and
Section IV-B-4-e-(4). Ensure the integrity of the physical
containment (e.g., biological safety cabinets) and the biological
containment (e.g., purity and genotypic and phenotypic
characteristics).
Section IV-C. Responsibilities of the National Institutes of Health
(NIH)
Section IV-C-1. NIH Director
The NIH Director is responsible for: (i) establishing the NIH
Guidelines, (ii) overseeing their implementation, and (iii) their final
interpretation. The NIH Director has responsibilities under the NIH
Guidelines that involve ORDA and the RAC. ORDA's responsibilities under
the NIH Guidelines are administrative. Advice from the RAC is primarily
scientific, technical, and ethical. In certain circumstances, there is
specific opportunity for public comment with published response prior
to final action.
Section IV-C-1-a. General Responsibilities
The NIH Director is responsible for:
Section IV-C-1-a-(1). Promulgating requirements as necessary to
implement the NIH Guidelines;
Section IV-C-1-a-(2). Establishing and maintaining the RAC to carry
out the responsibilities set forth in Section IV-C-2 (RAC membership is
specified in its charter and in Section IV-C-2); and
Section IV-C-1-a-(3). Establishing and maintaining ORDA to carry
out the responsibilities defined in Section IV-C-3.
Section IV-C-1-b. Specific Responsibilities
In carrying out the responsibilities set forth in this section, the
NIH Director, or a designee shall weigh each proposed action through
appropriate analysis and consultation to determine whether it complies
with the NIH Guidelines and presents no significant risk to health or
the environment.
Section IV-C-1-b-(1). Major Actions
To execute Major Actions, the NIH Director shall seek the advice of
the RAC and provide an opportunity for public and Federal agency
comment. Specifically, the Notice of Meeting and Proposed Actions to
the NIH Guidelines shall be published in the Federal Register at least
15 days before the RAC meeting (not applicable for Expedited Review
single patient human gene transfer experiments considered under
Appendix M-VI). The NIH Director's decision, at his/her discretion, may
be published in the Federal Register for 15 days of comment before
final action is taken. The NIH Director's final decision, along with
responses to public comments, shall be published in the Federal
Register. The RAC and Institutional Biosafety Committee Chairs shall be
notified of the following decisions:
Section IV-C-1-b-(1)-(a). Changing containment levels for types of
experiments that are specified in the NIH Guidelines when a Major
Action is involved;
Section IV-C-1-b-(1)-(b). Assigning containment levels for types of
experiments that are not explicitly considered in the NIH Guidelines
when a Major Action is involved;
Section IV-C-1-b-(1)-(c). Promulgating and amending a list of
classes of recombinant DNA molecules to be exempt from the NIH
Guidelines because they consist entirely of DNA segments from species
that exchange DNA by known physiological processes or otherwise do not
present a significant risk to health or the environment;
Section IV-C-1-b-(1)-(d). Permitting experiments specified by
Section III-A;
Section IV-C-1-b-(1)-(e). Certifying new host-vector systems with
the exception of minor modifications of already certified systems (the
standards and procedures for certification are described in Appendix I-
II). Minor modifications constitute (e.g., those of minimal or no
consequence to the properties relevant to containment); and
Section IV-C-1-b-(1)-(f). Adopting other changes in the NIH
Guidelines.
Section IV-C-1-b-(2). Minor Actions
NIH/ORDA shall carry out certain functions as delegated to it by
the NIH Director (see Section IV-C-3). Minor Actions (as determined by
NIH/ORDA in consultation with the RAC Chair and one or more RAC
members, as necessary) will be transmitted to the RAC and Institutional
Biosafety Committee Chairs:
Section IV-C-1-b-(2)-(a). Reviewing and approving certain
experiments involving the deliberate transfer of recombinant DNA or DNA
or RNA derived from recombinant DNA into one or more human subjects
that qualify for the Accelerated Review process (see Section III-B-2);
Section IV-C-1-b-(2)-(b). Reviewing and approving minor changes to
human gene transfer protocols under Section III-A-2 and III-B-2;
Section IV-C-1-b-(2)-(c). Changing containment levels for
experiments that are specified in Section III;
Section IV-C-1-b-(2)-(d). Assigning containment levels for
experiments not explicitly considered in the NIH Guidelines; and
Section IV-C-1-b-(2)-(e). Revising the Classification of Etiologic
Agents for the purpose of these NIH Guidelines (see Section V-A).
Section IV-C-1-b-(2)-(f). Interpreting the NIH Guidelines for
experiments to which the NIH Guidelines do not specifically assign
containment levels;
Section IV-C-1-b-(2)-(g). Setting containment under Sections III-C-
1-d and III-C-2- b;
Section IV-C-1-b-(2)-(h). Approving minor modifications of already
certified host-vector systems (the standards and procedures for such
modifications are described in Appendix I-II);
Section IV-C-1-b-(2)-(i). Decertifying already certified host-
vector systems;
Section IV-C-1-b-(2)-(j). Adding new entries to the list of
molecules toxic for vertebrates (see Appendix F); and
Section IV-C-1-b-(2)-(k). Determining appropriate containment
conditions for experiments according to case precedents developed under
Section IV-C-1-b-(2)-(c).
Section IV-C-1-b-(3). The NIH Director shall conduct, support, and
assist training programs in laboratory safety for Institutional
Biosafety Committee members, Biological Safety Officers, Principal
Investigators, and laboratory staff.
Section IV-C-2. Recombinant DNA Advisory Committee (RAC)
The RAC is responsible for carrying out specified functions cited
below as well as others assigned under its charter or by the DHHS
Secretary, the DHHS Assistant Secretary for Health, and the NIH
Director. The RAC consists of 25 members including the Chair, appointed
by the DHHS Secretary or his/her designee, at least fourteen of whom
are selected from authorities knowledgeable in the fields of molecular
genetics, molecular biology, recombinant DNA research, or other
scientific fields. At least six members of the RAC shall be persons
knowledgeable in applicable law, standards of professional conduct and
practice, public attitudes, the environment, public health,
occupational health, or related fields. Representatives from Federal
agencies shall serve as non-voting members.
Nominations for the RAC may be submitted to the Office of
Recombinant DNA Activities, National Institutes of Health, Building 31,
Room 4B11, Bethesda, Maryland 20892, (301) 496-9838.
All meetings of the RAC shall be announced in the Federal Register,
including tentative agenda items, 15 days before the meeting. Final
agendas, if modified, shall be available at least 72 hours before the
meeting. No item defined as a Major Action under Section IV-C-1-b-(1)
may be added to an agenda following Federal Register publication.
The RAC shall be responsible for advising the NIH Director on the
actions listed in Sections IV-C-1-b-(1).
Section IV-C-3. Office of Recombinant DNA Activities (ORDA)
ORDA shall serve as a focal point for information on recombinant
DNA activities and provide advice to all within and outside NIH
including institutions, Biological Safety Officers, Principal
Investigators, Federal agencies, state and local governments, and
institutions in the private sector. ORDA shall carry out such other
functions as may be delegated to it by the NIH Director, including
those authorities described in Section IV-C-1-b-(2). ORDA's
responsibilities include, but are not limited to the following:
Section IV-C-3-a. Reviewing and approving experiments in
conjunction with ad hoc experts involving the cloning of genes encoding
for toxin molecules that are lethal for vertebrates at an 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