# Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines); Notice DEPARTMENT OF HEALTH AND HUMAN SERVICES

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URL: https://www.frixlaw.com/law-library/documents/fr%3A94-16200

## Record

- **Collection:** Federal Register
- **Document type:** Uncategorized Document
- **Published:** July 5, 1994

## Text

[Federal Register Volume 59, Number 127 (Tuesday, July 5, 1994)]
[Unknown Section]
[Page 0]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 94-16200]

[[Page Unknown]]

[Federal Register: July 5, 1994]

_______________________________________________________________________

Part IV

Department of Health and Human Services

_______________________________________________________________________

National Institutes of Health

_______________________________________________________________________

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

National Institutes of Health

Guidelines for Research Involving Recombinant DNA Molecules (NIH
Guidelines)

June 1994.
These NIH Guidelines supersede all earlier versions and shall be in
effect until further notice.

Table of Contents

Section I. Scope of the NIH Guidelines
Section I-A. Purpose
Section I-B. Definition of Recombinant DNA Molecules
Section I-C. General Applicability
Section I-D. General Definitions
Section II. Containment
Section III. Experiments Covered by the NIH Guidelines
Section III-A. Experiments that Require Institutional Biosafety
Committee Approval, RAC Review, and NIH Approval Before Initiation
Section III-B. Experiments that Require NIH/ORDA and Institutional
Biosafety Committee Approval Before Initiation
Section III-B-1. Experiments Involving the Cloning of Toxin
Molecules with LD50 of Less than 100 Nanograms Per Kilogram
Body Weight
Section III-B-2. Accelerated Review of Human Gene Transfer
Experiments
Section III-B-3. Minor Modifications to Human Gene Transfer
Experiments
Section III-C. Experiments that Require Institutional Biosafety
Committee Approval Before Initiation
Section III-C-1. Experiments Using Human or Animal Pathogens (Class
2, Class 3, Class 4, or Class 5) Agents as Host-Vector Systems
Section III-C-2. Experiments in which DNA from Human or Animal
Pathogens (Class 2, Class 3, Class 4, or Class 5) Agents is Cloned
into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector
Systems
Section III-C-3. Experiments Involving the Use of Infectious Animal
or Plant DNA or RNA Viruses or Defective Animal or Plant DNA or RNA
Viruses in the Presence of Helper Virus in Tissue Culture Systems
Section III-C-4. Experiments Involving Whole Animals
Section III-C-5. Experiments Involving Whole Plants
Section III-C-6. Experiments Involving More than 10 Liters of
Culture
Section III-C-7. Human Gene Transfer Experiments Not Covered by
Section III-A-2, III-B-2, III-B-3, and Not Considered Exempt under
Section V-U
Section III-D. Experiments that Require Institutional Biosafety
Committee Notice Simultaneous with Initiation
Section III-D-1. Experiments Involving the Formation of Recombinant
DNA Molecules Containing No More than Two-Thirds of the Genome of
any Eukaryotic Virus
Section III-D-2. Experiments Involving Whole Plants
Section III-E. Exempt Experiments
Section IV. Roles and Responsibilities
Section IV-A. Policy
Section IV-B. Responsibilities of the Institution
Section IV-B-1. General Information
Section IV-B-2. Institutional Biosafety Committee (IBC)
Section IV-B-3. Biological Safety Officer (BSO)
Section IV-B-4. Principal Investigator (PI)
Section IV-C. Responsibilities of the National Institutes of Health
(NIH)
Section IV-C-1. NIH Director
Section IV-C-1-a. General Responsibilities
Section IV-C-1-b. Specific Responsibilities
Section IV-C-1-b-(1). Major Actions
Section IV-C-1-b-(2). Minor Actions
Section IV-C-2. Recombinant DNA Advisory Committee (RAC)
Section IV-C-3. Office of Recombinant DNA Activities (ORDA)
Section IV-C-4. Other NIH Components
Section IV-D. Compliance with the NIH Guidelines
Section IV-E. Voluntary Compliance
Section V. Footnotes and References of Sections I-IV
Appendix A. Exemptions under Section III-E-5--Sublists of Natural
Exchangers
Appendix B. Classification of Etiologic Agents and Oncogenic Viruses
on the Basis of Hazard
Appendix B-I. Class 1 Agents
Appendix B-II. Class 2 Agents
Appendix B-III. Class 3 Agents
Appendix B-IV. Class 4 Agents
Appendix B-V. Class 5 Agents
Appendix B-VI. Footnotes and References of Appendix B
Appendix C. Exemptions under Section III-E-6
Appendix C-I. Recombinant DNA in Tissue Culture
Appendix C-II. Escherichia coli K-12 Host-Vector Systems
Appendix C-III. Saccharomyces Host-Vector Systems
Appendix C-IV. Bacillus subtilis or Bacillus licheniformis Host-
Vector Systems
Appendix C-V. Extrachromosomal Elements of Gram Positive Organisms
Appendix C-VI. Footnotes and References of Appendix C
Appendix D. Major Actions Taken under the NIH Guidelines
Appendix E. Certified Host-Vector Systems
Appendix E-I. Bacillus subtilis
Appendix E-II. Saccharomyces cerevisiae
Appendix E-III. Escherichia coli
Appendix E-IV. Neurospora crassa
Appendix E-V. Streptomyces
Appendix E-VI. Pseudomonas putida
Appendix F. Containment Conditions for Cloning of Genes Coding for
the Biosynthesis of Molecules Toxic for Vertebrates
Appendix F-I. General Information
Appendix F-II. Cloning of Toxin Molecule Genes in Escherichia coli
K-12
Appendix F-III. Cloning of Toxic Molecule Genes in Organisms other
than Escherichia coli K-12
Appendix F-IV. Specific Approvals
Appendix G. Physical Containment
Appendix G-I. Standard Practices and Training
Appendix G-II. Physical Containment Levels
Appendix G-II-A. Biosafety Level 1 (BL1)
Appendix G-II-B. Biosafety Level 2 (BL2)
Appendix G-II-C. Biosafety Level 3 (BL3)
Appendix G-II-D. Biosafety Level 4 (BL4)
Appendix G-III. Footnotes and References of Appendix G
Appendix H. Shipment
Appendix I. Biological Containment
Appendix I-I. Levels of Biological Containment
Appendix I-I-A. Host-Vector 1 Systems
Appendix I-I-B. Host-Vector 2 Systems
Appendix I-II. Certification of Host-Vector Systems
Appendix I-III. Footnotes and References of Appendix I
Appendix J. Biotechnology Research Subcommittee
Appendix K. Physical Containment for Large Scale Uses of Organisms
Containing Recombinant DNA Molecules
Appendix K-I. Selection of Physical Containment Levels
Appendix K-II. Good Large Scale Practices (GLSP)
Appendix K-III. Biosafety Level 1 (BL1)--Large Scale
Appendix K-IV. Biosafety Level 2 (BL2)--Large Scale
Appendix K-V. Biosafety Level 3 (BL3)--Large Scale
Appendix K-VI. Footnotes of Appendix K
Appendix K-VII. Definitions to Accompany Containment Grid and
Appendix K
Appendix L. Release into the Environment of Certain Plants
Appendix M. Points to Consider in the Design and Submission of
Protocols for the Transfer of Recombinant DNA Molecules into the
Genome of One or More Human Subjects
Appendix M-I. Description of Proposal
Appendix M-I-A. Objectives and Rationale of the Proposed Research
Appendix M-I-B. Research Design, Anticipated Risks and Benefits
Appendix M-I-C. Selection of the Patients
Appendix M-I-D. Informed Consent
Appendix M-I-E. Privacy and Confidentiality
Appendix M-II. Special Issues
Appendix M-III. Guidelines for the Submission of Human Gene Transfer
Protocols
Appendix M-III-A. Principal Investigator-Submitted Material
Appendix M-III-B. Time Frame for Submissions
Appendix M-III-C. Oral Responses to the RAC
Appendix M-III-D. Primary Reviewers' Responses
Appendix M-IV. Reporting Requirements
Appendix M-V. Procedures to be Followed for Accelerated Review of
Human Gene Transfer Experiments by NIH/ORDA under Section III-B-2
Appendix M-VI. Procedures to be Followed for Expedited Review of
Single Patient Human Gene Transfer Experiments by the NIH Director
Under Section III-A-2
Appendix M-VII. Footnotes of Appendix M
Appendix P. Physical and Biological Containment for Recombinant DNA
Research Involving Plants
Appendix P-I. General Plant Biosafety Levels
Appendix P-II. Physical Containment Levels
Appendix P-II-A. Biosafety Level 1--Plants (BL1-P)
Appendix P-II-B. Biosafety Level 2--Plants (BL2-P)
Appendix P-II-C. Biosafety Level 3--Plants (BL3-P)
Appendix P-II-D. Biosafety Level 4--Plants (BL4-P)
Appendix P-III. Biological Containment Practices
Appendix P-III-A. Biological Containment Practices (Plants)
Appendix P-III-B. Biological Containment Practices (Microorganisms)
Appendix P-III-C. Biological Containment Practices (Macroorganisms)
Appendix Q. Physical and Biological Containment for Recombinant DNA
Research Involving Animals
Appendix Q-I. General Considerations
Appendix Q-I-A. Containment Levels
Appendix Q-I-B. Disposal of Animals (BL1-N through BL4-N)
Appendix Q-II. Physical and Biological Containment Levels
Appendix Q-II-A. Biosafety Level 1--Animals (BL1-N)
Appendix Q-II-B. Biosafety Level 2--Animals (BL2-N)
Appendix Q-II-C. Biosafety Level 3--Animals (BL3-N)
Appendix Q-II-D. Biosafety Level 4--Animals (BL4-N)
Appendix Q-III. Footnotes and References for Appendix Q

Section I. Scope of the NIH Guidelines

Section I-A. Purpose

The purpose of the NIH Guidelines is to specify practices for
constructing and handling: (i) Recombinant deoxyribonucleic acid (DNA)
molecules, and (ii) organisms and viruses containing recombinant DNA
molecules.
Section I-A-1. Any recombinant DNA experiment, which according to
the NIH Guidelines requires approval by the NIH, must be submitted to
the NIH or to another Federal agency that has jurisdiction for review
and approval. Once approval, or other applicable clearances, has been
obtained from a Federal agency other than the NIH (whether the
experiment is referred to that agency by the NIH or sent directly there
by the submitter), the experiment may proceed without the necessity for
NIH review or approval (see exceptions in Sections I-A-2 and I-A-3).
Section I-A-2. Certain experiments that involve the deliberate
transfer of recombinant DNA or DNA or RNA derived from recombinant DNA
into one or more human subjects (see Section V-U) shall be considered
Major Actions (see Section IV-C-1-b-(1)), and shall require RAC review
and NIH Director approval, if determined by NIH/ORDA in consultation
with the RAC Chair and/or one or more RAC members, as necessary, to:
(i) Represent novel characteristics (e.g., target disease or vector),
(ii) represent an uncertain degree of risk to human health or the
environment, or (iii) contain information determined to require further
public review (see Section III-A-2).
Section I-A-3. Experiments involving the transfer of recombinant
DNA to one or more human subjects that are not considered under Section
III-A-2 may qualify for Accelerated Review (see Section III-B-2 and
Appendix M-V) and will be considered as Minor Actions (see Section IV-
C-1-b-(2)-(a)). Actions that qualify for Accelerated Review will be
reviewed and approved by NIH/ORDA in consultation with the RAC Chair
and/or one or more RAC members, as necessary.
Certain experiments involving the transfer of recombinant DNA or
DNA or RNA derived from recombinant DNA into one or more human subjects
(see Section V-U) may be considered exempt from RAC and/or NIH/ORDA
review and/or NIH Director approval and only require registration with
NIH/ORDA (see Section III-C-7).

Section I-B. Definition of Recombinant DNA Molecules

In the context of the NIH Guidelines, recombinant DNA molecules are
defined as either: (i) Molecules that are constructed outside living
cells by joining natural or synthetic DNA segments to DNA molecules
that can replicate in a living cell, or (ii) molecules that result from
the replication of those described in (i) above.
Synthetic DNA segments which are likely to yield a potentially
harmful polynucleotide or polypeptide (e.g., a toxin or a
pharmacologically active agent) are considered as equivalent to their
natural DNA counterpart. If the synthetic DNA segment is not expressed
in vivo as a biologically active polynucleotide or polypeptide product,
it is exempt from the NIH Guidelines.
Genomic DNA of plants and bacteria that have acquired a
transposable element, even if the latter was donated from a recombinant
vector no longer present, are not subject to the NIH Guidelines unless
the transposon itself contains recombinant DNA.
Section I-C. General Applicability
Section I-C-1. The NIH Guidelines are applicable to:
Section I-C-1-a. All recombinant DNA research within the United
States (U.S.) or its territories that is conducted at or sponsored by
an institution that receives any support for recombinant DNA research
from the NIH, including research performed directly by the NIH. An
individual who receives support for research involving recombinant DNA
must be associated with or sponsored by an institution that assumes the
responsibilities assigned in the NIH Guidelines.
Section I-C-1-b. All recombinant DNA research performed abroad:
Specifically:
Section I-C-1-b-(1). Research supported by NIH funds.
Section I-C-1-b-(2). If they involve testing in humans of materials
containing recombinant DNA developed with NIH funds and if the
institution that developed those materials sponsors or participates in
those projects. Participation includes research collaboration or
contractual agreements, not mere provision of research materials.
Section I-C-1-b-(3). If the host country has established rules for
the conduct of recombinant DNA research, then the research must be in
compliance with those rules. If the host country does not have such
rules, the proposed research must be reviewed and approved by an NIH-
approved Institutional Biosafety Committee or equivalent review body
and accepted in writing by an appropriate national governmental
authority of the host country. The safety practices that are employed
abroad must be reasonably consistent with the NIH Guidelines.

Section I-D. General Definitions

The following terms, which are used throughout the NIH Guidelines,
are defined as follows:
Section I-D-1. An ``institution'' is any public or private entity
(including Federal, state, and local government agencies).
Section I-D-2. An ``Institutional Biosafety Committee'' is a
committee that: (i) Meets the requirements for membership specified in
Section IV-B-2, and (ii) reviews, approves, and oversees projects in
accordance with the responsibilities defined in Section IV-B-2.
Section I-D-3. The ``Office of Recombinant DNA Activities (ORDA)''
is the office within the NIH that is responsible for: (i) Reviewing and
coordinating all activities relating to the NIH Guidelines, and (ii)
performing other duties as defined in Section IV-C-3.
Section I-D-4. The ``Recombinant DNA Advisory Committee'' is the
public advisory committee that advises the Department of Health and
Human Services (DHHS) Secretary, the DHHS Assistant Secretary for
Health, and the NIH Director concerning recombinant DNA research. The
RAC shall be constituted as specified in Section IV-C-2.
Section I-D-5. The ``NIH Director'' is the Director of the National
Institutes of Health, or any other officer or employee of NIH to whom
authority has been delegated.
Section I-D-6. ``Deliberate release'' is defined as a planned
introduction of recombinant DNA-containing microorganisms, plants, or
animals into the environment.

Section II. Containment

Effective biological safety programs have been operative in a
variety of laboratories for many years. Considerable information
already exists about the design of physical containment facilities and
selection of laboratory procedures applicable to organisms carrying
recombinant DNA (see section V-A). The existing programs rely upon
mechanisms that can be divided into two categories: (i) A set of
standard practices that are generally used in microbiological
laboratories; and (ii) special procedures, equipment, and laboratory
installations that provide physical barriers that are applied in
varying degrees according to the estimated biohazard. Four biosafety
levels are described in Appendix G. These biosafety levels consist of
combinations of laboratory practices and techniques, safety equipment,
and laboratory facilities appropriate for the operations performed and
are based on the potential hazards imposed by the agents used and for
the laboratory function and activity. Biosafety Level 4 provides the
most stringent containment conditions, Biosafety Level 1 the least
stringent.
Experiments involving recombinant DNA lend themselves to a third
containment mechanism, namely, the application of highly specific
biological barriers. Natural barriers exist that limit either: (i) The
infectivity of a vector or vehicle (plasmid or virus) for specific
hosts, or (ii) its dissemination and survival in the environment.
Vectors, which provide the means for recombinant DNA and/or host cell
replication, can be genetically designed to decrease, by many orders of
magnitude, the probability of dissemination of recombinant DNA outside
the laboratory (see Appendix I).
Since these three means of containment are complementary, different
levels of containment can be established that apply various
combinations of the physical and biological barriers along with a
constant use of standard practices. Categories of containment are
considered separately in order that such combinations can be
conveniently expressed in the NIH Guidelines.
Physical containment conditions within laboratories, described in
Appendix G, may not always be appropriate for all organisms because of
their physical size, the number of organisms needed for an experiment,
or the particular growth requirements of the organism. Likewise,
biological containment for microorganisms described in Appendix I may
not be appropriate for all organisms, particularly higher eukaryotic
organisms. However, significant information exists about the design of
research facilities and experimental procedures that are applicable to
organisms containing recombinant DNA that is either integrated into the
genome or into microorganisms associated with the higher organism as a
symbiont, pathogen, or other relationship. This information describes
facilities for physical containment of organisms used in non-
traditional laboratory settings and special practices for limiting or
excluding the unwanted establishment, transfer of genetic information,
and dissemination of organisms beyond the intended location, based on
both physical and biological containment principles. Research conducted
in accordance with these conditions effectively confines the organism.
For research involving plants, four biosafety levels (BL1-P through
BL4-P) are described in Appendix P. BL1-P is designed to provide a
moderate level of containment for experiments for which there is
convincing biological evidence that precludes the possibility of
survival, transfer, or dissemination of recombinant DNA into the
environment, or in which there is no recognizable and predictable risk
to the environment in the event of accidental release. BL2-P is
designed to provide a greater level of containment for experiments
involving plants and certain associated organisms in which there is a
recognized possibility of survival, transmission, or dissemination of
recombinant DNA containing organisms, but the consequence of such an
inadvertent release has a predictably minimal biological impact. BL3-P
and BL4-P describe additional containment conditions for research with
plants and certain pathogens and other organisms that require special
containment because of their recognized potential for significant
detrimental impact on managed or natural ecosystems. BL1-P relies upon
accepted scientific practices for conducting research in most ordinary
greenhouse or growth chamber facilities and incorporates accepted
procedures for good pest control and cultural practices. BL1-P
facilities and procedures provide a modified and protected environment
for the propagation of plants and microorganisms associated with the
plants and a degree of containment that adequately controls the
potential for release of biologically viable plants, plant parts, and
microorganisms associated with them. BL2-P and BL3-P rely upon accepted
scientific practices for conducting research in greenhouses with
organisms infecting or infesting plants in a manner that minimizes or
prevents inadvertent contamination of plants within or surrounding the
greenhouse. BL4-P describes facilities and practices known to provide
containment of certain exotic plant pathogens.
For research involving animals, which are of a size or have growth
requirements that preclude the use of conventional primary containment
systems used for small laboratory animals, four biosafety levels (BL1-N
through BL4-N) are described in Appendix Q. BL1-N describes containment
for animals that have been modified by stable introduction of
recombinant DNA, or DNA derived therefrom, into the germ-line
(transgenic animals) and experiments involving viable recombinant DNA-
modified microorganisms and is designed to eliminate the possibility of
sexual transmission of the modified genome or transmission of
recombinant DNA-derived viruses known to be transmitted from animal
parent to offspring only by sexual reproduction. Procedures, practices,
and facilities follow classical methods of avoiding genetic exchange
between animals. BL2-N describes containment which is used for
transgenic animals associated with recombinant DNA-derived organisms
and is designed to eliminate the possibility of vertical or horizontal
transmission. Procedures, practices, and facilities follow classical
methods of avoiding genetic exchange between animals or controlling
arthropod transmission. BL3-N and BL4-N describe higher levels of
containment for research with certain transgenic animals involving
agents which pose recognized hazard.
In constructing the NIH Guidelines, it was necessary to define
boundary conditions for the different levels of physical and biological
containment and for the classes of experiments to which they apply.
These definitions do not take into account all existing and anticipated
information on special procedures that will allow particular
experiments to be conducted under different conditions than indicated
here without affecting risk. Individual investigators and Institutional
Biosafety Committees are urged to devise simple and more effective
containment procedures and to submit recommended changes in the NIH
Guidelines to permit the use of these procedures.

Section III. Experiments Covered by the NIH Guidelines

This section describes five categories of experiments involving
recombinant DNA: (i) Those that require RAC review and NIH and
Institutional Biosafety Committee approval before initiation (see
section III-A), (ii) those that require NIH/ORDA and Institutional
Biosafety Committee approval before initiation (see section III-B);
(iii) those that require Institutional Biosafety Committee approval
before initiation (see section III-C), (iv) those that require
Institutional Biosafety Committee notification simultaneous with
initiation (see section III-D), and (v) those that are exempt from the
NIH Guidelines (see section III-E).

Note: If an experiment falls into either section III-A or
section III-B and one of the other categories, the rules pertaining
to section III-A or section III-B shall be followed. If an
experiment falls into section III-E and into either sections III-C
or III-D categories as well, the experiment is considered exempt
from the NIH Guidelines.
Any change in containment level, which is different from those
specified in the NIH Guidelines, may not be initiated without the
express approval of NIH/ORDA (see Minor Actions, section IV-C-1-b-(2)
and its subsections).

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

Experiments in this category are considered Major Actions (see
section IV-C-1-b-(1)) and cannot be initiated without submission of
relevant information on the proposed experiment to the Office of
Recombinant DNA Activities, National Institutes of Health, Building 31,
room 4B11, Bethesda, Maryland 20892, (301) 496-9838, the publication of
the proposal in the Federal Register for 15 days of comment, reviewed
by the RAC, and specific approval by the NIH (not applicable for
Expedited Review single patient human gene transfer experiments
considered under Appendix M-VI). The containment conditions for such
experiments will be recommended by the RAC and set by the NIH at the
time of approval. Such experiments require Institutional Biosafety
Committee approval before initiation. Specific experiments already
approved are included in Appendix D which may be obtained from the
Office of Recombinant DNA Activities, National Institutes of Health,
Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-9838.
Section III-A-1. Deliberate transfer of a drug resistance trait to
microorganisms that are not known to acquire the trait naturally (see
section V-B), if such acquisition could compromise the use of the drug
to control disease agents in humans, veterinary medicine, or
agriculture.
Section III-A-2. Certain experiments involving the deliberate
transfer of recombinant DNA or DNA or RNA derived from recombinant DNA
into one or more human subjects (see section V-U) shall be considered
Major Actions (see section IV-C-1-b-(1) and Appendix M-III), and shall
require RAC review and NIH Director approval, if determined by NIH/
ORDA, in consultation with the RAC Chair and one or more RAC members,
as necessary, to: (i) Represent novel characteristics (e.g., target
disease or vector), (ii) represent an uncertain degree of risk to human
health or the environment, or (iii) contain information determined to
require further public review. The requirement for RAC review shall not
be considered to preempt any other required review or approval of
experiments with one or more human subjects. Relevant Institutional
Biosafety Committee and Institutional Review Board reviews and
approvals of the proposal should be completed before submission to NIH.
Certain experiments involving deliberate transfer of recombinant DNA or
DNA or RNA derived from recombinant DNA into one or more human subjects
may qualify for the Accelerated Review process (see section III-B-2).
Certain categories of experiments involving the deliberate transfer of
recombinant DNA or DNA or RNA derived from recombinant DNA into one or
more human subjects and that are not covered by section V-U, may be
considered exempt from RAC and/or NIH/ORDA review and/or NIH Director
approval and only require registration with NIH/ORDA (see section III-
C-7).

Section III-B. Experiments That Require NIH/ORDA and Institutional
Biosafety Committee Approval Before Initiation

Section III-B-1. Experiments Involving the Cloning of Toxin Molecules
with LD50 of Less than 100 Nanograms per Kilogram Body Weight

Deliberate formation of recombinant DNA containing genes for the
biosynthesis of toxin molecules lethal for vertebrates at an LD50
of less than 100 nanograms per kilogram body weight (e.g., microbial
toxins such as the botulinum toxins, tetanus toxin, diphtheria toxin,
and Shigella dysenteriae neurotoxin). Specific approval has been given
for the cloning in Escherichia coli K-12 of DNA containing genes coding
for the biosynthesis of toxic molecules which are lethal to vertebrates
at 100 nanograms to 100 micrograms per kilogram body weight. Specific
experiments already approved under this section may be obtained from
the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-
9838.
Section III-B-1-(a). Experiments in this category cannot be
initiated without submission of relevant information on the proposed
experiment to NIH/ORDA. The containment conditions for such experiments
will be determined by NIH/ORDA in consultation with ad hoc experts.
Such experiments require Institutional Biosafety Committee approval
before initiation (see section IV-B-2-b-(1)).

Section III-B-2. Accelerated Review of Human Gene Transfer Experiments

As determined by NIH/ORDA, in consultation with the RAC Chair and
one or more RAC members, as necessary, certain categories of human gene
transfer experiments may be considered as Minor Actions and qualify for
Accelerated Review and approval (see section IV-C-1-b-(2)-(a), Appendix
M-III-A, and Appendix M-V). The RAC Chair will present a report of all
NIH/ORDA approved human gene transfer protocols at the next regularly
scheduled RAC meeting. If NIH/ORDA determines that an experiment does
not qualify for the Accelerated Review process, the Principal
Investigator must submit the proposal for full RAC review 8
weeks prior to the next scheduled RAC meeting (See section III-A-2).
Section III-B-3. Minor Modifications to Human Gene Transfer Experiments
A minor modification in a human gene transfer protocol is a
modification that does not significantly alter the basic design of the
protocol and that does not increase risk to human subjects or the
environment. After approval has been obtained by the relevant
Institutional Biosafety Committee and Institutional Review Board, NIH/
ORDA will consider the change in consultation with the RAC Chair and
one or more RAC members, as necessary. Submit minor modifications to
the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
9838. The RAC Chair will provide a report on any such approvals at the
next regularly scheduled RAC meeting.
Section III-C. Experiments that Require Institutional Biosafety
Committee Approval Before Initiation
Prior to the initiation of an experiment that falls into this
category, the Principal Investigator must submit a registration
document to the Institutional Biosafety Committee which contains the
following information: (i) The source(s) of DNA; (ii) the nature of the
inserted DNA sequences; (iii) the host(s) and vector(s) to be used;
(iv) if an attempt will be made to obtain expression of a foreign gene,
and if so, indicate the protein that will be produced; and (v) the
containment conditions that will be implemented as specified in the NIH
Guidelines. For experiments in this category, the registration document
shall be dated, signed by the Principal Investigator, and filed with
the Institutional Biosafety Committee. The Institutional Biosafety
Committee shall review and approve all experiments in this category
prior to their initiation. Requests to decrease the level of
containment specified for experiments in this category will be
considered by NIH (see Section IV-C-1-b-(2)-(c)).
Section III-C-1. Experiments Using Human or Animal Pathogens (Class 2,
Class 3, Class 4, or Class 5 Agents (see Section V-A) as Host-Vector
Systems
Section III-C-1-a. Experiments involving the introduction of
recombinant DNA into Class 2 agents shall be conducted at Biosafety
Level (BL) 2 containment. Experiments with such agents shall be
conducted with whole animals at BL2 or BL2-N (Animals) containment.
Section III-C-1-b. Experiments involving the introduction of
recombinant DNA into Class 3 agents shall be conducted at BL3
containment. Experiments with such agents shall be conducted with whole
animals at BL3 or BL3-N containment.
Section III-C-1-c. Experiments involving the introduction of
recombinant DNA into Class 4 agents shall be conducted at BL4
containment. Experiments with such agents shall be conducted with whole
animals at BL4 or BL4-N containment.
Section III-C-1-d. Containment conditions for experiments involving
the introduction of recombinant DNA into Class 5 agents shall be set on
a case-by-case basis following NIH/ORDA review. A U.S. Department of
Agriculture permit is required for work with Class 5 agents (see
Sections V-R and V-T). Experiments with such agents shall be conducted
with whole animals at BL4 or BL4-N containment.
Section III-C-2. Experiments in Which DNA From Human or Animal
Pathogens (Class 2, Class 3, Class 4, or Class 5 Agents (see Section V-
A) is Cloned Into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-
Vector Systems
Section III-C-2-a. Experiments in which DNA from Class 2 or Class 3
agents (see Section V-A) is transferred into nonpathogenic prokaryotes
or lower eukaryotes may be performed under BL2 containment. Experiments
in which DNA from Class 4 agents is transferred into nonpathogenic
prokaryotes or lower eukaryotes may be performed under BL2 containment
after demonstration that only a totally and irreversibly defective
fraction of the agent's genome is present in a given recombinant. In
the absence of such a demonstration, BL4 containment shall be used. The
Institutional Biosafety Committee may approve the specific lowering of
containment for particular experiments to BL1. Many experiments in this
category are exempt from the NIH Guidelines (see Section III-E).
Experiments involving the formation of recombinant DNA for certain
genes coding for molecules toxic for vertebrates require NIH/ORDA
approval (see Section III-B-1) or shall be conducted under NIH
specified conditions as described in Appendix F.
Section III-C-2-b. Containment conditions for experiments in which
DNA from Class 5 agents is transferred into nonpathogenic prokaryotes
or lower eukaryotes shall be determined by NIH/ORDA following a case-
by-case review. A U.S. Department of Agriculture permit is required for
work with Class 5 agents (see Sections V-R and V-T).
Section III-C-3. Experiments Involving the Use of Infectious Animal or
Plant DNA or RNA Viruses or Defective Animal or Plant DNA or RNA
Viruses in the Presence of Helper Virus in Tissue Culture Systems
Caution: Special care should be used in the evaluation of
containment levels for experiments which are likely to either enhance
the pathogenicity (e.g., insertion of a host oncogene) or to extend the
host range (e.g., introduction of novel control elements) of viral
vectors under conditions that permit a productive infection. In such
cases, serious consideration should be given to increasing physical
containment by at least one level.

Note: Recombinant DNA or RNA molecules derived therefrom, which
contain less than two-thirds of the genome of any eukaryotic virus
(all viruses from a single Family (see Section V-Q) being considered
identical (see Section V-S), are considered defective and may be
used in the absence of helper under the conditions specified in
Section III-D-1.

Section III-C-3-a. Experiments involving the use of infectious or
defective Class 2 animal viruses (see Section V-A, Appendix B-II, and
Appendix B-II-E) in the presence of helper virus may be conducted at
BL2.
Section III-C-3-b. Experiments involving the use of infectious or
defective Class 3 animal viruses (see Section V-A and Appendix B-III-D)
in the presence of helper virus may be conducted at BL3.
Section III-C-3-c. Experiments involving the use of infectious or
defective Class 4 animal viruses (see Section V-A and Appendix B-IV-D)
in the presence of helper virus may be conducted at BL4.
Section III-C-3-d. Experiments involving the use of infectious or
defective Class 5 viruses (see Section V-A and Appendix B-V) in the
presence of helper virus shall be determined on a case-by-case basis
following NIH/ORDA review. A U.S. Department of Agriculture permit is
required for work with Class 5 agents (see Sections V-R and V-T).
Section III-C-3-e. Experiments involving the use of infectious or
defective animal or plant viruses in the presence of helper virus are
not covered in Sections III-C-3-a through III-C-3-d and may be
conducted at BL1.

Section III-C-4. Experiments Involving Whole Animals

This section covers experiments involving whole animals in which
the animal's genome has been altered by stable introduction of
recombinant DNA, or DNA derived therefrom, into the germ-line
(transgenic animals) and experiments involving viable recombinant DNA-
modified microorganisms tested on whole animals. For the latter, other
than viruses which are only vertically transmitted, the experiments may
not be conducted at BL1-N containment. A minimum containment of BL2 or
BL2-N is required.
Caution--Special care should be used in the evaluation of
containment conditions for some experiments with transgenic animals.
For example, such experiments might lead to the creation of novel
mechanisms or increased transmission of a recombinant pathogen or
production of undesirable traits in the host animal. In such cases,
serious consideration should be given to increasing the containment
conditions.
Section III-C-4-a. Recombinant DNA, or DNA or RNA molecules derived
therefrom, from any source except for greater than two-thirds of
eukaryotic viral genome may be transferred to any non-human vertebrate
or any invertebrate organism and propagated under conditions of
physical containment comparable to BL1 or BL1-N and appropriate to the
organism under study (see Section V-B). Animals that contain sequences
from viral vectors, which do not lead to transmissible infection either
directly or indirectly as a result of complementation or recombination
in animals, may be propagated under conditions of physical containment
comparable to BL1 or BL1-N and appropriate to the organism under study.
Experiments involving the introduction of other sequences from
eukaryotic viral genomes into animals are covered under Section III-C-
4-b. For experiments involving recombinant DNA-modified Class 2, 3, 4,
or 5 organisms, see Section V-A. It is important that the investigator
demonstrate that the fraction of the viral genome being utilized does
not lead to productive infection. A U.S. Department of Agriculture
permit is required for work with Class 5 agents (see Section V-R and V-
T).
Section III-C-4-b. For experiments involving recombinant DNA, or
DNA or RNA derived therefrom, involving whole animals, including
transgenic animals, and not covered by Sections III-C-1 or III-C-4-a,
the appropriate containment shall be determined by the Institutional
Biosafety Committee.

Section III-C-5. Experiments Involving Whole Plants

Experiments to genetically engineer plants by recombinant DNA
methods, to use such plants for other experimental purposes (e.g.,
response to stress), to propagate such plants, or to use plants
together with microorganisms or insects containing recombinant DNA, may
be conducted under the containment conditions described in Sections
III-C-5-a through III-C-5-e. If experiments involving whole plants are
not described in Section III-C-5 and do not fall under Sections III-A,
III-B, or III-E, they are included in Section III-D.

Note: For recombinant DNA experiments falling under Sections
III-C-5-a through III-C-5-d, physical containment requirements may
be reduced to the next lower level by appropriate biological
containment practices, such as conducting experiments on a virus
with an obligate insect vector in the absence of that vector or
using a genetically attenuated strain.

Section III-C-5-a. BL3-P (Plants) or BL2-P + biological containment
is recommended for experiments involving most exotic (see Section V-W)
infectious agents with recognized potential for serious detrimental
impact on managed or natural ecosystems when recombinant DNA techniques
are associated with whole plants.
Section III-C-5-b. BL3-P or BL2-P + biological containment is
recommended for experiments involving plants containing cloned genomes
of readily transmissible exotic (see Section V-W) infectious agents
with recognized potential for serious detrimental effects on managed or
natural ecosystems in which there exists the possibility of
reconstituting the complete and functional genome of the infectious
agent by genomic complementation in planta.
Section III-C-5-c. BL4-P containment is recommended for experiments
with a small number of readily transmissible exotic (see Section V-W)
infectious agents, such as the soybean rust fungus (Phakospora
pachyrhizi) and maize streak or other viruses in the presence of their
specific arthropod vectors, that have the potential of being serious
pathogens of major U.S. crops.
Section III-C-5-d. BL3-P containment is recommended for experiments
involving sequences encoding potent vertebrate toxins introduced into
plants or associated organisms. Recombinant DNA containing genes for
the biosynthesis of toxin molecules lethal for vertebrates at an
LD50 of 50
100 nanograms per kilogram body weight in organisms other
than Escherichia coli K-12 (see Section III-B-1 and Appendices F-I and
F-II);
Section IV-C-3-b. Reviewing and approving certain experiments
involving the deliberate transfer of recombinant DNA or DNA or RNA
derived from recombinant DNA into one or more human subjects, in
consultation with the RAC Chair and one or more RAC members, as
necessary, that qualify for the Accelerated Review process (see Section
III-B-2);
Section IV-C-3-c. Reviewing and approving minor changes to human
gene transfer protocols approved under Sections III-A-2 and III-B-2, in
consultation with the RAC Chair and one or more RAC members, as
necessary;
Section IV-C-3-d. Reviewing and approving the membership of an
institution's Institutional Biosafety Committee, and where it finds the
Institutional Biosafety Committee meets the requirements set forth in
Section IV-B-2 will give its approval to the Institutional Biosafety
Committee membership;
Section IV-C-3-e. Publishing in the Federal Register:
Section IV-C-3-e-(1). Announcements of RAC meetings and agendas at
least 15 days in advance (NOTE--If the agenda for a RAC meeting is
modified, ORDA shall make the revised agenda available to anyone upon
request at least 72 hours in advance of the meeting);
Section IV-C-3-e-(2). Proposed Major Actions to the NIH Guidelines
(see Section IV-C-1-b-(1)) at least 15 days prior to the RAC meeting;
Section IV-C-3-f. Serve as the focal point for data management of
NIH-approved human gene transfer protocols approved under Sections III-
A-2 and III-B-2 and registered with NIH/ORDA as required under Section
III-C-7;
Section IV-C-3-g. Serve as the executive secretary of the RAC; and
Section IV-C-3-h. Maintain a list of Major and Minor Actions
approved under Section III-A-2 and III-B-3 and a list of experiments
registered with NIH/ORDA as described in Section III-C-7.
Section IV-C-4. Other NIH Components
Other NIH components shall be responsible for certifying maximum
containment (BL4) facilities, inspecting them periodically, and
inspecting other recombinant DNA facilities as deemed necessary.
Section IV-D. Compliance with the NIH Guidelines
As a condition for NIH funding of recombinant DNA research,
institutions shall ensure that such research conducted at or sponsored
by the institution, irrespective of the source of funding, shall comply
with the NIH Guidelines. The policies on noncompliance are as follows:
All NIH-funded projects involving recombinant DNA techniques must
comply with the NIH Guidelines. Non-compliance may result in: (i)
suspension, limitation, or termination of financial assistance for such
projects and of NIH funds for other recombinant DNA research at the
institution, or (ii) a requirement for prior NIH approval of any or all
recombinant DNA projects at the institution.
All non-NIH funded projects involving recombinant DNA techniques
conducted at or sponsored by an institution that receives NIH funds for
projects involving such techniques must comply with the NIH Guidelines.
Noncompliance may result in: (i) suspension, limitation, or termination
of NIH funds for recombinant DNA research at the institution, or (ii) a
requirement for prior NIH approval of any or all recombinant DNA
projects at the institution.
Information concerning noncompliance with the NIH Guidelines may be
brought forward by any person. It should be delivered to both NIH/ORDA
and the relevant institution. The institution, generally through the
Institutional Biosafety Committee, shall take appropriate action. The
institution shall forward a complete report of the incident
recommending any further action to the Office of Recombinant DNA
Activities, National Institutes of Health, Building 31, Room 4B11,
Bethesda, Maryland 20892, (301) 496-9838.
In cases where NIH proposes to suspend, limit, or terminate
financial assistance because of noncompliance with the NIH Guidelines,
applicable DHHS and Public Health Service procedures shall govern.
Section IV-E. Voluntary Compliance
Section IV-E-1. Basic Policy
Individuals, corporations, and institutions not otherwise covered
by the NIH Guidelines are encouraged to do so by following the
standards and procedures set forth in Sections I through IV. In order
to simplify discussion, references hereafter to ``institutions'' are
intended to encompass corporations and individuals who have no
organizational affiliation. For purposes of complying with the NIH
Guidelines, an individual intending to carry out research involving
recombinant DNA is encouraged to affiliate with an institution that has
an Institutional Biosafety Committee approved under the NIH Guidelines.
Since commercial organizations have special concerns, such as
protection of proprietary data, some modifications and explanations of
the procedures are provided in Sections IV-E-2 through IV-E-5-b in
order to address these concerns.
Section IV-E-2. Institutional Biosafety Committee Approval
It should be emphasized that employment of an Institutional
Biosafety Committee member solely for purposes of membership on the
Institutional Biosafety Committee does not itself make the member an
institutionally affiliated member. Except for the unaffiliated members,
a member of an Institutional Biosafety Committee for an institution not
otherwise covered by the NIH Guidelines may participate in the review
and approval of a project in which the member has a direct financial
interest so long as the member has not been, and does not expect to be,
engaged in the project. Section IV-B-2-a-(4) is modified to that extent
for purposes of these institutions.
Section IV-E-3. Certification of Host-Vector Systems
A host-vector system may be proposed for certification by the NIH
Director in accordance with the procedures set forth in Appendix I-II.
In order to ensure protection for proprietary data, any public notice
regarding a host-vector system which is designated by the institution
as proprietary under Section IV-E-5-a will be issued only after
consultation with the institution as to the content of the notice.
Section IV-E-4. Requests for Exemptions and Approvals
Requests for exemptions or other approvals as required by the NIH
Guidelines should be submitted based on the procedures set forth in
Sections I through IV. In order to ensure protection for proprietary
data, any public notice regarding a request for an exemption or other
approval which is designated by the institution as proprietary under
Section IV-E-5-a will be issued only after consultation with the
institution as to the content of the notice.
Section IV-E-5. Protection of Proprietary Data
Section IV-E-5-a. General
In general, the Freedom of Information Act requires Federal
agencies to make their records available to the public upon request.
However, this requirement does not apply to, among other things,
``trade secrets and commercial or financial information that is
obtained from a person and that is privileged or confidential.'' Under
18 U.S.C. 1905, it is a criminal offense for an officer or employee of
the U.S. or any Federal department or agency to publish, divulge,
disclose, or make known ``in any manner or to any extent not authorized
by law any information coming to him in the course of his employment or
official duties or by reason of any examination or investigation made
by, or return, report or record made to or filed with, such department
or agency or officer or employee thereof, which information concerns or
relates to the trade secrets, (or) processes--of any person, firm,
partnership, corporation, or association.'' This provision applies to
all employees of the Federal Government, including special Government
employees. Members of the RAC are ``special Government employees.''
In submitting to NIH for purposes of voluntary compliance with the
NIH Guidelines, an institution may designate those items of information
which the institution believes constitute trade secrets, privileged,
confidential, commercial, or financial information. If NIH receives a
request under the Freedom of Information Act for information so
designated, NIH will promptly contact the institution to secure its
views as to whether the information (or some portion) should be
released. If the NIH decides to release this information (or some
portion) in response to a Freedom of Information request or otherwise,
the institution will be advised; and the actual release will not be
made until the expiration of 15 days after the institution is so
advised except to the extent that earlier release in the judgment of
the NIH Director is necessary to protect against an imminent hazard to
the public or the environment.
Section IV-E-5-b. Presubmission Review
Any institution not otherwise covered by the NIH Guidelines, which
is considering submission of data or information voluntarily to NIH,
may request presubmission review of the records involved to determine
if NIH will make all or part of the records available upon request
under the Freedom of Information Act.
A request for presubmission review should be submitted to NIH/ORDA
along with the records involved to the Office of Recombinant DNA
Activities, National Institutes of Health, Building 31, Room 4B11,
Bethesda, Maryland 20892, (301) 496-9838. These records shall be
clearly marked as being the property of the institution on loan to NIH
solely for the purpose of making a determination under the Freedom of
Information Act. NIH/ORDA will seek a determination from the
responsible official under DHHS regulations (45 Code of Federal
Regulations, Part 5) as to whether the records involved, (or some
portion) will be made available to members of the public under the
Freedom of Information Act. Pending such a determination, the records
will be kept separate from NIH/ORDA files, will be considered records
of the institution and not NIH/ORDA, and will not be received as part
of NIH/ORDA files. No copies will be made of such records.
NIH/ORDA will inform the institution of the DHHS Freedom of
Information Officer's determination and follow the institution's
instructions as to whether some or all of the records involved are to
be returned to the institution or to become a part of NIH/ORDA files.
If the institution instructs NIH/ORDA to return the records, no copies
or summaries of the records will be made or retained by DHHS, NIH, or
ORDA. The DHHS Freedom of Information Officer's determination will
represent that official's judgment at the time of the determination as
to whether the records involved (or some portion) would be exempt from
disclosure under the Freedom of Information Act if at the time of the
determination the records were in NIH/ORDA files and a request was
received for such files under the Freedom of Information Act.
Section V. Footnotes and References of Sections I Through IV
Section V-A. The original reference to organisms as Class 1, 2, 3,
4, or 5 refers to the classification in the publication Classification
of Etiologic Agents on the Basis of Hazard, 4th Edition, July 1974,
U.S. Department of Health, Education, and Welfare, Public Health
Services, Centers for Disease Control and Prevention, Office of
Biosafety, Atlanta, Georgia 30333. The NIH Director, with advice of the
RAC, may revise the classification for the purposes of the NIH
Guidelines (see Section IV-C-1-b-(2)-(e)). The revised list of
organisms in each class is reprinted in Appendix B.
Section V-B. Section III describes a number of places where
judgments are to be made. In all these cases, the Principal
Investigator shall make the judgment on these matters as part of his/
her responsibility to ``make the initial determination of the required
levels of physical and biological containment in accordance with the
NIH Guidelines'' (see Section IV-B-4-c-(1)). For cases falling under
Sections III-A through III-D, this judgment is to be reviewed and
approved by the Institutional Biosafety Committee as part of its
responsibility to make an ``independent assessment of the containment
levels required by the NIH Guidelines for the proposed research'' (see
Section IV-B-2-b-(1)). The Institutional Biosafety Committee may refer
specific cases to NIH/ORDA as part of NIH/ORDA's functions to ``provide
advice to all within and outside NIH'' (see Section IV-C-3). NIH/ORDA
may request advice from the RAC as part of the RAC's responsibility for
``interpreting the NIH Guidelines for experiments to which the NIH
Guidelines do not specifically assign containment levels'' (see Section
IV-C-1-b-(2)-(f)).
Section V-C. Laboratory Safety at the Centers for Disease Control,
September 1974, U.S. Department of Health, Education, and Welfare
Publication No. CDC 75-8118.
Section V-D. Classification of Etiologic Agents on the Basis of
Hazard, 4th Edition, July 1974, U.S. Department of Health, Education,
and Welfare, Public Health Service, Centers for Disease Control, Office
of Biosafety, Atlanta, Georgia 30333.
Section V-E. National Cancer Institute Safety Standards for
Research Involving Oncogenic Viruses, October 1974, U.S. Department of
Health, Education, and Welfare, Publication No. (NIH) 75-790.
Section V-F. National Institutes of Health Biohazards Safety Guide,
1974, U.S. Department of Health, Education, and Welfare, Public Health
Service, NIH, U.S. Government Printing Office, Stock No. 1740-00383.
Section V-G. A. Hellman, M. N. Oxman, and R. Pollack (eds.), 1973,
Biohazards in Biological Research, Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY.
Section V-H. Furr, A. K., Handbook of Laboratory Safety, 2nd ed,
The Chemical Rubber Co., Boca Raton, Florida, 1990.
Section V-I. American Public Health Association, Bodily, J. L.,
General Administration of the Laboratory, 6th ed., ``Diagnostic
Procedures for Bacterial, Mycotic, and Parasitic Infections,'' New
York, 1981.
Section V-J. H. M. Darlow, Safety in the Microbiological
Laboratory, in J. R. Norris and D. W. Robbins (eds.), Methods in
Microbiology, Academic Press, Inc, New York, New York, 1969, pp. 169-
204.
Section V-K. C. M. Collins, E. G. Hartley, and R. Pilsworth, The
Prevention of Laboratory Acquired Infection, Public Health Laboratory
Service, Monograph Series No. 6, 1974.
Section V-L. Chatigny, M. A, ``Protection Against Infection in the
Microbiological Laboratory: Devices and Procedures,'' in W.W. Umbreit
(ed.), Advances in Applied Microbiology, Academic Press, New York, New
York, 1961, 3:131-192.
Section V-M. Design Criteria for Viral Oncology Research
Facilities, U.S. Department of Health, Education, and Welfare, Public
Health Service, NIH, DHEW Publication No. (NIH) 75-891, 1975.
Section V-N. Kuehne, R. W., Biological Containment Facility for
Studying Infectious Disease, Appl. Microbiol. 26:239-243, 1973.
Section V-O. Runkle, R. B., and G. B. Phillips, Microbial
Containment Control Facilities, Van Nostrand Reinhold, New York, 1969.
Section V-P. Chatigny, M. A., and D. I. Clinger, ``Contamination
Control in Aerobiology,'' in R. L. Dimmick and A. B. Akers (eds.), An
Introduction to Experimental Aerobiology, John Wiley & Sons, New York,
1969, pp. 194-263.
Section V-Q. As classified in the Third Report of the International
Committee on Taxonomy of Viruses: Classification and Nomenclature of
Viruses, R. E. F. Matthews (ed.), Intervirology 12 (129-296), 1979.
Section V-R. A U.S. Department of Agriculture permit is required
for the importation, interstate movement, and release into the
environment of certain organisms that are plant or animal pathogens,
whether genetically engineered or not. Permits are required for
veterinary biologics and for certain plants or microorganisms derived
through genetic engineering using genetic sequences from plant pests
(pathogens). Specific information about regulated organisms and
procedures for obtaining a permit for regulated organisms may be
obtained from the Director, Biotechnology, Biologics, and Environmental
Protection, Animal and Plant Health Inspection Service, U.S. Department
of Agriculture, 6505 Belcrest Road, Room 850, Hyattsville, Maryland
20782, (301) 436-7601.
Section V-S. i.e., the total of all genomes within a family shall
not exceed two-thirds of the genome.
Section V-T. All activities, including storage of variola and
whitepox, are restricted to the single national facility (World Health
Organization Collaborating Center for Smallpox Research, Centers for
Disease Control and Prevention, Atlanta, Georgia).
Section V-U. Human studies in which the induction or enhancement of
an immune response to a vector-encoded microbial immunogen is the major
goal, such an immune response has been demonstrated in model systems,
and the persistence of the vector-encoded immunogen is not expected,
are not covered under Sections III-A-2, III-B-2, or III-B-3. Such
studies may be initiated without RAC review and NIH approval if
approved by another Federal agency.
Section V-V. For recombinant DNA experiments in which the intent is
to modify stably the genome of cells of one or more human subjects (see
Sections III-A-2, III-B-2, and III-B-3).
Section V-W. In accordance with accepted scientific and regulatory
practices of the discipline of plant pathology, an exotic plant
pathogen (e.g., virus, bacteria, or fungus) is one that is unknown to
occur within the U.S. (see Section V-R). Determination of whether a
pathogen has a potential for serious detrimental impact on managed
(agricultural, forest, grassland) or natural ecosystems should be made
by the Principal Investigator and the Institutional Biosafety
Committee, in consultation with scientists knowledgeable of plant
diseases, crops, and ecosystems in the geographic area of the research.

Appendix A. Exemptions Under Section III-E-5--Sublists of Natural
Exchangers

Certain specified recombinant DNA molecules that ``consist entirely
of DNA segments from different species that exchange DNA by known
physiological processes, though one or more of the segments may be a
synthetic equivalent are exempt from these NIH Guidelines (see Section
III-E-5). Institutional Biosafety Committee registration is not
required for these exempt experiments. A list of such exchangers will
be prepared and periodically revised by the NIH Director with advice
from the RAC after appropriate notice and opportunity for public
comment (see Section IV-C-1-b-(1)-(c)). See Appendices A-I through A-VI
for a list of natural exchangers that are exempt from the NIH
Guidelines.'' Section III-E-5 describes recombinant DNA molecules that
are: (1) composed entirely of DNA segments from one or more of the
organisms within a sublist, and (2) to be propagated in any of the
organisms within a sublist (see Classification of Bergey's Manual of
Determinative Bacteriology; 8th edition, R. E. Buchanan and N. E.
Gibbons, editors, Williams and Wilkins Company; Baltimore, Maryland
1984). Although these experiments are exempt, it is recommended that
they be performed at the appropriate biosafety level for the host or
recombinant organism (see Biosafety in Microbiological and Biomedical
Laboratories, 3rd edition, May 1993, U.S. DHHS, Public Health Service,
Centers for Disease Control, Atlanta, Georgia, and NIH Office of
Biosafety, Bethesda, Maryland).
Appendix A-I. Sublist A
Genus Escherichia
Genus Shigella
Genus Salmonella--including Arizona
Genus Enterobacter
Genus Citrobacter--including Levinea
Genus Klebsiella--including oxytoca
Genus Erwinia
Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens,
and Pseudomonas mendocina
Serratia marcescens
Yersinia enterocolitica
Appendix A-II. Sublist B
Bacillus subtilis
Bacillus licheniformis
Bacillus pumilus
Bacillus globigii
Bacillus niger
Bacillus nato
Bacillus amyloliquefaciens
Bacillus aterrimus
Appendix A-III. Sublist C
Streptomyces aureofaciens
Streptomyces rimosus
Streptomyces coelicolor
Appendix A-IV. Sublist D
Streptomyces griseus
Streptomyces cyaneus
Streptomyces venezuelae
Appendix A-V. Sublist E
One way transfer of Streptococcus mutans or Streptococcus lactis DNA
into Streptococcus sanguis
Appendix A-VI. Sublist F
Streptococcus sanguis
Streptococcus pneumoniae
Streptococcus faecalis
Streptococcus pyogenes
Streptococcus mutans
Appendix B. Classification of Etiologic Agents and Oncogenic Viruses on
the Basis of Hazard (See Appendix B-VI-A).
Appendix B-I. Class 1 Agents
All bacterial, parasitic, fungal, viral, rickettsial, and
chlamydial agents not included in higher classes shall be considered
Class 1 agents.
Appendix B-II. Class 2 Agents
Appendix B-II-A. Class 2 Bacterial Agents
Acinetobacter calcoaceticus
Actinobacillus--all species
Aeromonas hydrophila
Amycolata autotrophica
Arizona hinshawii--all serotypes
Bacillus anthracis
Bordetella--all species
Borrelia recurrentis, B. vincenti
Campylobacter fetus
Campylobacter jejuni
Chlamydia psittaci
Chlamydia trachomatis
Clostridium botulinum, Cl. chauvoei, Cl. haemolyticum, Cl.
histolyticum, Cl. novyi, Cl.
septicum, Cl. tetani
Corynebacterium diphtheriae, C. equi, C. haemolyticum, C.
pseudotuberculosis, C.
pyogenes, C. renale
Dermatophilus congolensis
Edwardsiella tarda
Erysipelothrix insidiosa
Escherichia coli--all enteropathogenic, enterotoxigenic, enteroinvasive
and strains
bearing K1 antigen
Haemophilus ducreyi, H. influenzae
Klebsiella--all species except oxytoca
Legionella pneumophila
Leptospira interrogans--all serotypes
Listeria--all species
Moraxella--all species
Mycobacteria--all species except those listed in Class 3
Mycobacterium avium
Mycoplasma--all species except Mycoplasma mycoides and Mycoplasma
agalactiae, which are in Class 5
Neisseria gonorrhoea, N. meningitides
Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N.
transvalensis
Pasteurella--all species except those listed in Class 3
Rhodococcus equi
Salmonella--all species and all serotypes
Shigella-all species and all serotypes
Sphaerophorus necrophorus
Staphylococcus aureus
Streptobacillus moniliformis
Streptococcus pneumoniae, S. pyogenes
Treponema carateum, T. pallidum, and T. pertenue
Vibrio cholerae, V. parahemolyticus
Yersinia enterocolitica

Appendix B-II-B. Class 2 Fungal Agents

Blastomyces dermatitidis
Cryptococcus neoformans
Paracoccidioides braziliensis

Appendix B-II-C. Class 2 Parasitic Agents

Endamoeba histolytica
Leishmania sp.
Naegleria gruberi
Schistosoma mansoni
Toxocara canis
Toxoplasma gondii
Trichinella spiralis
Trypanosoma cruzi

Appendix B-II-D. Class 2 Viral, Rickettsial, and Chlamydial Agents

Adenoviruses--human--all types
Cache Valley virus
Coronaviruses
Coxsackie A and B viruses
Cytomegaloviruses
Echoviruses--all types
Encephalomyocarditis virus (EMC)
Flanders virus
Hart Park virus
Hepatitis viruses--associated antigen material
Herpesviruses--except Herpesvirus simiae (Monkey B virus) which is in
Class 4
Influenza viruses--all types except A/PR8/34, which is in Class 1
Langat virus
Lymphogranuloma venereum agent
Measles virus
Mumps virus
Parainfluenza virus--all types except Parainfluenza virus 3, SF4
strain, which is in Class 1
Polioviruses --all types, wild and attenuated
Poxviruses--all types except Alastrim, Smallpox, and Whitepox which are
Class 5 and Monkey pox which depending on experiments is in Class 3 or
Class 4
Rabies virus--all strains except Rabies street virus which should be
classified in Class 3
Reoviruses--all types
Respiratory syncytial virus
Rhinoviruses--all types
Rubella virus
Simian viruses--all types except Herpesvirus simiae (Monkey B virus)
and Marburg virus which are in Class 4
Sindbis virus
Tensaw virus
Turlock virus
Vaccinia virus
Varicella virus
Vesicular stomatitis virus (see Appendix B-VI-B)
Vole rickettsia
Yellow fever virus, 17D vaccine strain

Appendix B-II-E. Class 2 Oncogenic Viruses (See Appendix B-VI-C)

Appendix B-II-E-1. Low-Risk Oncogenic Viruses

Adenovirus 7-Simian virus 40 (Ad7-SV40)
Adenovirus
Avian leukosis virus
Bovine leukemia virus
Bovine papilloma virus
Chick-embryo-lethal orphan (CELO) virus or fowl adenovirus 1
Dog sarcoma virus
Guinea pig herpes virus
Lucke (Frog) virus
Hamster leukemia virus
Marek's disease virus
Mason-Pfizer monkey virus
Mouse mammary tumor virus
Murine leukemia virus
Murine sarcoma virus
Polyoma virus
Rat leukemia virus
Rous sarcoma virus
Shope fibroma virus
Shope papilloma virus
Simian virus 40 (SV-40)

Appendix B-II-E-2. Moderate-Risk Oncogenic Viruses

Adenovirus 2-Simian virus 40 (Ad2-SV40)
Epstein-Barr virus (EBV)
Feline leukemia virus (FeLV)
Feline sarcoma virus (FeSV)
Gibbon leukemia virus (GaLV)
Herpesvirus (HV) ateles
Herpesvirus (HV) saimiri
Simian sarcoma virus (SSV)-1
Yaba

Appendix B-III. Class 3 Agents

Appendix B-III-A. Class 3 Bacterial Agents

Bartonella--all species
Brucella--all species
Francisella tularensis
Mycobacterium bovis, M. tuberculosis
Pasteurella multocide type--``buffalo'' and other foreign virulent
strains (see Appendix B-VI-B)
Pseudomonas mallei (see Appendix B-VI-B)
Pseudomonas pseudomallei (see Appendix B-VI-B)
Yersinia pestis

Appendix B-III-B. Class 3 Fungal Agents

Coccidioides immitis
Histoplasma capsulatum
Histoplasma capsulatum var. duboisii

Appendix B-III-C. Class 3 Parasitic Agents

None

Appendix B-III-D. Class 3 Viral, Rickettsial, and Chlamydial Agents

Monkey pox virus--when used in vitro (see Appendix B-VI-D)
Arboviruses--all strains except those in Class 2 and 4. (Arboviruses
indigenous to the United States are in Class 3 except those listed in
Class 2. West Nile and Semliki Forest viruses may be classified up or
down depending on the conditions of use and geographical location of
the laboratory).
Dengue virus--when used for transmission or animal inoculation
experiments
Lymphocytic choriomeningitis virus (LCM)
Rickettsia--all species except Vole rickettsia when used for
transmission or animal inoculation experiments
Yellow fever virus--wild, when used in vitro

Appendix B-IV. Class 4 Agents

Appendix B-IV-A. Class 4 Bacterial Agents

None

Appendix B-IV-B. Class 4 Fungal Agents

None

Appendix B-IV-C. Class 4 Parasitic Agents

None

Appendix B-IV-D. Class 4 Viral, Rickettsial, and Chlamydial Agents

Ebola fever virus
Monkey pox virus--when used for transmission or animal inoculation
experiments (see Appendix B-VI-D)
Hemorrhagic fever agents--including Crimean hemorrhagic fever, (Congo),
Junin, and Machupo viruses, and others as yet undefined
Herpesvirus simiae (Monkey B virus)
Lassa virus
Marburg virus
Tick-borne encephalitis virus complex--including Russian spring-summer
encephalitis, Kyasanur forest disease, Omsk hemorrhagic fever, and
Central European encephalitis viruses
Venezuelan equine encephalitis virus, epidemic strains--when used for
transmission or animal inoculation experiments
Yellow fever virus-wild--when used for transmission or animal
inoculation experiments

Appendix B-V. Class 5 Agents (see Appendix B-VI-E)

Appendix B-V-A. Animal Disease Organisms which are Forbidden Entry into
the United States by Law

Foot and mouth disease virus

Appendix B-V-B. Animal Disease Organisms and Vectors which are
Forbidden Entry into the United States by U.S. Department of
Agriculture Policy

African horse sickness virus
African swine fever virus
Besnoitia besnoiti
Borna disease virus
Bovine infectious petechial fever
Camel pox virus
Ephemeral fever virus
Fowl plague virus
Goat pox virus
Hog cholera virus
Louping ill virus
Lumpy skin disease virus
Mycoplasma mycoides--contagious bovine pleuropneumonia
Mycoplasma agalactiae--contagious agalactia of sheep
Nairobi sheep disease virus
Newcastle disease virus--Asiatic strains
Rhinderpest virus
Rickettsia ruminatium--heart water
Rift valley fever virus
Sheep pox virus
Swine vesicular disease virus
Teschen disease virus
Theileria annulata
Theileria bovis
Theileria hirci
Theileria lawrencei
Theileria parva--East Coast fever
Trypanosoma evansi
Trypanosoma vivax--Nagana
Vesicular exanthema virus
Wesselsbron disease virus
Zyonema

Appendix B-V-C. Organisms which may not be Studied in the United States
Except at Specified Facilities

Alastrim (see Appendix B-VI-D)
Small pox (see Appendix B-VI-D)
White pox (see Appendix B-VI-D)

Appendix B-VI. Footnotes and References of Appendix B

Appendix B-VI-A. The original reference for this classification was
the publication Classification of Etiologic Agents on the Basis of
Hazard, 4th edition, July 1974, U.S. DHHS, Public Health Service,
Centers for Disease Control and Prevention, Office of Biosafety,
Atlanta, Georgia 30333. For the purposes of these NIH Guidelines, this
list has been revised by the NIH.
Appendix B-VI-B. A U.S. Department of Agriculture permit, required
for import and interstate transport of pathogens, may be obtained from
the U.S. Department of Agriculture, ATTN: Animal and Plant Health
Inspection Service, Import-Export Products Office, Room 756, Federal
Building, 6505 Belcrest Road, Hyattsville, Maryland 20782.
Appendix B-VI-C. National Cancer Institute Safety Standards for
Research Involving Oncogenic Viruses, U.S. Department of Health,
Education, and Welfare Publication No. (NIH) 75-790, October 1974.
Appendix B-VI-D. All activities, including storage of variola and
whitepox, are restricted to the single national facility (World Health
Organization Collaborating Center for Smallpox Research, Centers for
Disease Control and Prevention, Atlanta, Georgia).
Appendix B-VI-E. U.S. Department of Agriculture, Animal and Plant
Health Inspection Service.

Appendix C. Exemptions Under Section III-E-6

Section III-E-6 states that exempt from these NIH Guidelines are
``those that do not present a significant risk to health or the
environment (see Section IV-C-1-b-(1)-(c)), as determined by the NIH
Director, with the advice of the RAC, and following appropriate notice
and opportunity for public comment. See Appendix C for other classes of
experiments which are exempt from the NIH Guidelines.'' The following
classes of experiments are exempt under Section III-E-6:

Appendix C-I. Recombinant DNA in Tissue Culture

Recombinant DNA molecules containing less than one-half of any
eukaryotic viral genome (all viruses from a single family (see Appendix
C-VI-D) being considered identical (see Appendix C-VI-E), that are
propagated and maintained in cells in tissue culture are exempt from
these NIH Guidelines with the exceptions listed in Appendix C-I-A.

Appendix C-I-A. Exceptions

The following categories are not exempt from the NIH Guidelines:
(i) experiments described in Section III-A which require specific RAC
review and NIH and Institutional Biosafety Committee approval before
initiation, (ii) experiments described in Section III-B which require
NIH/ORDA and Institutional Biosafety Committee approval before
initiation, (iii) experiments involving DNA from Class 3, 4, or 5
organisms (see Appendix C-VI-A) or cells known to be infected with
these agents, (iv) experiments involving the deliberate introduction of
genes coding for the biosynthesis of molecules that are toxic for
vertebrates (see Appendix F), and (v) whole plants regenerated from
plant cells and tissue cultures are covered by the exemption provided
they remain axenic cultures even though they differentiate into
embryonic tissue and regenerate into plantlets.
Appendix C-II. Escherichia coli K-12 Host-Vector Systems
Experiments which use Escherichia coli K-12 host-vector systems,
with the exception of those experiments listed in Appendix C-II-A, are
exempt from the NIH Guidelines provided that: (i) the Escherichia coli
host does not contain conjugation proficient plasmids or generalized
transducing phages; or (ii) lambda or lambdoid or Ff bacteriophages or
non-conjugative plasmids (see Appendix C-VI-B) shall be used as
vectors. However, experiments involving the insertion into Escherichia
coli K-12 of DNA from prokaryotes that exchange genetic information
(see Appendix C-VI-C) with Escherichia coli may be performed with any
Escherichia coli K-12 vector (e.g., conjugative plasmid). When a non-
conjugative vector is used, the Escherichia coli K-12 host may contain
conjugation-proficient plasmids either autonomous or integrated, or
generalized transducing phages. For these exempt laboratory
experiments, Biosafety Level (BL) 1 physical containment conditions are
recommended. For large scale fermentation experiments, the appropriate
physical containment conditions need be no greater than those for the
host organism unmodified by recombinant DNA techniques; the
Institutional Biosafety Committee can specify higher containment if
deemed necessary.

Appendix C-II-A. Exceptions

The following categories of experiments are not exempt from the NIH
Guidelines: (i) experiments described in Section III-A which require
Institutional Biosafety Committee approval, RAC review, and NIH
approval before initiation, (ii) experiments described in Section III-B
which require Institutional Biosafety Committee and NIH/ORDA approval
before initiation, (iii) experiments involving DNA from Class 3, 4, or
5 organisms (see Appendix C-VI-A) or cells known to be infected with
these agents may be conducted under containment conditions specified in
Section III-C-2 with prior Institutional Biosafety Committee review and
approval, (iv) large scale experiments (e.g., more than 10 liters of
culture), and (v) experiments involving the cloning of toxin molecule
genes coding for the biosynthesis of molecules toxic for vertebrates
(see Appendix F).

Appendix C-III. Saccharomyces Host-Vector Systems

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

Appendix C-III-A. Exceptions

The following categories are not exempt from the NIH Guidelines:
(i) Experiments described in Section III-A which require Institutional
Biosafety Committee approval, RAC review, and NIH approval before
initiation, (ii) experiments described in Section III-B which require
Institutional Biosafety Committee and NIH/ORDA approval before
initiation, (iii) experiments involving DNA from Class 3, 4, or 5
organisms (see Appendix C-VI-A) or cells known to be infected with
these agents may be conducted under containment conditions specified in
Section III-C-2 with prior Institutional Biosafety Committee review and
approval, (iv) large scale experiments (e.g., more than 10 liters of
culture), and (v) experiments involving the deliberate cloning of genes
coding for the biosynthesis of molecules toxic for vertebrates (see
Appendix F).

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

Any asporogenic Bacillus subtilis or asporogenic Bacillus
licheniformis strain which does not revert to a spore-former with a
frequency greater than 10-7 may be used for cloning DNA with the
exception of those experiments listed in Appendix C-IV-A. For these
exempt laboratory experiments, BL1 physical containment conditions are
recommended. For large scale fermentation experiments, the appropriate
physical containment conditions need be no greater than those for the
host organism unmodified by recombinant DNA techniques; the
Institutional Biosafety Committee can specify higher containment if it
deems necessary.

Appendix C-IV-A. Exceptions

The following categories are not exempt from the NIH Guidelines:
(i) Experiments described in Section III-A which require Institutional
Biosafety Committee approval, RAC review, and NIH approval before
initiation, (ii) experiments described in Section III-B which require
Institutional Biosafety Committee and NIH/ORDA approval before
initiation, (iii) experiments involving DNA from Class 3, 4, or 5
organisms (see Appendix C-VI-A) or cells known to be infected with
these agents may be conducted under containment conditions specified in
Section III-C-2 with prior Institutional Biosafety Committee review and
approval, (iv) large scale experiments (e.g., more than 10 liters of
culture), and (v) experiments involving the deliberate cloning of genes
coding for the biosynthesis of molecules toxic for vertebrates (see
Appendix F).

Appendix C-V. Extrachromosomal Elements of Gram Positive Organisms

Recombinant DNA molecules derived entirely from extrachromosomal
elements of the organisms listed below (including shuttle vectors
constructed from vectors described in Appendix C), propagated and
maintained in organisms listed below are exempt from these NIH
Guidelines.

Bacillus amyloliquefaciens
Bacillus amylosacchariticus
Bacillus anthracis
Bacillus aterrimus
Bacillus brevis
Bacillus cereus
Bacillus globigii
Bacillus licheniformis
Bacillus megaterium
Bacillus natto
Bacillus niger
Bacillus pumilus
Bacillus sphaericus
Bacillus stearothermophilis
Bacillus subtilis
Bacillus thuringiensis
Clostridium acetobutylicum
Lactobacillus casei
Listeria grayi
Listeria monocytogenes
Listeria murrayi
Pediococcus acidilactici
Pediococcus damnosus
Pediococcus pentosaceus
Staphylococcus aureus
Staphylcoccus carnosus
Staphylococcus epidermidis
Streptococcus agalactiae
Streptococcus anginosus
Streptococcus avium
Streptococcus cremoris
Streptococcus dorans
Streptococcus equisimilis
Streptococcus faecalis
Streptococcus ferus
Streptococcus lactis
Streptococcus ferns
Streptococcus mitior
Streptococcus mutans
Streptococcus pneumoniae
Streptococcus pyogenes
Streptococcus salivarious
Streptococcus sanguis
Streptococcus sobrinus
Streptococcus thermophylus

Appendix C-V-A. Exceptions

The following categories of experiments are not exempt from the NIH
Guidelines: (i) Experiments described in Section III-A which require
Institutional Biosafety Committee, specific RAC review, and NIH
approval before initiation, (ii) experiments described in Section III-B
which require Institutional Biosafety Committee and NIH/ORDA approval
before initiation, (iii) experiments involving DNA from Class 3, 4, or
5 organisms (see Appendix C-VI-A) or cells known to be infected with
these agents may be conducted under containment conditions specified in
Section III-C-2 with prior Institutional Biosafety Committee review and
approval, (iv) large scale experiments (e.g., more than 10 liters of
culture), and (v) experiments involving the deliberate cloning of genes
coding for the biosynthesis of molecules toxic for vertebrates (see
Appendix F).

Appendix C-VI. Footnotes and References of Appendix C

Appendix C-VI-A. The original reference to organisms as Class 1, 2,
3, 4, or 5 refers to the classification in the publication
Classification of Etiologic Agents on the Basis of Hazard, 4th Edition,
July 1974, U.S. Department of Health, Education, and Welfare, Public
Health Service, Centers for Disease Control and Prevention, Office of
Biosafety, Atlanta, Georgia 30333.
Appendix C-VI-A-1. The NIH Director, with advice of the RAC, may
revise the classification for the purposes of these NIH Guidelines (see
Section IV-C-1-b-(2)-(d)). The revised list of organisms in each class
is reprinted in Appendix B.
Appendix C-VI-B. A subset of non-conjugative plasmid vectors are
poorly mobilizable (e.g., pBR322, pBR313). Where practical, these
vectors should be employed.
Appendix C-VI-C. Defined as observable under optimal laboratory
conditions by transformation, transduction, phage infection, and/or
conjugation with transfer of phage, plasmid, and/or chromosomal genetic
information. Note that this definition of exchange may be less
stringent than that applied to exempt organisms under Section III-E-5.
Appendix C-VI-D. As classified in the Third Report of the
International Committee on Taxonomy of Viruses: Classification and
Nomenclature of Viruses, R.E.F. Matthews (ed.), Intervirology 12 (129-
296), 1979.
Appendix C-VI-E. i.e., the total of all genomes within a Family
shall not exceed one-half of the genome.

Appendix D. Major Actions Taken Under the NIH Guidelines

Under Section IV-C-1-b-(1), the NIH Director may take certain
actions with regard to the NIH Guidelines after the issues have been
considered by the RAC. An updated list of these actions are available
from the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
9838.

Appendix E. Certified Host-Vector Systems (see Appendix I)

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

Appendix E-I. Bacillus subtilis

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

Appendix E-II. Saccharomyces Cerevisiae

Appendix E-II-A. Saccharomyces Cerevisiae Host-Vector 2 Systems
The following sterile strains of Saccharomyces cerevisiae, all of
which have the ste-VC9 mutation, SHY1, SHY2, SHY3, and SHY4. The
following plasmids are certified for use: YIp1, YEp2, YEp4, YIp5, YEp6,
YRp7, YEp20, YEp21, YEp24, YIp25, YIp26, YIp27, YIp28, YIp29, YIp30,
YIp31, YIp32, and YIp33.

Appendix E-III. Escherichia coli

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

------------------------------------------------------------------------
Vector Host
------------------------------------------------------------------------
gt WESB'......... DP50supF
gt WESB*......... DP50supF
gt ZJ virB'...... Escherichia coli K-12
gtALOB... DP50supF
Charon 3A.......................... DP50 or DP50supF
Charon 4A.......................... DP50 or DP50supF
Charon 16A......................... DP50 or DP50supF
Charon 21A......................... DP50supF
Charon 23A......................... DP50 or DP50supF
Charon 24A......................... DP50 or DP50supF
------------------------------------------------------------------------

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

Appendix E-IV. Neurospora crassa

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

Appendix E-V. Streptomyces

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

Appendix E-VI. Pseudomonas Putida

Appendix E-VI-A. Pseudomonas putida Host-Vector 1 Systems
Pseudomonas putida strains KT2440 with plasmid vectors pKT262,
pKT263, and pKT264.

Appendix F. Containment Conditions for Cloning of Genes Coding for
the Biosynthesis of Molecules Toxic for Vertebrates

Appendix F-I. General Information

Appendix F specifies the containment to be used for the deliberate
cloning of genes coding for the biosynthesis of molecules toxic for
vertebrates. The cloning of genes coding for molecules toxic for
vertebrates that have an LD50 of 50 100 micrograms per kilograms of body weight.
Experiments involving genes coding for toxin molecules with an
LD50 of 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
50 of >1 microgram per
kilogram and 50 of 50 of 8 under specified conditions. Specific Host-Vector
2 systems are:
Appendix I-I-B-1. For Escherichia coli K-12 Host-Vector 2 systems
(EK2) in which the vector is a plasmid, no more than 1/108 host
cells shall perpetuate a cloned DNA fragment under the specified non-
permissive laboratory conditions designed to represent the natural
environment, either by survival of the original host or as a
consequence of transmission of the cloned DNA fragment.
Appendix I-I-B-2. For Escherichia coli K-12 Host-Vector 2 systems
(EK2) in which the vector is a phage, no more than 1/10\8\ phage
particles shall perpetuate a cloned DNA fragment under the specified
non-permissive laboratory conditions designed to represent the natural
environment, either as a prophage (in the inserted or plasmid form) in
the laboratory host used for phage propagation, or survival in natural
environments and transferring a cloned DNA fragment to other hosts (or
their resident prophages).
Appendix I-II. Certification of Host-Vector Systems
Appendix I-II-A. Responsibility. Host-Vector 1 systems (other than
Escherichia coli K-12) and Host-Vector 2 systems may not be designated
as such until they have been certified by the NIH Director. Requests
for certification of host-vector systems may be submitted to the Office
of Recombinant DNA Activities, National Institutes of Health, Building
31, room 4B11, Bethesda, Maryland 20892, (301) 496-9838. Proposed host-
vector systems will be reviewed by the RAC (see section IV-C-1-b-(1)-
(e)). Initial review will based on the construction, properties, and
testing of the proposed host-vector system by a subcommittee composed
of one or more RAC members and/or ad hoc experts. The RAC will evaluate
the subcommittee's report and any other available information at the
next scheduled RAC meeting. The NIH Director is responsible for
certification of host-vector systems, following advice of the RAC.
Minor modifications to existing host-vector systems (i.e., those that
are of minimal or no consequence to the properties relevant to
containment), may be certified by the NIH Director without prior RAC
review (see section IV-C-1-b-(2)-(h)). Once a host-vector system has
been certified by the NIH Director, a notice of certification will be
sent by NIH/ORDA to the applicant and to the Institutional Biosafety
Committee Chairs. A list of all currently certified host-vector systems
is available from the Office of Recombinant DNA Activities, National
Institutes of Health, Building 31, room 4B11, Bethesda, Maryland 20892,
(301) 496-9838. The NIH Director may rescind the certification of a
host-vector system (see section IV-C-1-b-(2)-(i)). If certification is
rescinded, NIH will instruct investigators to transfer cloned DNA into
a different system or use the clones at a higher level of physical
containment level, unless NIH determines that the already constructed
clones incorporate adequate biological containment. Certification of an
host-vector system does not extend to modifications of either the host
or vector component of that system. Such modified systems shall be
independently certified by the NIH Director. If modifications are
minor, it may only be necessary for the investigator to submit data
showing that the modifications have either improved or not impaired the
major phenotypic traits on which the containment of the system depends.
Substantial modifications to a certified host-vector system requires
submission of complete testing data.
Appendix I-II-B. Data To Be Submitted for Certification
Appendix I-II-B-1. Host-Vector 1 Systems Other than Escherichia
coli K-12. The following types of data shall be submitted, modified as
appropriate for the particular system under consideration: (i) a
description of the organism and vector; the strain's natural habitat
and growth requirements; its physiological properties, particularly
those related to its reproduction, survival, and the mechanisms by
which it exchanges genetic information; the range of organisms with
which this organism normally exchanges genetic information and the type
of information is exchanged; and any relevant information about its
pathogenicity or toxicity; (ii) a description of the history of the
particular strains and vectors to be used, including data on any
mutations which render this organism less able to survive or transmit
genetic information; and (iii) a general description of the range of
experiments contemplated with emphasis on the need for developing such
an Host-Vector 1 system.
Appendix I-II-B-2. Host-Vector 2 Systems. Investigators planning to
request Host-Vector 2 systems certification may obtain instructions
from NIH/ORDA concerning data to be submitted (see Appendices I-III-N
and O). In general, the following types of data are required: (i)
description of construction steps with indication of source,
properties, and manner of introduction of genetic traits; (ii)
quantitative data on the stability of genetic traits that contribute to
the containment of the system; (iii) data on the survival of the host-
vector system under non-permissive laboratory conditions designed to
represent the relevant natural environment; (iv) data on
transmissibility of the vector and/or a cloned DNA fragment under both
permissive and non-permissive conditions; (v) data on all other
properties of the system which affect containment and utility,
including information on yields of phage or plasmid molecules, ease of
DNA isolation, and ease of transfection or transformation; and (vi) in
some cases, the investigator may be asked to submit data on survival
and vector transmissibility from experiments in which the host-vector
is fed to laboratory animals or one or more human subjects. Such in
vivo data may be required to confirm the validity of predicting in vivo
survival on the basis of in vitro experiments. Data shall be submitted
12 weeks prior to the RAC meeting at which such data will be considered
by the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-
9838. Investigators are encouraged to publish their data on the
construction, properties, and testing of proposed Host Vector 2 systems
prior to consideration of the system by the RAC and its subcommittee.
Specific instructions concerning the submission of data for proposed
Escherichia coli K-12 Host-Vector 2 system (EK2) involving either
plasmids or bacteriophage in Escherichia coli K-12, are available from
the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-
9838.

Appendix I-III. Footnotes and References of Appendix I

Appendix I-III-A. Hersfield, V., H.W. Boyer, C. Yanofsky, M.A.
Lovett, and D.R. Helinski, Plasmid Co1E1 as a Molecular Vehicle for
Cloning and Amplification of DNA. Proc. Nat. Acad. Sci., 1974, 71, pp.
3455-3459.
Appendix I-III-B. Wensink, P.C., D.J. Finnegan, J.E. Donelson, and
D.S. Hogness, A System for Mapping DNA Sequences in the Chromosomes of
Drosophila Melanogaster. Cell, 1974, 3, pp. 315-335.
Appendix I-III-C. Tanaka, T., and B. Weisblum, Construction of a
Colicin El-R Factor Composite Plasmid in Vitro: Means for Amplification
of Deoxyribonucleic Acid. J. Bacteriol., 1975, 121, pp. 354-362.
Appendix I-III-D. Armstrong, K.A., V. Hershfield, and D.R.
Helinski, Gene Cloning and Containment Properties of Plasmid Col E1 and
Its Derivatives, Science, 1977, 196, pp. 172-174.
Appendix I-III-E. Bolivar, F., R.L. Rodriguez, M.C. Betlack, and
H.W. Boyer, Construction and Characterization of New Cloning Vehicles:
I. Ampicillin-Resistant Derivative of PMB9, Gene, 1977, 2, pp. 75-93.
Appendix I-III-F. Cohen, S.N., A.C.W. Chang, H. Boyer, and R.
Helling. Construction of Biologically Functional Bacterial Plasmids in
Vitro. Proc. Natl. Acad, Sci., 1973, 70, pp. 3240-3244.
Appendix I-III-G. Bolivar, F., R.L. Rodriguez, R.J. Greene, M.C.
Batlack, H.L. Reyneker, H.W. Boyer, J.H. Cross, and S. Falkow, 1977,
Construction and Characterization of New Cloning Vehicles II. A Multi-
Purpose Cloning System, Gene, 1977, 2, pp. 95-113.
Appendix I-III-H. Thomas, M., J.R. Cameron, and R.W. Davis (1974).
Viable Molecular Hybrids of Bacteriophage Lambda and Eukaryotic DNA.
Proc. Nat. Acad. Sci., 1974, 71, pp. 4579-4583.
Appendix I-III-I. Murray, N.E., and K. Murray, Manipulation of
Restriction Targets in Phage Lambda to Form Receptor Chromosomes for
DNA Fragments. Nature, 1974, 51, pp. 476-481.
Appendix I-III-J. Ramback, A., and P. Tiollais (1974).
Bacteriophage Having EcoRI Endonuclease Sites Only in the Non-Essential
Region of the Genome. Proc. Nat. Acad. Sci., 1974, 71, pp. 3927-3820.
Appendix I-III-K. Blattner, F.R., B.G. Williams, A.E. Bleche, K.
Denniston-Thompson, H.E. Faber, L.A. Furlong, D.J. Gunwald, D.O.
Kiefer, D.D. Moore, J.W. Shumm, E.L. Sheldon, and O. Smithies, Charon
Phages: Safer Derivatives of Bacteriophage Lambda for DNA Cloning,
Science 1977, 196, pp. 163-169.
Appendix I-III-L. Donoghue, D.J., and P.A. Sharp, An Improved
Lambda Vector: Construction of Model Recombinants Coding for Kanamycin
Resistance, Gene, 1977, 1, pp. 209-227.
Appendix I-III-M. Leder, P., D. Tiemeier and L. Enquist (1977), EK2
Derivatives of Bacteriophage Lambda Useful in the Cloning of DNA from
Higher Organisms: The gt WES System, Science, 1977, 196, pp.
175-177.
Appendix I-III-N. Skalka, A., Current Status of Coliphage AEK2
Vectors, Gene, 1978, 3, pp. 29-35.
Appendix I-III-O. Szybalski, W., A. Skalka, S. Gottesman, A.
Campbell, and D. Botstein, Standardized Laboratory Tests for EK2
Certification, Gene, 1978, 3, pp. 36-38.

Appendix J. Biotechnology Research Subcommittee

The National Science and Technology Council's Committee on
Fundamental Science determined that a subcommittee should be continued
to identify and coordinate Federal research efforts, identify research
needs, stimulating international cooperation, and assess national and
international policy issues concerning biotechnology sciences. The
primary emphasis will be on scientific issues to increase the overall
effectiveness and productivity of the Federal investment in
biotechnology sciences, especially regarding issues which cut across
agency boundaries. This subcommittee is called the Biotechnology
Research Subcommittee.
Membership of the Biotechnology Research Subcommittee will include
Federal agencies that support biotechnology research. Agencies
represented are: U.S. Department of Agriculture, Department of
Commerce, Department of Defense, Department of Energy, Department of
Health and Human Services, Department of Interior, Department of
Justice, Department of State, Department of Veterans Affairs, Agency
for International Development, Environmental Protection Agency,
National Aeronautics and Space Administration, and National Science
Foundation. The Biotechnology Research Subcommittee will function in an
advisory capacity to the Committee on Fundamental Science, the Director
of the Office of Science and Technology Policy, and the Executive
Office of the President. The Biotechnology Research Subcommittee will
review the scientific aspects of proposed regulations and guidelines as
they are developed.
The primary responsibilities of the Biotechnology Research
Subcommittee are to: (i) Describe and review current Federal efforts in
biotechnology research; (ii) identify and define the priority areas for
future Federal biotechnology research, including areas needing greater
emphasis, describing the role of each agency in those areas, and
delineate where interagency cooperation would enhance progress in the
biotechnology sciences, with an emphasis on integrated research
efforts, where appropriate; (iii) assess major international efforts in
the biotechnology sciences and develop mechanisms for international
collaboration. For example, activities of the U.S.-European Community
Task Force on Biotechnology have been coordinated through the
Biotechnology Research Subcommittee; (iv) identify and review national
and international policy issues (such as public education) associated
with biotechnology; and (v) provide reviews, analyses, and
recommendations to the Chairs of the Committee on Fundamental Science
on scientific issues related to regulations and the applications of
biotechnology research and biotechnology policies and issues.
In 1990, the Biotechnology Research Subcommittee replaced the
Biotechnology Sciences Coordinating Committee. Both the Biotechnology
Research Subcommittee and the Biotechnology Sciences Coordinating
Committee previously functioned under the Federal Coordinating Council
on Science, Engineering, and Technology (FCCSET). While regulatory
issues became the primary focus of the Biotechnology Sciences
Coordinating Committee, the Biotechnology Research Subcommittee focuses
on scientific issues, although it will still provide scientific support
for regulatory responsibilities.

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

Appendix K specifies physical containment guidelines for large
scale (greater than 10 liters of culture) research or production
involving viable organisms containing recombinant DNA molecules. It
shall apply to large scale research or production activities as
specified in Section III-C-6. It is important to note that this
appendix addresses only the biological hazard associated with organisms
containing recombinant DNA. Other hazards accompanying the large scale
cultivation of such organisms (e.g., toxic properties of products;
physical, mechanical, and chemical aspects of downstream processing)
are not addressed and shall be considered separately, albeit in
conjunction with this appendix.
All provisions shall apply to large scale research or production
activities with the following modifications: (i) Appendix K shall
supersede Appendix G when quantities in excess of 10 liters of culture
are involved in research or production. Appendix K-II applies to Good
Large Scale Practice; (ii) the institution shall appoint a Biological
Safety Officer if it engages in large scale research or production
activities involving viable organisms containing recombinant DNA
molecules. The duties of the Biological Safety Officer shall include
those specified in Section IV-B-3; (iii) the institution shall
establish and maintain a health surveillance program for personnel
engaged in large scale research or production activities involving
viable organisms containing recombinant DNA molecules which require
Biosafety Level (BL) 3 containment at the laboratory scale. The program
shall include: preassignment and periodic physical and medical
examinations; collection, maintenance, and analysis of serum specimens
for monitoring serologic changes that may result from the employee's
work experience; and provisions for the investigation of any serious,
unusual, or extended illnesses of employees to determine possible
occupational origin.

Appendix K-I. Selection of Physical Containment Levels

The selection of the physical containment level required for
recombinant DNA research or production involving more than 10 liters of
culture is based on the containment guidelines established in Section
III. For purposes of large scale research or production, four physical
containment levels are established. The four levels set containment
conditions at those appropriate for the degree of hazard to health or
the environment posed by the organism, judged by experience with
similar organisms unmodified by recombinant DNA techniques and
consistent with Good Large Scale Practice. The four biosafety levels of
large scale physical containment are referred to as Good Large Scale
Practice, BL1-Large Scale, BL2-Large Scale, and BL3-Large Scale. Good
Large Scale Practice is recommended for large scale research or
production involving viable, non-pathogenic, and non-toxigenic
recombinant strains derived from host organisms that have an extended
history of safe large scale use. Good Large Scale Practice is
recommended for organisms such as those included in Appendix C which
have built-in environmental limitations that permit optimum growth in
the large scale setting but limited survival without adverse
consequences in the environment. BL1-Large Scale is recommended for
large scale research or production of viable organisms containing
recombinant DNA molecules that require BL1 containment at the
laboratory scale and that do not qualify for Good Large Scale Practice.
BL2-Large Scale is recommended for large scale research or production
of viable organisms containing recombinant DNA molecules that require
BL2 containment at the laboratory scale. BL3-Large Scale is recommended
for large scale research or production of viable organisms containing
recombinant DNA molecules that require BL3 containment at the
laboratory scale. No provisions are made for large scale research or
production of viable organisms containing recombinant DNA molecules
that require BL4 containment at the laboratory scale. If necessary,
these requirements will be established by NIH on an individual basis.

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

Appendix K-II-A. Institutional codes of practice shall be
formulated and implemented to assure adequate control of health and
safety matters.
Appendix K-II-B. Written instructions and training of personnel
shall be provided to assure that cultures of viable organisms
containing recombinant DNA molecules are handled prudently and that the
workplace is kept clean and orderly.
Appendix K-II-C. In the interest of good personal hygiene,
facilities (e.g., hand washing sink, shower, changing room) and
protective clothing (e.g., uniforms, laboratory coats) shall be
provided that are appropriate for the risk of exposure to viable
organisms containing recombinant DNA molecules. Eating, drinking,
smoking, applying cosmetics, and mouth pipetting shall be prohibited in
the work area.
Appendix K-II-D. Cultures of viable organisms containing
recombinant DNA molecules shall be handled in facilities intended to
safeguard health during work with microorganisms that do not require
containment.
Appendix K-II-E. Discharges containing viable recombinant organisms
shall be handled in accordance with applicable governmental
environmental regulations.
Appendix K-II-F. Addition of materials to a system, sample
collection, transfer of culture fluids within/between systems, and
processing of culture fluids shall be conducted in a manner that
maintains employee's exposure to viable organisms containing
recombinant DNA molecules at a level that does not adversely affect the
health and safety of employees.
Appendix K-II-G. The facility's emergency response plan shall
include provisions for handling spills. Spills and accidents which
result in overt exposures to organisms containing recombinant DNA
molecules are immediately reported to the Biological Safety Officer,
Institutional Biosafety Committee, NIH/ORDA, and other appropriate
authorities (if applicable). Reports to NIH/ORDA shall be sent to the
Office of Recombinant DNA Activities, National Institutes of Health,
Building 31, room 4B11, Bethesda, Maryland 20892, (301) 496-9838.

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

Appendix K-III-A. Spills and accidents which result in overt
exposures to organisms containing recombinant DNA molecules are
immediately reported to the Biological Safety Officer, Institutional
Biosafety Committee, NIH/ORDA, and other appropriate authorities (if
applicable). Reports to NIH/ORDA shall be sent to the Office of
Recombinant DNA Activities, National Institutes of Health, Building 31,
room 4B11, Bethesda, Maryland 20892, (301) 496-9838. Medical
evaluation, surveillance, and treatment are provided as appropriate and
written records are maintained.
Appendix K-III-B. Cultures of viable organisms containing
recombinant DNA molecules shall be handled in a closed system (e.g.,
closed vessel used for the propagation and growth of cultures) or other
primary containment equipment (e.g., biological safety cabinet
containing a centrifuge used to process culture fluids) which is
designed to reduce the potential for escape of viable organisms.
Volumes less than 10 liters may be handled outside of a closed system
or other primary containment equipment provided all physical
containment requirements specified in Appendix G-II-A are met.
Appendix K-III-C. Culture fluids (except as allowed in Appendix K-
III-D) shall not be removed from a closed system or other primary
containment equipment unless the viable organisms containing
recombinant DNA molecules have been inactivated by a validated
inactivation procedure. A validated inactivation procedure is one which
has been demonstrated to be effective using the organism that will
serve as the host for propagating the recombinant DNA molecules.
Appendix K-III-D. Sample collection from a closed system, the
addition of materials to a closed system, and the transfer of culture
fluids from one closed system to another shall be conducted in a manner
which minimizes the release of aerosols or contamination of exposed
surfaces.
Appendix K-III-E. Exhaust gases removed from a closed system or
other primary containment equipment shall be treated by filters which
have efficiencies equivalent to high efficiency particulate air/HEPA
filters or by other equivalent procedures (e.g., incineration) to
minimize the release of viable organisms containing recombinant DNA
molecules to the environment.
Appendix K-III-F. A closed system or other primary containment
equipment that has contained viable organisms containing recombinant
DNA molecules shall not be opened for maintenance or other purposes
unless it has been sterilized by a validated sterilization procedure. A
validated sterilization procedure is one which has been demonstrated to
be effective using the organism that will serve as the host for
propagating the recombinant DNA molecules.
Appendix K-III-G. Emergency plans required by Sections IV-B-2-b-(6)
and IV-B-3-c-(3) shall include methods and procedures for handling
large losses of culture on an emergency basis.
Appendix K-IV. Biosafety Level 2 (BL2)--Large Scale
Appendix K-IV-A. Spills and accidents which result in overt
exposures to organisms containing recombinant DNA molecules are
immediately reported to the Biological Safety Officer, Institutional
Biosafety Committee, NIH/ORDA, and other appropriate authorities (if
applicable). Reports to NIH/ORDA shall be sent to the Office of
Recombinant DNA Activities, National Institutes of Health, Building 31,
Room 4B11, Bethesda, Maryland 20892, (301) 496-9838. Medical
evaluation, surveillance, and treatment are provided as appropriate and
written records are maintained.
Appendix K-IV-B. Cultures of viable organisms containing
recombinant DNA molecules shall be handled in a closed system (e.g.,
closed vessel used for the propagation and growth of cultures) or other
primary containment equipment (e.g., Class III biological safety
cabinet containing a centrifuge used to process culture fluids) which
is designed to prevent the escape of viable organisms. Volumes less
than 10 liters may be handled outside of a closed system or other
primary containment equipment provided all physical containment
requirements specified in Appendix G-II-B are met.
Appendix K-IV-C. Culture fluids (except as allowed in Appendix K-
IV-D) shall not be removed from a closed system or other primary
containment equipment unless the viable organisms containing
recombinant DNA molecules have been inactivated by a validated
inactivation procedure. A validated inactivation procedure is one which
has been demonstrated to be effective using the organism that will
serve as the host for propagating the recombinant DNA molecules.
Appendix K-IV-D. Sample collection from a closed system, the
addition of materials to a closed system, and the transfer of cultures
fluids from one closed system to another shall be conducted in a manner
which prevents the release of aerosols or contamination of exposed
surfaces.
Appendix K-IV-E. Exhaust gases removed from a closed system or
other primary containment equipment shall be treated by filters which
have efficiencies equivalent to high efficiency particulate air/HEPA
filters or by other equivalent procedures (e.g., incineration) to
prevent the release of viable organisms containing recombinant DNA
molecules to the environment.
Appendix K-IV-F. A closed system or other primary containment
equipment that has contained viable organisms containing recombinant
DNA molecules shall not be opened for maintenance or other purposes
unless it has been sterilized by a validated sterilization procedure. A
validated sterilization procedure is one which has been demonstrated to
be effective using the organisms that will serve as the host for
propagating the recombinant DNA molecules.
Appendix K-IV-G. Rotating seals and other mechanical devices
directly associated with a closed system used for the propagation and
growth of viable organisms containing recombinant DNA molecules shall
be designed to prevent leakage or shall be fully enclosed in ventilated
housings that are exhausted through filters which have efficiencies
equivalent to high efficiency particulate air/HEPA filters or through
other equivalent treatment devices.
Appendix K-IV-H. A closed system used for the propagation and
growth of viable organisms containing recombinant DNA molecules and
other primary containment equipment used to contain operations
involving viable organisms containing sensing devices that monitor the
integrity of containment during operations.
Appendix K-IV-I. A closed system used for the propagation and
growth of viable organisms containing the recombinant DNA molecules
shall be tested for integrity of the containment features using the
organism that will serve as the host for propagating recombinant DNA
molecules. Testing shall be accomplished prior to the introduction of
viable organisms containing recombinant DNA molecules and following
modification or replacement of essential containment features.
Procedures and methods used in the testing shall be appropriate for the
equipment design and for recovery and demonstration of the test
organism. Records of tests and results shall be maintained on file.
Appendix K-IV-J. A closed system used for the propagation and
growth of viable organisms containing recombinant DNA molecules shall
be permanently identified. This identification shall be used in all
records reflecting testing, operation, and maintenance and in all
documentation relating to use of this equipment for research or
production activities involving viable organisms containing recombinant
DNA molecules.
Appendix K-IV-K. The universal biosafety sign shall be posted on
each closed system and primary containment equipment when used to
contain viable organisms containing recombinant DNA molecules.
Appendix K-IV-L. Emergency plans required by Sections IV-B-2-b-(6)
and IV-B-3-c-(3) shall include methods and procedures for handling
large losses of culture on an emergency basis.
Appendix K-V. Biosafety Level 3 (BL3)--Large Scale
Appendix K-V-A. Spills and accidents which result in overt
exposures to organisms containing recombinant DNA molecules are
immediately reported to the Biological Safety Officer, Institutional
Biosafety Committee, NIH/ORDA, and other appropriate authorities (if
applicable). Reports to NIH/ORDA shall be sent to the Office of
Recombinant DNA Activities, National Institutes of Health, Building 31,
Room 4B11, Bethesda, Maryland 20892, (301) 496-9838. Medical
evaluation, surveillance, and treatment are provided as appropriate and
written records are maintained.
Appendix K-V-B. Cultures of viable organisms containing recombinant
DNA molecules shall be handled in a closed system (e.g., closed vessels
used for the propagation and growth of cultures) or other primary
containment equipment (e.g., Class III biological safety cabinet
containing a centrifuge used to process culture fluids) which is
designed to prevent the escape of viable organisms. Volumes less than
10 liters may be handled outside of a closed system provided all
physical containment requirements specified in Appendix G-II-C are met.
Appendix K-V-C. Culture fluids (except as allowed in Appendix K-V-
D) shall not be removed from a closed system or other primary
containment equipment unless the viable organisms containing
recombinant DNA molecules have been inactivated by a validated
inactivation procedure. A validated inactivation procedure is one which
has been demonstrated to be effective using the organisms that will
serve as the host for propagating the recombinant DNA molecules.
Appendix K-V-D. Sample collection from a closed system, the
addition of materials to a closed system, and the transfer of culture
fluids from one closed system to another shall be conducted in a manner
which prevents the release of aerosols or contamination of exposed
surfaces.
Appendix K-V-E. Exhaust gases removed from a closed system or other
primary containment equipment shall be treated by filters which have
efficiencies equivalent to high efficiency particulate air/HEPA filters
or by other equivalent procedures (e.g., incineration) to prevent the
release of viable organisms containing recombinant DNA molecules to the
environment.
Appendix K-V-F. A closed system or other primary containment
equipment that has contained viable organisms containing recombinant
DNA molecules shall not be opened for maintenance or other purposes
unless it has been sterilized by a validated sterilization procedure. A
validated sterilization procedure is one which has been demonstrated to
be effective using the organisms that will serve as the host for
propagating the recombinant DNA molecules.
Appendix K-V-G. A closed system used for the propagation and growth
of viable organisms containing recombinant DNA molecules shall be
operated so that the space above the culture level will be maintained
at a pressure as low as possible, consistent with equipment design, in
order to maintain the integrity of containment features.
Appendix K-V-H. Rotating seals and other mechanical devices
directly associated with a closed system used to contain viable
organisms containing recombinant DNA molecules shall be designed to
prevent leakage or shall be fully enclosed in ventilated housings that
are exhausted through filters which have efficiencies equivalent to
high efficiency particulate air/HEPA filters or through other
equivalent treatment devices.
Appendix K-V-I. A closed system used for the propagation and growth
of viable organisms containing recombinant DNA molecules and other
primary containment equipment used to contain operations involving
viable organisms containing recombinant DNA molecules shall include
monitoring or sensing devices that monitor the integrity of containment
during operations.
Appendix K-V-J. A closed system used for the propagation and growth
of viable organisms containing recombinant DNA molecules shall be
tested for integrity of the containment features using the organisms
that will serve as the host for propagating the recombinant DNA
molecules. Testing shall be accomplished prior to the introduction of
viable organisms containing recombinant DNA molecules and following
modification or replacement of essential containment features.
Procedures and methods used in the testing shall be appropriate for the
equipment design and for recovery and demonstration of the test
organism. Records of tests and results shall be maintained on file.
Appendix K-V-K. A closed system used for the propagation and growth
of viable organisms containing recombinant DNA molecules shall be
permanently identified. This identification shall be used in all
records reflecting testing, operation, maintenance, and use of this
equipment for research production activities involving viable organisms
containing recombinant DNA molecules.
Appendix K-V-L. The universal biosafety sign shall be posted on
each closed system and primary containment equipment when used to
contain viable organisms containing recombinant DNA molecules.
Appendix K-V-M. Emergency plans required by Sections IV-B-2-b-(6)
and IV-B-3-c-(3) shall include methods and procedures for handling
large losses of culture on an emergency basis.
Appendix K-V-N. Closed systems and other primary containment
equipment used in handling cultures of viable organisms containing
recombinant DNA molecules shall be located within a controlled area
which meets the following requirements:
Appendix K-V-N-1. The controlled area shall have a separate entry
area. The entry area shall be a double-doored space such as an air
lock, anteroom, or change room that separates the controlled area from
the balance of the facility.
Appendix K-V-N-2. The surfaces of walls, ceilings, and floors in
the controlled area shall be such as to permit ready cleaning and
decontamination.
Appendix K-V-N-3. Penetrations into the controlled area shall be
sealed to permit liquid or vapor phase space decontamination.
Appendix K-V-N-4. All utilities and service or process piping and
wiring entering the controlled area shall be protected against
contamination.
Appendix K-V-N-5. Hand washing facilities equipped with foot,
elbow, or automatically operated valves shall be located at each major
work area and near each primary exit.
Appendix K-V-N-6. A shower facility shall be provided. This
facility shall be located in close proximity to the controlled area.
Appendix K-V-N-7. The controlled area shall be designed to preclude
release of culture fluids outside the controlled area in the event of
an accidental spill or release from the closed systems or other primary
containment equipment.
Appendix K-V-N-8. The controlled area shall have a ventilation
system that is capable of controlling air movement. The movement of air
shall be from areas of lower contamination potential to

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Source: Frix Law Library, https://www.frixlaw.com/law-library/documents/fr%3A94-16200. Public record. Not legal advice.