[Federal Register Volume 59, Number 127 (Tuesday, July 5, 1994)]
[Unknown Section]
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From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 94-16199]
[[Page Unknown]]
[Federal Register: July 5, 1994]
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Part III
Department of Health and Human Services
_______________________________________________________________________
National Institutes of Health
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Recombinant DNA Research: Actions Under the Guidelines; Notice
DEPARTMENT OF HEALTH AND HUMAN SERVICES
National Institutes of Health
Recombinant DNA Research: Actions Under the Guidelines
AGENCY: National Institutes of Health, PHS, DHHS.
ACTION: Notice of actions under the National Institutes of Health
Guidelines for Research Involving Recombinant DNA Molecules (NIH
Guidelines).
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SUMMARY: This notice sets forth four actions to be taken by the
Director, National Institutes of Health (NIH), under the May 7, 1986,
NIH Guidelines (51 FR 16958).
FOR FURTHER INFORMATION CONTACT: Additional information can be obtained
from Dr. Nelson A. Wivel, Director, Office of Recombinant DNA
Activities (ORDA), Office of Science Policy and Technology Transfer,
National Institutes of Health, Building 31, room 4B11, Bethesda,
Maryland 20892, (301) 496-9838.
SUPPLEMENTARY INFORMATION: Today four actions are being promulgated
under the NIH Guidelines. These four proposed actions were published
for comment in the Federal Register announcements of August 11, 1987
(52 FR 29800); April 18, 1988 (53 FR 12752); December 30, 1988 (53 FR
53262); April 29, 1991 (56 FR 19776); November 9, 1993 (58 FR 59612);
and February 11, 1994 (59 FR 6702). These proposed actions were
reviewed and recommended for approval by the NIH Recombinant DNA
Advisory Committee (RAC) at its meetings on September 21, 1987;
December 3, 1988; January 30, 1989; May 30-31, 1991; December 2-3,
1993; and March 3-4, 1994.
In accordance with Section IV-C-1-b of the NIH Guidelines, these
actions have been found to comply with the NIH Guidelines and to
present no significant risk to health or the environment.
A revised version of the NIH Guidelines is published in a separate
section of the Federal Register following this announcement. These
revised NIH Guidelines differ from the previous version promulgated on
May 7, 1986 (51 FR 16958) by incorporating within them the major
actions to the NIH Guidelines that were promulgated on August 24, 1987
(52 FR 31848); July 29, 1988 (53 FR 28819); October 26, 1988 (53 FR
43410); March 13, 1989 (54 FR 10508); March 1, 1990 (55 FR 7438);
September 12, 1990 (55 FR 37565); July 18, 1991 (56 FR 33174); October
15, 1991 (56 FR 51784); November 21, 1991 (56 FR 58800); January 28,
1992 (57 FR 3212); April 22, 1992 (57 FR 14774); August 26, 1992 (57 FR
38734); February 18, 1993 (58 FR 9102); April 23, 1993 (58 FR 21738);
September 13, 1993 (58 FR 47906); October 18, 1993 (58 FR 53814); and
the changes that are promulgated in this announcement.
I. Background Information and Decision on Action Under the NIH
Guidelines
A. Amendment to Sections II, III-C, III-D, V, Appendices C-I, and G and
Addition of Appendix P, Physical and Biological Containment for
Recombinant DNA Research Involving Plants, and Appendix Q, Physical and
Biological Containment for Recombinant DNA Research Involving Animals
of the NIH Guidelines
The NIH Guidelines were originally developed to cover research in
laboratories in which recombinant DNA techniques were used. It is
recognized today that these techniques are being used by scientists
working with plants and large animals, and that procedures for
containment of these plants and animals have not been specifically
described in the NIH Guidelines. Institutional Biosafety Committees
(IBCs) have requested guidance on the containment procedures that
should be recommended for specific experiments with these organisms
since they have the responsibility of approving such experiments under
containment appropriate for the organisms. The principles of biological
safety that are used to categorize experiments involving
microorganisms, for example, are equally applicable to plants and
animals. These safety procedures have been employed successfully for
many years and have been recognized for their efficacy in biological
containment.
Appendices P and Q are the result of several years of meetings and
discussions involving research scientists and representatives from
university, government, and industrial research sectors with expertise
in several disciplines, including plant genetics, plant physiology,
plant pathology, entomology, animal (including arthropod and aquatic
species) physiology and reproduction, molecular biology, veterinary
medicine, and human biomedical research. The Federal agencies involved
in the development of Appendices P and Q include the NIH, the National
Science Foundation (NSF), and the U.S. Department of Agriculture
(USDA).
The process of developing Appendices P and Q was initiated when the
USDA published an Advanced Notice of Proposed USDA Guidelines (USDA
Guidelines) in the Federal Register on June 26, 1986 (51 FR 23367).
This notice was followed by an announcement by the USDA regarding its
intent to propose new guidelines for conducting all phases of research
with domestic agriculture species, including both plants and animals
modified through the application of genetic engineering techniques, in
the Federal Register on December 9, 1986 (51 FR 44397). At that time,
the NIH Guidelines did not include specific descriptions for
containment conditions for research involving recombinant DNA
containing whole plants and animals. The USDA convened a working group
composed of university, government, and industrial scientists on
December 13-14, 1986, with the purpose of discussing and redrafting
guidelines for physical and biological containment of transgenic plant
and animal species, and associated microorganisms. This meeting came to
be known as the ``Arlington House Workshop.''
Participants of the ``Arlington House Workshop,'' including former
members of the RAC, agreed that the USDA Guidelines should be
incorporated into the NIH Guidelines. The workshop participants noted
that merging these two documents would offer the distinct advantage of
providing a single comprehensive source of information regarding
conduct of research involving organisms containing recombinant DNA and
plants and animals exposed to microorganisms containing recombinant
DNA.
A staff working group representing the Office of Recombinant DNA
Activities, NIH, and the Cooperative State Research Service, USDA, held
meetings during the following six months. This working group met with
the purpose of revising the containment section and developing a final
incorporated document for RAC review, approval by the NIH Director, and
incorporation into the NIH Guidelines.
On June 28, 1987, and July 16, 1987, the RAC appointed the Working
Group on Revision of the NIH Guidelines to meet and consider the draft
documents, Appendices P and Q, and minor modifications to the NIH
Guidelines, that would accommodate the proposed appendices. Appendices
P and Q and the proposed revisions to the NIH Guidelines were published
for public comment in the Federal Register on August 11, 1987 (52 FR
29800). Additional revisions to Appendices P and Q were proposed by the
RAC and the Agricultural Research Service, USDA, at the September 17,
1987, RAC meeting. These modifications were published for public
comment in the Federal Register on December 30, 1988 (53 FR 53262). The
RAC Working Group on Transgenic Animals proposed additional
modifications to Appendices P and Q which were published for public
comment in the Federal Register on April 18, 1988 (53 FR 12752).
Further revisions were approved by the RAC at its January 30, 1989,
meeting.
Throughout all of the meetings, discussions, and revisions, the
intent of the Federal agencies and interested parties has been to
describe working conditions that would minimize the risk to both the
researcher and the environment from any possible harm or adverse
effects due to the conduct of research involving recombinant DNA
containing organisms.
On June 24, 1994, an Environmental Assessment of Appendices P and Q
was completed by the NIH and USDA, and there was a finding of no
significant impact. Copies of the Environmental Assessment are
available from the Office of Recombinant DNA Activities, National
Institutes of Health, Building 31, room 4B11, Bethesda, Maryland 20892,
(301) 496-9838.
The actions are detailed in Section II--Summary of Actions. I
accept these recommendations, and the NIH Guidelines will be amended
accordingly.
B. Amendment to Sections I-C-1-b-(2) and Deletion of Section III-A-2 of
the NIH Guidelines Regarding Deliberate Release
On December 6, 1990, the RAC Planning Subcommittee recommended that
the requirement for RAC review of experiments involving deliberate
environmental release of organisms containing recombinant DNA be
eliminated from the NIH Guidelines. This recommendation reflects the
fact that the Federal regulatory agencies, the USDA, and the
Environmental Protection Agency (EPA), are responsible for the review
and approval of environmental release experiments. The proposed
amendment was published for public comment in the Federal Register on
April 29, 1991 (56 FR 19776). The RAC reviewed and recommended approval
of the proposed amendment at its May 30-31, 1991, meeting.
The actions are detailed in Section II--Summary of Actions. I
accept these recommendations, and the NIH Guidelines will be amended
accordingly.
C. Amendments to Sections I, III, IV, and V, and Appendix M of the NIH
Guidelines Regarding NIH/ORDA Review and Approval of Certain Categories
of Human Gene Transfer Experiments That Qualify for the Accelerated
Review Process
On December 3, 1993, and March 3-4, 1994, the Working Group on
Accelerated Review Protocols presented an overview of the proposed
amendments to the NIH Guidelines. The proposed amendments will: (1)
Establish an accelerated review process for certain categories of human
gene transfer experiments, (2) allow the NIH/Office of Recombinant DNA
Activities to assign the appropriate review category to all human gene
transfer proposals that are submitted in compliance with the NIH
Guidelines, (3) allow the NIH/Office of Recombinant DNA Activities to
approve those categories of human gene transfer experiments that
qualify for the accelerated review process in consultation with the
Chair and one or more RAC members, as necessary, and (4) exempt certain
experiments involving the transfer of recombinant DNA or DNA or RNA
derived from recombinant DNA into one or more human subjects which are
not covered by Section V-U. All human gene transfer experiments
approved by the NIH/Office of Recombinant DNA Activities through the
accelerated review process will be provided in a report by the RAC
Chair at the next regularly scheduled RAC meeting and will be included
in the list of approved experiments which is available from the Office
of Recombinant DNA Activities, National Institutes of Health, Building
31, Room 4B11, Bethesda, Maryland 20892, (301) 496-9838.
The proposed amendments were published for public comment in the
Federal Register on November 9, 1993 (58 FR 59612) and February 11,
1994 (59 FR 6702). The RAC reviewed and unanimously recommended
approval of the proposed amendments at its March 3-4, 1994, meeting.
The actions are detailed in Section II--Summary of Actions. I
accept these recommendations, and the NIH Guidelines will be amended
accordingly.
D. Amendments to Section V-U of the NIH Guidelines Regarding
Recombinant DNA Vaccines
On March 3, 1994, the Working Group on Vaccines presented an
overview of the proposed amendment to the footnote in Section V-U. The
proposed amendment will define those categories of experiments
involving the administration of recombinant DNA vaccines that are
exempt from RAC review and NIH and Institutional Biosafety Committee
approval.
The proposed amendment was published for public comments in the
Federal Register on February 11, 1994 (59 FR 6702). The proposed
amendment was revised by the RAC at its March 3-4, 1994, meeting. The
revised amendment was unanimously approved.
The action is detailed in Section II--Summary of Actions. I accept
this recommendation, and the NIH Guidelines will be amended
accordingly.
II. Summary of Actions
A. Amendment to Section I, Scope of the NIH Guidelines
The amended version of Section I reads as follows:
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.
B. Amendment to Section II, Containment, of the NIH Guidelines
The amended version of Section II reads as follows:
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.''
C. Amendment to Section III, Experiments Covered by the NIH Guidelines
The previous version of Section III-A-2 will be deleted as follows:
Section III-A-2. Deliberate release into the environment of any
organism containing recombinant DNA except those listed below. The term
`deliberate release' is defined as a planned introduction of
recombinant DNA-containing microorganisms, plants, or animals into the
environment.
Section III-A-2-a. Introduction conducted under conditions
considered to be accepted scientific practices in which there is
adequate evidence of biological and/or physical control of the
recombinant DNA-containing organisms. The nature of such evidence is
described in Appendix L.
Section III-A-2-b. Deletion derivatives and single base changes not
otherwise covered by the NIH Guidelines.
Section III-A-2-c. For extrachromosomal elements and microorganisms
(including viruses), rearrangements and amplifications within a single
genome. Rearrangements involving the introduction of DNA from different
strains of the same species would not be covered by this exemption.''
The amended version of Section III reads as follows:
Section III. Experiments Covered by the NIH Guidelines.
This section describes five categories of experiments involving
recombinant DNA: (i) Those that require RAC review and NIH and
Institutional Biosafety Committee approval before initiation (see
Section III-A), (ii) those that require NIH/ORDA and Institutional
Biosafety Committee approval before initiation (see Section III-B);
(iii) those that require Institutional Biosafety Committee approval
before initiation (see Section III-C), (iv) those that require
Institutional Biosafety Committee notification simultaneous with
initiation (see Section III-D), and (v) those that are exempt from the
NIH Guidelines (see Section III-E).
Note: If an experiment falls into either Section III-A or
Section III-B and one of the other categories, the rules pertaining
to Section III-A or Section III-B shall be followed. If an
experiment falls into Section III-E and into either Sections III-C
or III-D categories as well, the experiment is considered exempt
from the NIH Guidelines.
Any change in containment level, which is different from those
specified in the NIH Guidelines, may not be initiated without the
express approval of NIH/ORDA (see Minor Actions, Section IV-C-1-b-(2)
and its subsections).
Section III-A. Experiments That Require Institutional Biosafety
Committee Approval, RAC Review, and NIH Approval Before Initiation
Experiments in this category are considered Major Actions (see
Section IV-C-1-b-(1)) and cannot be initiated without submission of
relevant information on the proposed experiment to the Office of
Recombinant DNA Activities, National Institutes of Health, Building 31,
Room 4B11, Bethesda, Maryland 20892, (301) 496-9838, the publication of
the proposal in the Federal Register for 15 days of comment, reviewed
by the RAC, and specific approval by the NIH (not applicable for
Expedited Review single patient human gene transfer experiments
considered under Appendix M-VI). The containment conditions for such
experiments will be recommended by the RAC and set by the NIH at the
time of approval. Such experiments require Institutional Biosafety
Committee approval before initiation. Specific experiments already
approved are included in Appendix D which may be obtained from the
Office of Recombinant DNA Activities, National Institutes of Health,
Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-9838.
Section III-A-1. Deliberate transfer of a drug resistance trait to
microorganisms that are not known to acquire the trait naturally (see
Section V-B), if such acquisition could compromise the use of the drug
to control disease agents in humans, veterinary medicine, or
agriculture.
Section III-A-2. Certain experiments involving the deliberate
transfer of recombinant DNA or DNA or RNA derived from recombinant DNA
into one or more human subjects (see Section V-U) shall be considered
Major Actions (see Section IV-C-1-b-(1) and Appendix M-III), and shall
require RAC review and NIH Director approval, if determined by NIH/
ORDA, in consultation with the RAC Chair and one or more RAC members,
as necessary, to: (i) represent novel characteristics (e.g., target
disease or vector), (ii) represent an uncertain degree of risk to human
health or the environment, or (iii) contain information determined to
require further public review. The requirement for RAC review shall not
be considered to preempt any other required review or approval of
experiments with one or more human subjects. Relevant Institutional
Biosafety Committee and Institutional Review Board reviews and
approvals of the proposal should be completed before submission to NIH.
Certain experiments involving deliberate transfer of recombinant DNA or
DNA or RNA derived from recombinant DNA into one or more human subjects
may qualify for the Accelerated Review process (see Section III-B-2).
Certain categories of experiments involving the deliberate transfer of
recombinant DNA or DNA or RNA derived from recombinant DNA into one or
more human subjects and that are not covered by Section V-U, may be
considered exempt from RAC and/or NIH/ORDA review and/or NIH Director
approval and only require registration with NIH/ORDA (see Section III-
C-7).
Section III-B. Experiments That Require NIH/ORDA and Institutional
Biosafety Committee Approval Before Initiation
Section III-B-1. Experiments Involving the Cloning of Toxin Molecules
with LD50 of Less Than 100 Nanograms per Kilogram Body Weight
Deliberate formation of recombinant DNA containing genes for the
biosynthesis of toxin molecules lethal for vertebrates at an LD50
of less than 100 nanograms per kilogram body weight (e.g., microbial
toxins such as the botulinum toxins, tetanus toxin, diphtheria toxin,
and Shigella dysenteriae neurotoxin). Specific approval has been given
for the cloning in Escherichia coli K-12 of DNA containing genes coding
for the biosynthesis of toxic molecules which are lethal to vertebrates
at 100 nanograms to 100 micrograms per kilogram body weight. Specific
experiments already approved under this section may be obtained from
the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
9838.
Section III-B-1-(a). Experiments in this category cannot be
initiated without submission of relevant information on the proposed
experiment to NIH/ORDA. The containment conditions for such experiments
will be determined by NIH/ORDA in consultation with ad hoc experts.
Such experiments require Institutional Biosafety Committee approval
before initiation (see Section IV-B-2-b-(1)).
Section III-B-2. Accelerated Review of Human Gene Transfer Experiments
As determined by NIH/ORDA, in consultation with the RAC Chair and
one or more RAC members, as necessary, certain categories of human gene
transfer experiments may be considered as Minor Actions and qualify for
Accelerated Review and approval (see Section IV-C-1-b-(2)-(a), Appendix
M-III-A, and Appendix M- V). The RAC Chair will present a report of all
NIH/ORDA approved human gene transfer protocols at the next regularly
scheduled RAC meeting. If NIH/ORDA determines that an experiment does
not qualify for the Accelerated Review process, the Principal
Investigator must submit the proposal for full RAC review 8
weeks prior to the next scheduled RAC meeting (See Section III-A-2).
Section III-B-3. Minor Modifications to Human Gene Transfer Experiments
A minor modification in a human gene transfer protocol is a
modification that does not significantly alter the basic design of the
protocol and that does not increase risk to human subjects or the
environment. After approval has been obtained by the relevant
Institutional Biosafety Committee and Institutional Review Board, NIH/
ORDA will consider the change in consultation with the RAC Chair and
one or more RAC members, as necessary. Submit minor modifications to
the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
9838. The RAC Chair will provide a report on any such approvals at the
next regularly scheduled RAC meeting.
Section III-C. Experiments That Require Institutional Biosafety
Committee Approval Before Initiation
Prior to the initiation of an experiment that falls into this
category, the Principal Investigator must submit a registration
document to the Institutional Biosafety Committee which contains the
following information: (i) The source(s) of DNA; (ii) the nature of the
inserted DNA sequences; (iii) the host(s) and vector(s) to be used;
(iv) if an attempt will be made to obtain expression of a foreign gene,
and if so, indicate the protein that will be produced; and (v) the
containment conditions that will be implemented as specified in the NIH
Guidelines. For experiments in this category, the registration document
shall be dated, signed by the Principal Investigator, and filed with
the Institutional Biosafety Committee. The Institutional Biosafety
Committee shall review and approve all experiments in this category
prior to their initiation. Requests to decrease the level of
containment specified for experiments in this category will be
considered by NIH (see Section IV-C-1-b-(2)-(c)).
Section III-C-1. Experiments Using Human or Animal Pathogens (Class 2,
Class 3, Class 4, or Class 5 Agents (See Section V-A) as Host-Vector
Systems
Section III-C-1-a. Experiments involving the introduction of
recombinant DNA into Class 2 agents shall be conducted at Biosafety
Level (BL) 2 containment. Experiments with such agents shall be
conducted with whole animals at BL2 or BL2-N (Animals) containment.
Section III-C-1-b. Experiments involving the introduction of
recombinant DNA into Class 3 agents shall be conducted at BL3
containment. Experiments with such agents shall be conducted with whole
animals at BL3 or BL3-N containment.
Section III-C-1-c. Experiments involving the introduction of
recombinant DNA into Class 4 agents shall be conducted at BL4
containment. Experiments with such agents shall be conducted with whole
animals at BL4 or BL4-N containment.
Section III-C-1-d. Containment conditions for experiments involving
the introduction of recombinant DNA into Class 5 agents shall be set on
a case-by-case basis following NIH/ORDA review. A U.S. Department of
Agriculture permit is required for work with Class 5 agents (see
Sections V-R and V-T). Experiments with such agents shall be conducted
with whole animals at BL4 or BL4-N containment.
Section III-C-2. Experiments in Which DNA From Human or Animal
Pathogens (Class 2, Class 3, Class 4, or Class 5 Agents (See Section V-
A) is Cloned Into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-
Vector Systems
Section III-C-2-a. Experiments in which DNA from Class 2 or Class 3
agents (see Section V-A) is transferred into nonpathogenic prokaryotes
or lower eukaryotes may be performed under BL2 containment. Experiments
in which DNA from Class 4 agents is transferred into nonpathogenic
prokaryotes or lower eukaryotes may be performed under BL2 containment
after demonstration that only a totally and irreversibly defective
fraction of the agent's genome is present in a given recombinant. In
the absence of such a demonstration, BL4 containment shall be used. The
Institutional Biosafety Committee may approve the specific lowering of
containment for particular experiments to BL1. Many experiments in this
category are exempt from the NIH Guidelines (see Section III-E).
Experiments involving the formation of recombinant DNA for certain
genes coding for molecules toxic for vertebrates require NIH/ORDA
approval (see Section III-B-1) or shall be conducted under NIH
specified conditions as described in Appendix F.
Section III-C-2-b. Containment conditions for experiments in which
DNA from Class 5 agents is transferred into nonpathogenic prokaryotes
or lower eukaryotes shall be determined by NIH/ORDA following a case-
by-case review. A U.S. Department of Agriculture permit is required for
work with Class 5 agents (see Sections V-R and V-T).
Section III-C-3. Experiments Involving the Use of Infectious Animal or
Plant DNA or RNA Viruses or Defective Animal or Plant DNA or RNA
Viruses in the Presence of Helper Virus in Tissue Culture Systems
Caution: Special care should be used in the evaluation of
containment levels for experiments which are likely to either enhance
the pathogenicity (e.g., insertion of a host oncogene) or to extend the
host range (e.g., introduction of novel control elements) of viral
vectors under conditions that permit a productive infection. In such
cases, serious consideration should be given to increasing physical
containment by at least one level.
Note: Recombinant DNA or RNA molecules derived therefrom, which
contain less than two-thirds of the genome of any eukaryotic virus
(all viruses from a single Family (see Section V-Q) being considered
identical (see Section V-S), are considered defective and may be
used in the absence of helper under the conditions specified in
Section III-D-1.
Section III-C-3-a. Experiments involving the use of infectious or
defective Class 2 animal viruses (see Section V-A, Appendix B-II, and
Appendix B-II-E) in the presence of helper virus may be conducted at
BL2.
Section III-C-3-b. Experiments involving the use of infectious or
defective Class 3 animal viruses (see Section V-A and Appendix B-III-D)
in the presence of helper virus may be conducted at BL3.
Section III-C-3-c. Experiments involving the use of infectious or
defective Class 4 animal viruses (see Section V-A and Appendix B-IV-D)
in the presence of helper virus may be conducted at BL4.
Section III-C-3-d. Experiments involving the use of infectious or
defective Class 5 viruses (see Section V-A and Appendix B-V) in the
presence of helper virus shall be determined on a case-by-case basis
following NIH/ORDA review. A U.S. Department of Agriculture permit is
required for work with Class 5 agents (see Sections V-R and V-T).
Section III-C-3-e. Experiments involving the use of infectious or
defective animal or plant viruses in the presence of helper virus are
not covered in Sections III-C-3-a through III-C-3-d and may be
conducted at BL1.
Section III-C-4. Experiments Involving Whole Animals
This section covers experiments involving whole animals in which
the animal's genome has been altered by stable introduction of
recombinant DNA, or DNA derived therefrom, into the germ-line
(transgenic animals) and experiments involving viable recombinant DNA-
modified microorganisms tested on whole animals. For the latter, other
than viruses which are only vertically transmitted, the experiments may
not be conducted at BL1-N containment. A minimum containment of BL2 or
BL2-N is required.
Caution--Special care should be used in the evaluation of
containment conditions for some experiments with transgenic animals.
For example, such experiments might lead to the creation of novel
mechanisms or increased transmission of a recombinant pathogen or
production of undesirable traits in the host animal. In such cases,
serious consideration should be given to increasing the containment
conditions.
Section III-C-4-a. Recombinant DNA, or DNA or RNA molecules derived
therefrom, from any source except for greater than two-thirds of
eukaryotic viral genome may be transferred to any non-human vertebrate
or any invertebrate organism and propagated under conditions of
physical containment comparable to BL1 or BL1-N and appropriate to the
organism under study (see Section V-B).
Animals that contain sequences from viral vectors, which do not
lead to transmissible infection either directly or indirectly as a
result of complementation or recombination in animals, may be
propagated under conditions of physical containment comparable to BL1
or BL1-N and appropriate to the organism under study. Experiments
involving the introduction of other sequences from eukaryotic viral
genomes into animals are covered under Section III-C-4-b. For
experiments involving recombinant DNA-modified Class 2, 3, 4, or 5
organisms, see Section V-A. It is important that the investigator
demonstrate that the fraction of the viral genome being utilized does
not lead to productive infection. A U.S. Department of Agriculture
permit is required for work with Class 5 agents (see Section V-R and V-
T).
Section III-C-4-b. For experiments involving recombinant DNA, or
DNA or RNA derived therefrom, involving whole animals, including
transgenic animals, and not covered by Sections III-C-1 or III-C-4-a,
the appropriate containment shall be determined by the Institutional
Biosafety Committee.
Section III-C-5. Experiments Involving Whole Plants
Experiments to genetically engineer plants by recombinant DNA
methods, to use such plants for other experimental purposes (e.g.,
response to stress), to propagate such plants, or to use plants
together with microorganisms or insects containing recombinant DNA, may
be conducted under the containment conditions described in Sections
III-C-5-a through III-C-5-e. If experiments involving whole plants are
not described in Section III-C-5 and do not fall under Sections III-A,
III-B, or III-E, they are included in Section III-D.
Note. For recombinant DNA experiments falling under Sections
III-C-5-a through III-C-5-d, physical containment requirements may
be reduced to the next lower level by appropriate biological
containment practices, such as conducting experiments on a virus
with an obligate insect vector in the absence of that vector or
using a genetically attenuated strain.
Section III-C-5-a. BL3-P (Plants) or BL2-P + biological containment
is recommended for experiments involving most exotic (see Section V-W)
infectious agents with recognized potential for serious detrimental
impact on managed or natural ecosystems when recombinant DNA techniques
are associated with whole plants.
Section III-C-5-b. BL3-P or BL2-P + biological containment is
recommended for experiments involving plants containing cloned genomes
of readily transmissible exotic (see Section V-W) infectious agents
with recognized potential for serious detrimental effects on managed or
natural ecosystems in which there exists the possibility of
reconstituting the complete and functional genome of the infectious
agent by genomic complementation in planta.
Section III-C-5-c. BL4-P containment is recommended for experiments
with a small number of readily transmissible exotic (see Section V-W)
infectious agents, such as the soybean rust fungus (Phakospora
pachyrhizi) and maize streak or other viruses in the presence of their
specific arthropod vectors, that have the potential of being serious
pathogens of major U.S. crops.
Section III-C-5-d. BL3-P containment is recommended for experiments
involving sequences encoding potent vertebrate toxins introduced into
plants or associated organisms. Recombinant DNA containing genes for
the biosynthesis of toxin molecules lethal for vertebrates at an
LD50 of <100 nanograms="" per="" kilogram="" body="" weight="" fall="" under="" section="" iii-b-1="" and="" require="" nih/orda="" and="" institutional="" biosafety="" committee="" approval="" before="" initiation.="" section="" iii-c-5-e.="" bl3-p="" or="" bl2-p="" +="" biological="" containment="" is="" recommended="" for="" experiments="" with="" microbial="" pathogens="" of="" insects="" or="" small="" animals="" associated="" with="" plants="" if="" the="" recombinant="" dna-modified="" organism="" has="" a="" recognized="" potential="" for="" serious="" detrimental="" impact="" on="" managed="" or="" natural="" ecosystems.="" section="" iii-c-6.="" experiments="" involving="" more="" than="" 10="" liters="" of="" culture="" the="" appropriate="" containment="" will="" be="" decided="" by="" the="" institutional="" biosafety="" committee.="" where="" appropriate,="" appendix="" k,="" physical="" containment="" for="" large="" scale="" uses="" of="" organisms="" containing="" recombinant="" dna="" molecules,="" shall="" be="" used.="" appendix="" k="" describes="" containment="" conditions="" good="" large="" scale="" practice="" through="" bl3-large="" scale.="" section="" iii-c-7.="" human="" gene="" transfer="" experiments="" not="" covered="" by="" sections="" iii-a-2,="" iii-b-2,="" iii-b-3,="" and="" not="" considered="" exempt="" under="" section="" v-u="" certain="" experiments="" involving="" the="" transfer="" of="" recombinant="" dna="" or="" dna="" or="" rna="" derived="" from="" recombinant="" dna="" into="" one="" or="" more="" human="" subjects="" that="" are="" not="" covered="" by="" sections="" iii-a-2,="" iii-b-2,="" iii-b-3,="" and="" that="" are="" not="" considered="" exempt="" under="" section="" v-u="" must="" be="" registered="" with="" nih/orda.="" the="" relevant="" institutional="" biosafety="" committee="" and="" institutional="" review="" board="" must="" review="" and="" approve="" all="" experiments="" in="" this="" category="" prior="" to="" their="" initiation.="" section="" iii-d.="" experiments="" that="" require="" institutional="" biosafety="" committee="" notice="" simultaneous="" with="" initiation="" experiments="" not="" included="" in="" sections="" iii-a,="" iii-b,="" iii-c,="" iii-e,="" and="" their="" subsections="" are="" considered="" in="" section="" iii-d.="" all="" such="" experiments="" may="" be="" conducted="" at="" bl1="" containment.="" for="" experiments="" in="" this="" category,="" a="" registration="" document="" (see="" section="" iii-c)="" shall="" be="" dated="" and="" signed="" by="" the="" investigator="" and="" filed="" with="" the="" local="" institutional="" biosafety="" committee="" at="" the="" time="" the="" experiment="" is="" initiated.="" the="" institutional="" biosafety="" committee="" reviews="" and="" approves="" all="" such="" proposals,="" but="" institutional="" biosafety="" committee="" review="" and="" approval="" prior="" to="" initiation="" of="" the="" experiment="" is="" not="" required="" (see="" section="" iv-a).="" for="" example,="" experiments="" in="" which="" all="" components="" derived="" from="" non-pathogenic="" prokaryotes="" and="" non-pathogenic="" lower="" eukaryotes="" fall="" under="" section="" iii-d="" and="" may="" be="" conducted="" at="" bl1="" containment.="" section="" iii-d-1.="" experiments="" involving="" the="" formation="" of="" recombinant="" dna="" molecules="" containing="" no="" more="" than="" two-thirds="" of="" the="" genome="" of="" any="" eukaryotic="" virus="" recombinant="" dna="" molecules="" containing="" no="" more="" than="" two-thirds="" of="" the="" genome="" of="" any="" eukaryotic="" virus="" (all="" viruses="" from="" a="" single="" family="" (see="" section="" v-q)="" being="" considered="" identical="" (see="" section="" v-s))="" may="" be="" propagated="" and="" maintained="" in="" cells="" in="" tissue="" culture="" using="" bl1="" containment.="" for="" such="" experiments,="" it="" must="" be="" demonstrated="" that="" the="" cells="" lack="" helper="" virus="" for="" the="" specific="" families="" of="" defective="" viruses="" being="" used.="" if="" helper="" virus="" is="" present,="" procedures="" specified="" under="" section="" iii-c-3="" should="" be="" used.="" the="" dna="" may="" contain="" fragments="" of="" the="" genome="" of="" viruses="" from="" more="" than="" one="" family="" but="" each="" fragment="" shall="" be="" less="" than="" two-thirds="" of="" a="" genome.="" section="" iii-d-2.="" experiments="" involving="" whole="" plants="" this="" section="" covers="" experiments="" involving="" recombinant="" dna-modified="" whole="" plants,="" and/or="" experiments="" involving="" recombinant="" dna-modified="" organisms="" associated="" with="" whole="" plants,="" except="" those="" that="" fall="" under="" section="" iii-a,="" iii-b,="" iii-c,="" or="" iii-e.="" it="" should="" be="" emphasized="" that="" knowledge="" of="" the="" organisms="" and="" judgment="" based="" on="" accepted="" scientific="" practices="" should="" be="" used="" in="" all="" cases="" in="" selecting="" the="" appropriate="" level="" of="" containment.="" for="" example,="" if="" the="" genetic="" modification="" has="" the="" objective="" of="" increasing="" pathogenicity="" or="" converting="" a="" non-pathogenic="" organism="" into="" a="" pathogen,="" then="" a="" higher="" level="" of="" containment="" may="" be="" appropriate="" depending="" on="" the="" organism,="" its="" mode="" of="" dissemination,="" and="" its="" target="" organisms.="" by="" contrast,="" a="" lower="" level="" of="" containment="" may="" be="" appropriate="" for="" small="" animals="" associated="" with="" many="" types="" of="" recombinant="" dna-modified="" plants.="" section="" iii-d-2-a.="" bl1-p="" is="" recommended="" for="" all="" experiments="" with="" recombinant="" dna-containing="" plants="" and="" plant-associated="" microorganisms="" not="" covered="" in="" section="" iii-d-2-b="" or="" other="" sections="" of="" the="" nih="" guidelines.="" examples="" of="" such="" experiments="" are="" those="" involving="" recombinant="" dna-modified="" plants="" that="" are="" not="" noxious="" weeds="" or="" that="" cannot="" interbreed="" with="" noxious="" weeds="" in="" the="" immediate="" geographic="" area,="" and="" experiments="" involving="" whole="" plants="" and="" recombinant="" dna-modified="" non-exotic="" (see="" section="" v-w)="" microorganisms="" that="" have="" no="" recognized="" potential="" for="" rapid="" and="" widespread="" dissemination="" or="" for="" serious="" detrimental="" impact="" on="" managed="" or="" natural="" ecosystems="" (e.g.,="" rhizobium="" spp.="" and="" agrobacterium="" spp.).="" section="" iii-d-2-b.="" bl2-p="" or="" bl1-p="" +="" biological="" containment="" is="" recommended="" for="" the="" following="" experiments:="" section="" iii-d-2-b-(1).="" plants="" modified="" by="" recombinant="" dna="" that="" are="" noxious="" weeds="" or="" can="" interbreed="" with="" noxious="" weeds="" in="" the="" immediate="" geographic="" area.="" section="" iii-d-2-b-(2).="" plants="" in="" which="" the="" introduced="" dna="" represents="" the="" complete="" genome="" of="" a="" non-exotic="" infectious="" agent="" (see="" section="" v-w).="" section="" iii-d-2-b-(3).="" plants="" associated="" with="" recombinant="" dna-="" modified="" non-exotic="" microorganisms="" that="" have="" a="" recognized="" potential="" for="" serious="" detrimental="" impact="" on="" managed="" or="" natural="" ecosystems="" (see="" section="" v-w).="" section="" iii-d-2-b-(4).="" plants="" associated="" with="" recombinant="" dna-="" modified="" exotic="" microorganisms="" that="" have="" no="" recognized="" potential="" for="" serious="" natural="" ecosystems="" (see="" section="" v-w).="" section="" iii-d-2-b-(5).="" experiments="" with="" recombinant="" dna-modified="" arthropods="" or="" small="" animals="" associated="" with="" plants,="" or="" with="" arthropods="" or="" small="" animals="" with="" recombinant="" dna-modified="" microorganisms="" associated="" with="" them="" if="" the="" recombinant="" dna-modified="" microorganisms="" have="" no="" recognized="" potential="" for="" serious="" detrimental="" impact="" on="" managed="" or="" natural="" ecosystems="" (see="" section="" v-w).="" section="" iii-e.="" exempt="" experiments="" the="" following="" recombinant="" dna="" molecules="" are="" exempt="" from="" the="" nih="" guidelines="" and="" registration="" with="" the="" institutional="" biosafety="" committee="" is="" not="" required:="" section="" iii-e-1.="" those="" that="" are="" not="" in="" organisms="" or="" viruses.="" section="" iii-e-2.="" those="" that="" consist="" entirely="" of="" dna="" segments="" from="" a="" single="" nonchromosomal="" or="" viral="" dna="" source,="" though="" one="" or="" more="" of="" the="" segments="" may="" be="" a="" synthetic="" equivalent.="" section="" iii-e-3.="" those="" that="" consist="" entirely="" of="" dna="" from="" a="" prokaryotic="" host="" including="" its="" indigenous="" plasmids="" or="" viruses="" when="" propagated="" only="" in="" that="" host="" (or="" a="" closely="" related="" strain="" of="" the="" same="" species),="" or="" when="" transferred="" to="" another="" host="" by="" well="" established="" physiological="" means.="" section="" iii-e-4.="" those="" that="" consist="" entirely="" of="" dna="" from="" an="" eukaryotic="" host="" including="" its="" chloroplasts,="" mitochondria,="" or="" plasmids="" (but="" excluding="" viruses)="" when="" propagated="" only="" in="" that="" host="" (or="" a="" closely="" related="" strain="" of="" the="" same="" species).="" section="" iii-e-5.="" those="" that="" consist="" entirely="" of="" dna="" segments="" from="" different="" species="" that="" exchange="" dna="" by="" known="" physiological="" processes,="" though="" one="" or="" more="" of="" the="" segments="" may="" be="" a="" synthetic="" equivalent.="" a="" list="" of="" such="" exchangers="" will="" be="" prepared="" and="" periodically="" revised="" by="" the="" nih="" director="" with="" advice="" of="" the="" rac="" after="" appropriate="" notice="" and="" opportunity="" for="" public="" comment="" (see="" section="" iv-c-1-b-(1)-(c)).="" see="" appendices="" a-i="" through="" a-vi="" for="" a="" list="" of="" natural="" exchangers="" that="" are="" exempt="" from="" the="" nih="" guidelines.="" section="" iii-e-6.="" those="" that="" do="" not="" present="" a="" significant="" risk="" to="" health="" or="" the="" environment="" (see="" section="" iv-c-1-b-(1)-(c)),="" as="" determined="" by="" the="" nih="" director,="" with="" the="" advice="" of="" the="" rac,="" and="" following="" appropriate="" notice="" and="" opportunity="" for="" public="" comment.="" see="" appendix="" c="" for="" other="" classes="" of="" experiments="" which="" are="" exempt="" from="" the="" nih="" guidelines.''="" d.="" amendment="" to="" section="" iv,="" roles="" and="" responsibilities="" of="" the="" nih="" guidelines="" the="" amended="" version="" of="" section="" iv-c-1-b="" reads="" as="" follows:="" section="" iv-c-1-b.="" specific="" responsibilities="" [nih="" director]="" in="" carrying="" out="" the="" responsibilities="" set="" forth="" in="" this="" section,="" the="" nih="" director,="" or="" a="" designee="" shall="" weigh="" each="" proposed="" action="" through="" appropriate="" analysis="" and="" consultation="" to="" determine="" whether="" it="" complies="" with="" the="" nih="" guidelines="" and="" presents="" no="" significant="" risk="" to="" health="" or="" the="" environment.="" section="" iv-c-1-b-(1).="" major="" actions="" to="" execute="" major="" actions,="" the="" nih="" director="" shall="" seek="" the="" advice="" of="" the="" rac="" and="" provide="" an="" opportunity="" for="" public="" and="" federal="" agency="" comment.="" specifically,="" the="" notice="" of="" meeting="" and="" proposed="" actions="" to="" the="" nih="" guidelines="" shall="" be="" published="" in="" the="" federal="" register="" at="" least="" 15="" days="" before="" the="" rac="" meeting="" (not="" applicable="" for="" expedited="" review="" single="" patient="" human="" gene="" transfer="" experiments="" considered="" under="" appendix="" m-vi).="" the="" nih="" director's="" decision,="" at="" his/her="" discretion,="" may="" be="" published="" in="" the="" federal="" register="" for="" 15="" days="" of="" comment="" before="" final="" action="" is="" taken.="" the="" nih="" director's="" final="" decision,="" along="" with="" responses="" to="" public="" comments,="" shall="" be="" published="" in="" the="" federal="" register.="" the="" rac="" and="" institutional="" biosafety="" committee="" chairs="" shall="" be="" notified="" of="" the="" following="" decisions:="" section="" iv-c-1-b-(1)-(a).="" changing="" containment="" levels="" for="" types="" of="" experiments="" that="" are="" specified="" in="" the="" nih="" guidelines="" when="" a="" major="" action="" is="" involved;="" section="" iv-c-1-b-(1)-(b).="" assigning="" containment="" levels="" for="" types="" of="" experiments="" that="" are="" not="" explicitly="" considered="" in="" the="" nih="" guidelines="" when="" a="" major="" action="" is="" involved;="" section="" iv-c-1-b-(1)-(c).="" promulgating="" and="" amending="" a="" list="" of="" classes="" of="" recombinant="" dna="" molecules="" to="" be="" exempt="" from="" the="" nih="" guidelines="" because="" they="" consist="" entirely="" of="" dna="" segments="" from="" species="" that="" exchange="" dna="" by="" known="" physiological="" processes="" or="" otherwise="" do="" not="" present="" a="" significant="" risk="" to="" health="" or="" the="" environment;="" section="" iv-c-1-b-(1)-(d).="" permitting="" experiments="" specified="" by="" section="" iii-a;="" section="" iv-c-1-b-(1)-(e).="" certifying="" new="" host-vector="" systems="" with="" the="" exception="" of="" minor="" modifications="" of="" already="" certified="" systems="" (the="" standards="" and="" procedures="" for="" certification="" are="" described="" in="" appendix="" i-="" ii).="" minor="" modifications="" constitute="" (e.g.,="" those="" of="" minimal="" or="" no="" consequence="" to="" the="" properties="" relevant="" to="" containment);="" and="" section="" iv-c-1-b-(1)-(f).="" adopting="" other="" changes="" in="" the="" nih="" guidelines.="" section="" iv-c-1-b-(2).="" minor="" actions="" nih/orda="" shall="" carry="" out="" certain="" functions="" as="" delegated="" to="" it="" by="" the="" nih="" director="" (see="" section="" iv-c-3).="" minor="" actions="" (as="" determined="" by="" nih/orda="" in="" consultation="" with="" the="" rac="" chair="" and="" one="" or="" more="" rac="" members,="" as="" necessary)="" will="" be="" transmitted="" to="" the="" rac="" and="" institutional="" biosafety="" committee="" chairs:="" section="" iv-c-1-b-(2)-(a).="" reviewing="" and="" approving="" certain="" experiments="" involving="" the="" deliberate="" transfer="" of="" recombinant="" dna="" or="" dna="" or="" rna="" derived="" from="" recombinant="" dna="" into="" one="" or="" more="" human="" subjects="" that="" qualify="" for="" the="" accelerated="" review="" process="" (see="" section="" iii-b-2);="" section="" iv-c-1-b-(2)-(b).="" reviewing="" and="" approving="" minor="" changes="" to="" human="" gene="" transfer="" protocols="" under="" section="" iii-a-2="" and="" iii-b-2;="" section="" iv-c-1-b-(2)-(c).="" changing="" containment="" levels="" for="" experiments="" that="" are="" specified="" in="" section="" iii;="" section="" iv-c-1-b-(2)-(d).="" assigning="" containment="" levels="" for="" experiments="" not="" explicitly="" considered="" in="" the="" nih="" guidelines;="" and="" section="" iv-c-1-b-(2)-(e).="" revising="" the="" classification="" of="" etiologic="" agents="" for="" the="" purpose="" of="" these="" nih="" guidelines="" (see="" section="" v-a).="" section="" iv-c-1-b-(2)-(f).="" interpreting="" the="" nih="" guidelines="" for="" experiments="" to="" which="" the="" nih="" guidelines="" do="" not="" specifically="" assign="" containment="" levels;="" section="" iv-c-1-b-(2)-(g).="" setting="" containment="" under="" sections="" iii-c-="" 1-d="" and="" iii-c-2-b;="" section="" iv-c-1-b-(2)-(h).="" approving="" minor="" modifications="" of="" already="" certified="" host-vector="" systems="" (the="" standards="" and="" procedures="" for="" such="" modifications="" are="" described="" in="" appendix="" i-ii);="" section="" iv-c-1-b-(2)-(i).="" decertifying="" already="" certified="" host-="" vector="" systems;="" section="" iv-c-1-b-(2)-(j).="" adding="" new="" entries="" to="" the="" list="" of="" molecules="" toxic="" for="" vertebrates="" (see="" appendix="" f);="" and="" section="" iv-c-1-b-(2)-(k).="" determining="" appropriate="" containment="" conditions="" for="" experiments="" according="" to="" case="" precedents="" developed="" under="" section="" iv-c-1-b-(2)-(c).="" the="" amended="" version="" of="" section="" iv-c-2="" reads="" as="" follows:="" section="" iv-c-2.="" recombinant="" dna="" advisory="" committee="" (rac).="" the="" rac="" shall="" be="" responsible="" for="" advising="" the="" director,="" nih,="" on="" the="" actions="" listed="" in="" section="" iv-c-1-b-(1).="" the="" amended="" version="" of="" section="" iv-c-3="" reads="" as="" follows:="" section="" iv-c-3.="" office="" of="" recombinant="" dna="" activities="" (orda)="" orda="" shall="" serve="" as="" a="" focal="" point="" for="" information="" on="" recombinant="" dna="" activities="" and="" provide="" advice="" to="" all="" within="" and="" outside="" nih="" including="" institutions,="" biological="" safety="" officers,="" principal="" investigators,="" federal="" agencies,="" state="" and="" local="" governments,="" and="" institutions="" in="" the="" private="" sector.="" orda="" shall="" carry="" out="" such="" other="" functions="" as="" may="" be="" delegated="" to="" it="" by="" the="" nih="" director,="" including="" those="" authorities="" described="" in="" section="" iv-c-1-b-(2).="" orda's="" responsibilities="" include,="" but="" are="" not="" limited="" to="" the="" following:="" section="" iv-c-3-a.="" reviewing="" and="" approving="" experiments="" in="" conjunction="" with="" ad="" hoc="" experts="" involving="" the="" cloning="" of="" genes="" encoding="" for="" toxin="" molecules="" that="" are="" lethal="" for="" vertebrates="" at="" an="">50
100 nanograms per kilogram body weight in organisms other
than Escherichia coli K-12 (see Section III-B-1 and Appendices F-I and
F-II);
Section IV-C-3-b. Reviewing and approving certain experiments
involving the deliberate transfer of recombinant DNA or DNA or RNA
derived from recombinant DNA into one or more human subjects, in
consultation with the RAC Chair and one or more RAC members, as
necessary, that qualify for the Accelerated Review process (see Section
III-B-2);
Section IV-C-3-c. Reviewing and approving minor changes to human
gene transfer protocols approved under Sections III-A-2 and III-B-2, in
consultation with the RAC Chair and one or more RAC members, as
necessary;
Section IV-C-3-d. Reviewing and approving the membership of an
institution's Institutional Biosafety Committee, and where it finds the
Institutional Biosafety Committee meets the requirements set forth in
Section IV-B-2 will give its approval to the Institutional Biosafety
Committee membership;
Section IV-C-3-e. Publishing in the Federal Register:
Section IV-C-3-e-(1). Announcements of RAC meetings and agendas at
least 15 days in advance (Note--If the agenda for a RAC meeting is
modified, ORDA shall make the revised agenda available to anyone upon
request at least 72 hours in advance of the meeting);
Section IV-C-3-e-(2). Proposed Major Actions to the NIH Guidelines
(see Section IV-C-1-b-(1)) at least 15 days prior to the RAC meeting;
Section IV-C-3-f. Serve as the focal point for data management of
NIH-approved human gene transfer protocols approved under Sections III-
A-2 and III-B-2 and registered with NIH/ORDA as required under Section
III-C-7;
Section IV-C-3-g. Serve as the executive secretary of the RAC; and
Section IV-C-3-h. Maintain a list of Major and Minor Actions
approved under Section III-A-2 and III-B-3 and a list of experiments
registered with NIH/ORDA as described in Section III-C-7.
E. Amendment and Addition to Section V, Footnotes and References of
Sections I-IV of the NIH Guidelines
The amended version of Section V-U reads as follows:
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.''
The following new footnote, V-W is added to Section V:
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.
F. Addition to Appendix C-I, Recombinant DNA in Tissue Culture, of the
NIH Guidelines
The amended version of Appendix C-I reads as follows:
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.
G. Addition to Appendix G, Physical Containment, of the NIH Guidelines
Appendix G through G-I is amended to read as follows:
Appendix G specifies physical containment for standard laboratory
experiments and defines Biosafety Level 1 through Biosafety Level 4.
For large scale (over 10 liters) research or production, Appendix K
supersedes Appendix G. Appendix K defines Good Large Scale Practice
through Biosafety Level 3--Large Scale. For certain work with plants,
Appendix P supersedes Appendix G. Appendix P defines Biosafety Levels 1
through 4--Plants. For certain work with animals, Appendix Q supersedes
Appendix G. Appendix Q defines Biosafety Levels 1 through 4--Animals.
Appendix G-I. Standard Practices and Training
The first principle of containment is strict adherence to good
microbiological practices (see Appendices G-III-A through G-III-J).
Consequently, all personnel directly or indirectly involved in
experiments using recombinant DNA shall receive adequate instruction
(see Sections IV-B-1-e and IV-B-4-d). At a minimum, these instructions
include training in aseptic techniques and in the biology of the
organisms used in the experiments so that the potential biohazards can
be understood and appreciated.
Any research group working with agents that are known or potential
biohazards shall have an emergency plan that describes the procedures
to be followed if an accident contaminates personnel or the
environment. The Principal Investigator shall ensure that everyone in
the laboratory is familiar with both the potential hazards of the work
and the emergency plan (see Sections IV-B-4-d and IV-B-4-e). If a
research group is working with a known pathogen for which there is an
effective vaccine, the vaccine should be made available to all workers.
Serological monitoring, when clearly appropriate, will be provided (see
Section IV-B-1-f).
The Laboratory Safety Monograph (see Appendix G-III-O) and
Biosafety in Microbiological and Biomedical Laboratories (see Appendix
G-III-B) describe practices, equipment, and facilities in detail.
H. Addition of Appendix P, Physical and Biological Containment for
Recombinant DNA Research Involving Plants, to the NIH Guidelines
The following new appendix, Appendix P, reads as follows:
Appendix P specifies physical and biological containment conditions
and practices suitable to the greenhouse conduct of experiments
involving recombinant DNA-containing plants, plant-associated
microorganisms, and small animals. All provisions of the NIH Guidelines
apply to plant research activities with the following modifications:
Appendix P shall supersede Appendix G when the research plants are
of a size, number, or have growth requirements that preclude the use of
containment conditions described in Appendix G. The plants covered in
Appendix P include but are not limited to mosses, liverworts,
macroscopic algae, and vascular plants including terrestrial crops,
forest, and ornamental species.
Plant-associated microorganisms include viroids, virusoids,
viruses, bacteria, fungi, protozoans, certain small algae, and
microorganisms that have a benign or beneficial association with
plants, such as certain Rhizobium species and microorganisms known to
cause plant diseases. The appendix applies to microorganisms which are
being modified with the objective of fostering an association with
plants.
Plant-associated small animals include those arthropods that: (i)
Are in obligate association with plants, (ii) are plant pests, (iii)
are plant pollinators, or (iv) transmit plant disease agents, as well
as other small animals such as nematodes for which tests of biological
properties necessitate the use of plants. Microorganisms associated
with such small animals (e.g., pathogens or symbionts) are included.
The Institutional Biosafety Committee shall include at least one
individual with expertise in plant, plant pathogen, or plant pest
containment principles when experiments utilizing Appendix P require
prior approval by the Institutional Biosafety Committee.
Appendix P-I. General Plant Biosafety Levels
Appendix P-I-A. The principal purpose of plant containment is to
avoid the unintentional transmission of a recombinant DNA-containing
plant genome, including nuclear or organelle hereditary material or
release of recombinant DNA-derived organisms associated with plants.
Appendix P-I-B. The containment principles are based on the
recognition that the organisms that are used pose no health threat to
humans or higher animals (unless deliberately modified for that
purpose), and that the containment conditions minimize the possibility
of an unanticipated deleterious effect on organisms and ecosystems
outside of the experimental facility, e.g., the inadvertent spread of a
serious pathogen from a greenhouse to a local agricultural crop or the
unintentional introduction and establishment of an organism in a new
ecosystem.
Appendix P-I-C. Four biosafety levels, referred to as Biosafety
Level (BL) 1--Plants (P), BL2-P, BL3-P, and BL4-P, are established in
Section II. The selection of containment levels required for research
involving recombinant DNA molecules in plants or associated with plants
is specified in Section III. These biosafety levels are described in
Appendix P-II. This appendix describes greenhouse practices and special
greenhouse facilities for physical containment.
Appendix P-I-D. BL1-P through BL4-P are designed to provide
differential levels of biosafety for plants in the absence or presence
of other experimental organisms that contain recombinant DNA. These
biosafety levels, in conjunction with biological containment conditions
described in Appendix P-III, provide flexible approaches to ensure the
safe conduct of research.
Appendix P-I-E. For experiments in which plants are grown at the
BL1 through BL4 laboratory settings, containment practices shall be
followed as described in Appendix G. These containment practices
include the use of plant tissue culture rooms, growth chambers within
laboratory facilities, or experiments performed on open benches.
Additional biological containment practices should be added by the
Greenhouse Director or Institutional Biosafety Committee as necessary
(see Appendix P-III), if botanical reproductive structures are produced
that have the potential of being released.
Appendix P-II. Physical Containment Levels
Appendix P-II-A. Biosafety Level 1--Plants (BL1-P)
Appendix P-II-A-1. Standard Practices (BL1-P)
Appendix P-II-A-1-a. Greenhouse Access (BL1-P)
Appendix P-II-A-1-a-(1). Access to the greenhouse shall be limited
or restricted, at the discretion of the Greenhouse Director, when
experiments are in progress.
Appendix P-II-A-1-a-(2). Prior to entering the greenhouse,
personnel shall be required to read and follow instructions on BL1-P
greenhouse practices and procedures. All procedures shall be performed
in accordance with accepted greenhouse practices that are appropriate
to the experimental organism.
Appendix P-II-A-1-b. Records (BL1-P)
Appendix P-II-A-1-b-(1). A record shall be kept of experiments
currently in progress in the greenhouse facility.
Appendix P-II-A-1-c. Decontamination and Inactivation (BL1-P)
Appendix P-II-A-1-c-(1). Experimental organisms shall be rendered
biologically inactive by appropriate methods before disposal outside of
the greenhouse facility.
Appendix P-II-A-1-d. Control of Undesired Species and Motile
Macroorganisms (BL1-P)
Appendix P-II-A-1-d-(1). A program shall be implemented to control
undesired species (e.g., weed, rodent, or arthropod pests and
pathogens), by methods appropriate to the organisms and in accordance
with applicable state and Federal laws.
Appendix P-II-A-1-d-(2). Arthropods and other motile macroorganisms
shall be housed in appropriate cages. If macroorganisms (e.g., flying
arthropods or nematodes) are released within the greenhouse,
precautions shall be taken to minimize escape from the greenhouse
facility.
Appendix P-II-A-1-e. Concurrent Experiments Conducted in the Greenhouse
(BL1-P)
Appendix P-II-A-1-e-(1). Experiments involving other organisms that
require a containment level lower than BL1-P may be conducted in the
greenhouse concurrently with experiments that require BL1-P
containment, provided that all work is conducted in accordance with
BL1-P greenhouse practices.
Appendix P-II-A-2. Facilities (BL1-P)
Appendix P-II-A-2-a. Definitions (BL1-P)
Appendix P-II-A-2-a-(1). The term `greenhouse' refers to a
structure with walls, a roof, and a floor designed and used principally
for growing plants in a controlled and protected environment. The walls
and roof are usually constructed of transparent or translucent material
to allow passage of sunlight for plant growth.
Appendix P-II-A-2-a-(2). The term `greenhouse facility' includes
the actual greenhouse rooms or compartments for growing plants,
including all immediately contiguous hallways and head-house areas, and
is considered part of the confinement area.
Appendix P-II-A-2-b. Greenhouse Design (BL1-P)
Appendix P-II-A-2-b-(1). The greenhouse floor may be composed of
gravel or other porous material. At a minimum, impervious (e.g.,
concrete) walkways are recommended.
Appendix P-II-A-2-b-(2). Windows and other openings in the walls
and roof of the greenhouse facility may be open for ventilation as
needed for proper operation and do not require any special barrier to
contain or exclude pollen, microorganisms, or small flying animals
(e.g., arthropods and birds); however, screens are recommended.
Appendix P-II-B. Biosafety Level 2--Plants (BL2-P)
Appendix P-II-B-1. Standard Practices (BL2-P)
Appendix P-II-B-1-a. Greenhouse Access (BL2-P)
Appendix P-II-B-1-a-(1). Access to the greenhouse shall be limited
or restricted, at the discretion of the Greenhouse Director, to
individuals directly involved with the experiments when they are in
progress.
Appendix P-II-B-1-a-(2). Personnel shall be required to read and
follow instructions on BL2-P practices and procedures. All procedures
shall be conducted in accordance with accepted greenhouse practices
that are appropriate to the experimental organisms.
Appendix P-II-B-1-b. Records (BL2-P)
Appendix P-II-B-1-b-(1). A record shall be kept of experimental
plants, microorganisms, or small animals that are brought into or
removed from the greenhouse facility.
Appendix P-II-B-1-b-(2). A record shall be kept of experiments
currently in progress in the greenhouse facility.
Appendix P-II-B-1-b-(3). The Principal Investigator shall report
any greenhouse accident involving the inadvertent release or spill of
microorganisms to the Greenhouse Director, Institutional Biosafety
Committee, NIH/ORDA and other appropriate authorities immediately (if
applicable). Reports to the NIH/ORDA shall be sent to the Office of
Recombinant DNA Activities, National Institutes of Health, Building 31,
Room 4B11, Bethesda, Maryland 20892, (301) 496-9838. Documentation of
any such accident shall be prepared and maintained.
Appendix P-II-B-1-c. Decontamination and Inactivation (BL2-P)
Appendix P-II-B-1-c-(1). Experimental organisms shall be rendered
biologically inactive by appropriate methods before disposal outside of
the greenhouse facility.
Appendix P-II-B-1-c-(2). Decontamination of run-off water is not
necessarily required. If part of the greenhouse is composed of gravel
or similar material, appropriate treatments should be made periodically
to eliminate, or render inactive, any organisms potentially entrapped
by the gravel.
Appendix P-II-B-1-d. Control of Undesired Species and Motile
Macroorganisms (BL2-P)
Appendix P-II-B-1-d-(1). A program shall be implemented to control
undesired species (e.g., weed, rodent, or arthropod pests and
pathogens) by methods appropriate to the organisms and in accordance
with applicable state and Federal laws.
Appendix P-II-B-1-d-(2). Arthropods and other motile macroorganisms
shall be housed in appropriate cages. If macroorganisms (e.g., flying
arthropods or nematodes) are released within the greenhouse,
precautions shall be taken to minimize escape from the greenhouse
facility.
Appendix P-II-B-1-e. Concurrent Experiments Conducted in the Greenhouse
(BL2-P)
Appendix P-II-B-1-e-(1). Experiments involving other organisms that
require a containment level lower than BL2-P may be conducted in the
greenhouse concurrently with experiments that require BL2-P containment
provided that all work is conducted in accordance with BL2-P greenhouse
practices.
Appendix P-II-B-1-f. Signs (BL2-P)
Appendix P-II-B-1-f-(1). A sign shall be posted indicating that a
restricted experiment is in progress. The sign shall indicate the
following: (i) the name of the responsible individual, (ii) The plants
in use, and (iii) any special requirements for using the area.
Appendix P-II-B-1-f-(2). If organisms are used that have a
recognized potential for causing serious detrimental impacts on managed
or natural ecosystems, their presence shall be indicated on a sign
posted on the greenhouse access doors.
Appendix P-II-B-1-f-(3). If there is a risk to human health, a sign
shall be posted incorporating the universal biosafety symbol.
Appendix P-II-B-1-g. Transfer of Materials (BL2-P)
Appendix P-II-B-1-g-(1). Materials containing experimental
microorganisms, which are brought into or removed from the greenhouse
facility in a viable or intact state, shall be transferred in a closed
non-breakable container.
Appendix P-II-B-1-h. Greenhouse Practices Manual (BL2-P)
Appendix P-II-B-1-h-(1). A greenhouse practices manual shall be
prepared or adopted. This manual shall: (i) Advise personnel of the
potential consequences if such practices are not followed, and (ii)
outline contingency plans to be implemented in the event of the
unintentional release of organisms.
Appendix P-II-B-2. Facilities (BL2-P)
Appendix P-II-B-2-a. Definitions (BL2-P)
Appendix P-II-B-2-a-(1). The term `greenhouse' refers to a
structure with walls, a roof, and a floor designed and used principally
for growing plants in a controlled and protected environment. The walls
and roof are usually constructed of transparent or translucent material
to allow passage of sunlight for plant growth.
Appendix P-II-B-2-a-(2). The term `greenhouse facility' includes
the actual greenhouse rooms or compartments for growing plants,
including all immediately contiguous hallways and head-house areas and
is considered part of the confinement area.
Appendix P-II-B-2-b. Greenhouse Design (BL2-P)
Appendix P-II-B-2-b-(1). A greenhouse floor composed of an
impervious material. Concrete is recommended, but gravel or other
porous material under benches is acceptable unless propagules of
experimental organisms are readily disseminated through soil. Soil beds
are acceptable unless propagules of experimental organisms are readily
disseminated through soil.
Appendix P-II-B-2-b-(2). Windows and other openings in the walls
and roof of the greenhouse facility may be open for ventilation as
needed for proper operation and do not require any special barrier to
exclude pollen or microorganisms; however, screens are required to
exclude small flying animals (e.g., arthropods and birds).
Appendix P-II-B-2-c. Autoclaves (BL2-P)
Appendix P-II-B-2-c-(1). An autoclave shall be available for the
treatment of contaminated greenhouse materials.
Appendix P-II-B-2-d. Supply and Exhaust Air Ventilation Systems (BL2-P)
Appendix P-II-B-2-d-(1). If intake fans are used, measures shall be
taken to minimize the ingress of arthropods. Louvers or fans shall be
constructed such that they can only be opened when the fan is in
operation.
Appendix P-II-B-2-e. Other (BL2-P)
Appendix P-II-B-2-e-(1). BL2-P greenhouse containment requirements
may be satisfied by using a growth chamber or growth room within a
building provided that the external physical structure limits access
and escape of microorganisms and macroorganisms in a manner that
satisfies the intent of the foregoing clauses.
Appendix P-II-C. Biosafety Level 3--Plants (BL3-P)
Appendix P-II-C-1. Standard Practices (BL3-P)
Appendix P-II-C-1-a. Greenhouse Access (BL3-P)
Appendix P-II-C-1-a-(1). Authorized entry into the greenhouse shall
be restricted to individuals who are required for program or support
purposes. The Greenhouse Director shall be responsible for assessing
each circumstance and determining those individuals who are authorized
to enter the greenhouse facility.
Appendix P-II-C-1-a-(2). Prior to entering the greenhouse,
personnel shall be required to read and follow instructions on BL3-P
practices and procedures. All procedures shall be conducted in
accordance with accepted greenhouse practices that are appropriate to
the experimental organisms.
Appendix P-II-C-1-b. Records (BL3-P)
Appendix P-II-C-1-b-(1). A record shall be kept of experimental
plants, microorganisms, or small animals that are brought into or
removed from the greenhouse facility.
Appendix P-II-C-1-b-(2). A record shall be kept of experiments
currently in progress in the greenhouse facility.
Appendix P-II-C-1-b-(3). The Principal Investigator shall report
any greenhouse accident involving the inadvertent release or spill of
microorganisms to the Biological Safety Officer, Greenhouse Director,
Institutional Biosafety Committee, NIH/ORDA, and other appropriate
authorities immediately (if applicable).
Reports to the NIH/ORDA shall be sent to the Office of Recombinant
DNA Activities, National Institutes of Health, Building 31, Room 4B11,
Bethesda, Maryland 20892, (301) 496-9838. Documentation of any such
accident shall be prepared and maintained.
Appendix P-II-C-1-c. Decontamination and Inactivation (BL3-P)
Appendix P-II-C-1-c-(1). All experimental materials shall be
sterilized in an autoclave or rendered biologically inactive by
appropriate methods before disposal, except those that are to remain in
a viable or intact state for experimental purposes; including water
that comes in contact with experimental microorganisms or with material
exposed to such microorganisms, and contaminated equipment and
supplies.
Appendix P-II-C-1-d. Control of Undesired Species and Motile
Macroorganisms (BL3-P)
Appendix P-II-C-1-d-(1). A program shall be implemented to control
undesired species (e.g., weed, rodent, or arthropod pests and
pathogens) by methods appropriate to the organisms and in accordance
with applicable state and Federal laws.
Appendix P-II-C-1-d-(2). Arthropods and other motile macroorganisms
shall be housed in appropriate cages. When appropriate to the organism,
experiments shall be conducted within cages designed to contain the
motile organisms.
Appendix P-II-C-1-e. Concurrent Experiments Conducted in the Greenhouse
(BL3-P)
Appendix P-II-C-1-e-(1). Experiments involving organisms that
require a containment level lower than BL3-P may be conducted in the
greenhouse concurrently with experiments that require BL3-P containment
provided that all work is conducted in accordance with BL3-P greenhouse
practices.
Appendix P-II-C-1-f. Signs (BL3-P)
Appendix P-II-C-1-f-(1). A sign shall be posted indicating that a
restricted experiment is in progress. The sign shall indicate the
following: (i) The name of the responsible individual, (ii) the plants
in use, and (iii) any special requirements for using the area.
Appendix P-II-C-1-f-(2). If organisms are used that have a
recognized potential for causing serious detrimental impacts on managed
or natural ecosystems, their presence should be indicated on a sign
posted on the greenhouse access doors.
Appendix P-II-C-1-f-(3). If there is a risk to human health, a sign
shall be posted incorporating the universal biosafety symbol.
Appendix P-II-C-1-g. Transfer of Materials (BL3-P)
Appendix P-II-C-1-g-(1). Experimental materials that are brought
into or removed from the greenhouse facility in a viable or intact
state shall be transferred to a non-breakable sealed secondary
container. At the time of transfer, if the same plant species, host, or
vector are present within the effective dissemination distance of
propagules of the experimental organism, the surface of the secondary
container shall be decontaminated. Decontamination may be accomplished
by passage through a chemical disinfectant or fumigation chamber or by
an alternative procedure that has demonstrated effective inactivation
of the experimental organism.
Appendix P-II-C-1-h. Greenhouse Practices Manual (BL3-P)
Appendix P-II-C-1-h-(1). A greenhouse practices manual shall be
prepared or adopted. This manual shall: (i) Advise personnel of the
potential consequences if such practices are not followed, and (ii)
outline contingency plans to be implemented in the event of the
unintentional release of organisms with recognized potential for
serious detrimental impact.
Appendix P-II-C-1-i. Protective Clothing (BL3-P)
Appendix P-II-C-1-i-(1). Disposable clothing (e.g., solid front or
wrap-around gowns, scrub suits, or other appropriate clothing) shall be
worn in the greenhouse if deemed necessary by the Greenhouse Director
because of potential dissemination of the experimental microorganisms.
Appendix P-II-C-1-i-(2). Protective clothing shall be removed
before exiting the greenhouse and decontaminated prior to laundering or
disposal.
Appendix P-II-C-1-j. Other (BL3-P)
Appendix P-II-C-1-j-(1). Personnel are required to thoroughly wash
their hands upon exiting the greenhouse.
Appendix P-II-C-1-j-(2). All procedures shall be performed
carefully to minimize the creation of aerosols and excessive splashing
of potting material/soil during watering, transplanting, and all
experimental manipulations.
Appendix P-II-C-2. Facilities (BL3-P)
Appendix P-II-C-2-a. Definitions (BL3-P)
Appendix P-II-C-2-a-(1). The term 'greenhouse' refers to a
structure with walls, roof, and floor designed and used principally for
growing plants in a controlled and protected environment. The walls and
roof are usually constructed of transparent or translucent material to
allow passage of sunlight for plant growth.
Appendix P-II-C-2-a-(2). The term 'greenhouse facility' includes
the actual greenhouse rooms or compartments for growing plants,
including all immediately contiguous hallways and head-house areas, and
is considered part of the confinement area. The need to maintain
negative pressure should be considered when constructing or renovating
the greenhouse.
Appendix P-II-C-2-b. Greenhouse Design (BL3-P)
Appendix P-II-C-2-b-(1). The greenhouse floor shall be composed of
concrete or other impervious material with provision for collection and
decontamination of liquid run-off.
Appendix P-II-C-2-b-(2). Windows shall be closed and sealed. All
glazing shall be resistant to breakage (e.g., double-pane tempered
glass or equivalent).
Appendix P-II-C-2-b-(3). The greenhouse shall be a closed self-
contained structure with a continuous covering that is separated from
areas that are open to unrestricted traffic flow. The minimum
requirement for greenhouse entry shall be passage through two sets of
self-closing locking doors.
Appendix P-II-C-2-b-(4). The greenhouse facility shall be
surrounded by a security fence or protected by equivalent security
measures.
Appendix P-II-C-2-b-(5). Internal walls, ceilings, and floors shall
be resistant to penetration by liquids and chemicals to facilitate
cleaning and decontamination of the area. All penetrations into these
structures and surfaces (e.g., plumbing and utilities) shall be sealed.
Appendix P-II-C-2-b-(6). Bench tops and other work surfaces should
have seamless surfaces that are impervious to water and resistant to
acids, alkalis, organic solvents, and moderate heat.
Appendix P-II-C-2-b-(7). The greenhouse contains a foot, elbow, or
automatically operated sink, which is located near the exit door for
hand washing.
Appendix P-II-C-2-c. Autoclaves (BL3-P)
Appendix P-II-C-2-c-(1). An autoclave shall be available for
decontaminating materials within the greenhouse facility. A double-door
autoclave is recommended (not required) for the decontamination of
materials passing out of the greenhouse facility.
Appendix P-II-C-2-d. Supply and Exhaust Air Ventilation Systems (BL3-P)
Appendix P-II-C-2-d-(1). An individual supply and exhaust air
ventilation system shall be provided. The system maintains pressure
differentials and directional airflow, as required, to assure inward
(or zero) airflow from areas outside of the greenhouse.
Appendix P-II-C-2-d-(2). The exhaust air from the greenhouse
facility shall be filtered through high efficiency particulate air-HEPA
filters and discharged to the outside. The filter chambers shall be
designed to allow in situ decontamination before filters are removed
and to facilitate certification testing after they are replaced. Air
filters shall be 80-85% average efficiency by the American Society of
Heating, Refrigerating, and Air Conditioning Engineers (ASHRAE)
Standard 52- 68 test method using atmosphere dust. Air supply fans
shall be equipped with a back-flow damper that closes when the air
supply fan is off. Alternatively, a HEPA filter may be used on the air
supply system instead of the filters and damper. The supply and exhaust
airflow shall be interlocked to assure inward (or zero) airflow at all
times.
Appendix P-II-C-2-e. Other (BL3-P)
Appendix P-II-C-2-e-(1). BL3-P greenhouse containment requirements
may be satisfied using a growth chamber or growth room within a
building provided that the location, access, airflow patterns, and
provisions for decontamination of experimental materials and supplies
meet the intent of the foregoing clauses.
Appendix P-II-C-2-e-(2). Vacuum lines shall be protected with high
efficiency particulate air/HEPA or equivalent filters and liquid
disinfectant traps.
Appendix P-II-D. Biosafety Level 4--Plants (BL4-P)
Appendix P-II-D-1. Standard Practices (BL4-P)
Appendix P-II-D-1-a. Greenhouse Access (BL4-P)
Appendix P-II-D-1-a-(1). Authorized entry into the greenhouse shall
be restricted to individuals who are required for program or support
purposes. The Greenhouse Director shall be responsible for assessing
each circumstance and determining those individuals who are authorized
to enter the greenhouse facility or work in the greenhouse during
experiments.
Appendix P-II-D-1-a-(2). Access shall be managed by the Greenhouse
Director, Biological Safety Officer, or other individual responsible
for physical security of the greenhouse facility; and access limited by
means of secure, locked doors.
Appendix P-II-D-1-a-(3). Prior to entering, individuals shall be
advised of the potential environmental hazards and instructed on
appropriate safeguards for ensuring environmental safety. Individuals
authorized to enter the greenhouse facility shall comply with the
instructions and all other applicable entry/exit procedures.
Appendix P-II-D-1-a-(4). Personnel shall enter and exit the
greenhouse facility only through the clothing change and shower rooms
and shall shower each time they exit the greenhouse facility. Personnel
shall use the airlocks to enter or exit the laboratory only in an
emergency. In the event of an emergency, every reasonable effort should
be made to prevent the possible transport of viable propagules from
containment.
Appendix P-II-D-1-a-(5). Prior to entering the greenhouse,
personnel shall be required to read and follow instructions on BL4-P
practices and procedures.
Appendix P-II-D-1-b. Records (BL4-P)
Appendix P-II-D-1-b-(1). A record shall be kept of all experimental
materials brought into or removed from the greenhouse.
Appendix P-II-D-1-b-(2). A record shall be kept of experiments
currently in progress in the greenhouse facility.
Appendix P-II-D-1-b-(3). A record shall be kept of all personnel
entering and exiting the greenhouse facility, including the date and
time of each entry.
Appendix P-II-D-1-b-(4). The Principal Investigator shall report
any greenhouse accident involving the inadvertent release or spill of
microorganisms to the Biological Safety Officer, Greenhouse Director,
Institutional Biosafety Committee, NIH/ORDA, and other appropriate
authorities immediately (if applicable). Reports to the NIH/ORDA shall
be sent to the Office of Recombinant DNA Activities, National
Institutes of Health, Building 31, Room 4B11, Bethesda, Maryland 20892,
(301) 496-9838. Documentation of any such accident shall be prepared
and maintained.
Appendix P-II-D-1-c. Decontamination and Inactivation (BL4-P)
Appendix P-II-D-1-c-(1). All materials, except for those that are
to remain in a viable or intact state for experimental purposes, shall
be autoclaved prior to removal from the maximum containment greenhouse.
Equipment or material that could be damaged by high temperatures or
steam shall be decontaminated by alternative methods (e.g., gas or
vapor sterilization) in an airlock or chamber designed for this
purpose.
Appendix P-II-D-1-c-(2). Water that comes in contact with
experimental microorganisms or with material exposed to such
microorganisms (e.g., run-off from watering plants) shall be collected
and decontaminated before disposal.
Appendix P-II-D-1-c-(3). Standard microbiological procedures shall
be followed for decontamination of equipment and materials. Spray or
liquid waste or rinse water from containers used to apply the
experimental microorganisms shall be decontaminated before disposal.
Appendix P-II-D-1-d. Control of Undesired Species and Motile
Macroorganisms (BL4-P)
Appendix P-II-D-1-d-(1). A chemical control program shall be
implemented to eliminate undesired pests and pathogens in accordance
with applicable state and Federal laws.
Appendix P-II-D-1-d-(2). Arthropods and other motile macroorganisms
used in conjunction with experiments requiring BL4-P level physical
containment shall be housed in appropriate cages. When appropriate to
the organism, experiments shall be conducted within cages designed to
contain the motile organisms.
Appendix P-II-D-1-e. Concurrent Experiments Conducted in the Greenhouse
(BL4-P)
Appendix P-II-D-1-e-(1). Experiments involving organisms that
require a containment level lower than BL4-P may be conducted in the
greenhouse concurrently with experiments that require BL4-P containment
provided that all work is conducted in accordance with BL4-P greenhouse
practices. When the experimental microorganisms in use require a
containment level lower than BL4-P, greenhouse practices reflect the
level of containment required by the highest containment level
microorganisms being tested.
Appendix P-II-D-1-f. Signs (BL4-P)
Appendix P-II-D-1-f-(1). A sign shall be posted indicating that a
restricted experiment is in progress. The sign shall indicate the
following: (i) The name of the responsible individual, (ii) the plants
in use, and (iii) any special requirements for using the area.
Appendix P-II-D-1-f-(2). If organisms are used that have a
recognized potential for causing serious detrimental impacts on managed
or natural ecosystems, their presence shall be indicated by a sign
posted on the greenhouse access doors.
Appendix P-II-D-1-f-(3). If there is a risk to human health, a sign
shall be posted incorporating the universal biosafety symbol.
Appendix P-II-D-1-g. Transfer of Materials (BL4-P)
Appendix P-II-D-1-g-(1). Experimental materials that are brought
into or removed from the greenhouse in a viable or intact state shall
be transferred to a non-breakable, sealed, primary container then
enclosed in a non-breakable, sealed secondary container. These
containers shall be removed from the greenhouse facility through a
chemical disinfectant, fumigation chamber, or an airlock designed for
this purpose.
Appendix P-II-D-g-(2). Supplies and materials shall be brought into
the greenhouse facility through a double-door autoclave, fumigation
chamber, or airlock that is appropriately decontaminated between each
use. After securing the outer doors, personnel within the greenhouse
facility shall retrieve the materials by opening the interior door of
the autoclave, fumigation chamber, or airlock. These doors shall be
secured after the materials are brought into the greenhouse facility.
Appendix P-II-D-1-h. Greenhouse Practices Manual (BL4-P)
Appendix P-II-D-1-h-(1). A greenhouse practices manual shall be
prepared or adopted. This manual shall include contingency plans to be
implemented in the event of the unintentional release of experimental
organisms.
Appendix P-II-D-1-i. Protective Clothing (BL4-P)
Appendix P-II-D-1-i-(1). Street clothing shall be removed in the
outer clothing change room. Complete laboratory clothing (may be
disposable) including undergarments, pants, and shirts, jump suits,
shoes, and hats shall be provided and worn by all personnel entering
the greenhouse facility.
Appendix P-II-D-1-i-(2). Personnel shall remove laboratory clothing
when exiting the greenhouse facility and before entering the shower
area. This clothing shall be stored in a locker or hamper in the inner
change room.
Appendix P-II-D-1-i-(3). All laboratory clothing shall be
autoclaved before laundering.
Appendix P-II-D-2. Facilities (BL4-P)
Appendix P-II-D-2-a. Greenhouse Design (BL4-P)
Appendix P-II-D-2-a-(1). The maximum containment greenhouse
facility shall consist of a separate building or a clearly demarcated
and isolated area within a building. The need to maintain negative
pressure should be considered when constructing or renovating the
greenhouse facility.
Appendix P-II-D-2-a-(2). Outer and inner change rooms, separated by
a shower, shall be provided for personnel entering and exiting the
greenhouse facility.
Appendix P-II-D-2-a-(3). Windows shall be closed and sealed. All
glazing shall be resistant to breakage (e.g., double-pane tempered
glass or equivalent).
Appendix P-II-D-2-a-(4). Access doors to the greenhouse shall be
self-closing and locking.
Appendix P-II-D-2-a-(5). The greenhouse facility shall be
surrounded by a security fence or protected by equivalent security
measures.
Appendix P-II-D-2-a-(6). The walls, floors, and ceilings of the
greenhouse shall be constructed to form a sealed internal shell that
facilitates fumigation and is animal and arthropod-proof. These
internal surfaces shall be resistant to penetration and degradation by
liquids and chemicals to facilitate cleaning and decontamination of the
area. All penetrations into these structures and surfaces (e.g.,
plumbing and utilities) shall be sealed.
Appendix P-II-D-2-a-(7). Bench tops and other work surfaces shall
have seamless surfaces impervious to water and resistant to acids,
alkalis, organic solvents, and moderate heat.
Appendix P-II-D-2-a-(8). A double-door autoclave, fumigation
chamber, or ventilated airlock shall be provided for passage of all
materials, supplies, or equipment that are not brought into the
greenhouse facility through the change room.
Appendix P-II-D-2-b. Autoclaves (BL4-P)
Appendix P-II-D-2-b-(1). A double-door autoclave shall be provided
for the decontamination of materials removed from the greenhouse
facility. The autoclave door, which opens to the area external to the
greenhouse facility, shall be sealed to the outer wall and
automatically controlled so that it can only be opened upon completion
of the sterilization cycle.
Appendix P-II-D-2-c. Supply and Exhaust Air Ventilation Systems
(BL4-P)
Appendix P-II-D-2-c-(1). An individual supply and exhaust air
ventilation system shall be provided. The system shall maintain
pressure differentials and directional airflow as required to assure
inward (or zero) airflow from areas outside of the greenhouse.
Differential pressure transducers shall be used to sense pressure
levels. If a system malfunctions, the transducers shall sound an alarm.
A backup source of power should be considered. The supply and exhaust
airflow shall be interlocked to assure inward (or zero) airflow at all
times. The integrity of the greenhouse shall have an air leak rate
(decay rate) not to exceed 7 percent per minute (logarithm of pressure
against time) over a 20-minute period at 2 inches of water gauge
pressure. Nominally, this is 0.05 inches of water gauge pressure loss
in 1 minute at 2 inches water gauge pressure.
Appendix P-II-D-2-c-(2). Exhaust air from the greenhouse facility
shall be filtered through high efficiency particulate air/HEPA filters
and discharged to the outside and dispersed away from occupied
buildings and air intakes. Filter chambers shall be designed to allow
in situ decontamination before filters are removed and to facilitate
certification testing after they are replaced. HEPA filters shall be
provided to treat air supplied to the greenhouse facility. HEPA filters
shall be certified annually.
Appendix P-II-D-2-d. Other (BL4-P)
Appendix P-II-D-2-d-(1). Sewer vents and other ventilation lines
contain high efficiency particulate air/HEPA filters. HEPA filters
shall be certified annually.
Appendix P-II-D-2-d-(2). A pass-through dunk tank, fumigation
chamber, or an equivalent method of decontamination shall be provided
to ensure decontamination of materials and equipment that cannot be
decontaminated in the autoclave.
Appendix P-II-D-2-d-(3). Liquid effluent from sinks, floors, and
autoclave chambers shall be decontaminated by heat or chemical
treatment before being released from the maximum containment greenhouse
facility. Liquid wastes from shower rooms and toilets may be
decontaminated by heat or chemical treatment. Autoclave and chemical
decontamination of liquid wastes shall be evaluated by appropriate
standard procedures for autoclaved wastes. Decontamination shall be
evaluated mechanically and biologically using a recording thermometer
and an indicator microorganism with a defined heat susceptibility
pattern. If liquid wastes are decontaminated with chemical
disinfectants, the chemicals used must have demonstrated efficacy
against the target or indicator microorganisms.
Appendix P-II-D-2-d-(4). If there is a central vacuum system, it
shall not serve areas outside of the greenhouse facility. In-line high
efficiency particulate air/HEPA filters shall be placed as near as
practicable to each use point or vacuum service cock. Other liquid and
gas services to the greenhouse facility shall be protected by devices
that prevent back-flow. HEPA filters shall be certified annually.
Appendix P-III. Biological Containment Practices
Appropriate selection of the following biological containment
practices may be used to meet the containment requirements for a given
organism. The present list is not exhaustive; there may be other ways
of preventing effective dissemination that could possibly lead to the
establishment of the organism or its genetic material in the
environment resulting in deleterious consequences to managed or natural
ecosystems.
Appendix P-III-A. Biological Containment Practices (Plants)
Appendix P-III-A-1. Effective dissemination of plants by pollen or
seed can be prevented by one or more of the following procedures: (i)
Cover the reproductive structures to prevent pollen dissemination at
flowering and seed dissemination at maturity; (ii) remove reproductive
structures by employing male sterile strains, or harvest the plant
material prior to the reproductive stage; (iii) ensure that
experimental plants flower at a time of year when cross-fertile plants
are not flowering within the normal pollen dispersal range of the
experimental plant; or (iv) ensure that cross-fertile plants are not
growing within the known pollen dispersal range of the experimental
plant.
Appendix P-III-B. Biological Containment Practices (Microorganisms)
Appendix P-III-B-1. Effective dissemination of microorganisms
beyond the confines of the greenhouse can be prevented by one or more
of the following procedures: (i) Confine all operations to injections
of microorganisms or other biological procedures (including genetic
manipulation) that limit replication or reproduction of viruses and
microorganisms or sequences derived from microorganisms, and confine
these injections to internal plant parts or adherent plant surfaces;
(ii) ensure that organisms, which can serve as hosts or promote the
transmission of the virus or microorganism, are not present within the
farthest distance that the airborne virus or microorganism may be
expected to be effectively disseminated; (iii) conduct experiments at a
time of year when plants that can serve as hosts are either not growing
or are not susceptible to productive infection; (iv) use viruses and
other microorganisms or their genomes that have known arthropod or
animal vectors, in the absence of such vectors; (v) use microorganisms
that have an obligate association with the plant; or (vi) use
microorganisms that are genetically disabled to minimize survival
outside of the research facility and whose natural mode of transmission
requires injury of the target organism, or assures that inadvertent
release is unlikely to initiate productive infection of organisms
outside of the experimental facility.
Appendix P-III-C. Biological Containment Practices (Macroorganisms)
Appendix P-III-C-1. Effective dissemination of arthropods and other
small animals can be prevented by using one or more of the following
procedures: (i) Use non-flying, flight-impaired, or sterile arthropods;
(ii) use non-motile or sterile strains of small animals; (iii) conduct
experiments at a time of year that precludes the survival of escaping
organisms; (iv) use animals that have an obligate association with a
plant that is not present within the dispersal range of the organism;
or (v) prevent the escape of organisms present in run-off water by
chemical treatment or evaporation of run-off water.
I. Addition of Appendix Q, Physical and Biological Containment for
Recombinant DNA Research Involving Animals, to the NIH Guidelines
The following new appendix, Appendix Q, reads as follows:
Appendix Q specifies containment and confinement practices for
research involving whole animals, both those in which the animal's
genome has been altered by stable introduction of recombinant DNA, or
DNA derived therefrom, into the germ-line (transgenic animals) and
experiments involving viable recombinant DNA-modified microorganisms
tested on whole animals. The appendix applies to animal research
activities with the following modifications:
Appendix Q shall supersede Appendix G when research animals are of
a size or have growth requirements that preclude the use of containment
for laboratory animals. Some animals may require other types of
containment (see Appendix Q-III-D). The animals covered in Appendix Q
are those species normally categorized as animals including but not
limited to cattle, swine, sheep, goats, horses, and poultry.
The Institutional Biosafety Committee shall include at least one
scientist with expertise in animal containment principles when
experiments utilizing Appendix Q require Institutional Biosafety
Committee prior approval.
The institution shall establish and maintain a health surveillance
program for personnel engaged in animal research involving viable
recombinant DNA-containing microorganisms that require Biosafety Level
(BL) 3 or greater containment in the laboratory.
Appendix Q-I. General Considerations
Appendix Q-I-A. Containment Levels
The containment levels required for research involving recombinant
DNA associated with or in animals is based on classification of
experiments in Section III. For the purpose of animal research, four
levels of containment are established. These are referred to as BL1-
Animals (N), BL2-N, BL3-N, and BL4-N and are described in the following
sections of Appendix Q. The descriptions include: (i) standard
practices for physical and biological containment, and (ii) animal
facilities.
Appendix Q-I-B. Disposal of Animals (BL1-N through BL4-N)
Appendix Q-I-B-1. When an animal covered by Appendix Q containing
recombinant DNA or a recombinant DNA-derived organism is euthanized or
dies, the carcass shall be disposed of to avoid its use as food for
human beings or animals unless food use is specifically authorized by
an appropriate Federal agency.
Appendix Q-I-B-2. A permanent record shall be maintained of the
experimental use and disposal of each animal or group of animals.
Appendix Q-II. Physical and Biological Containment Levels
Appendix Q-II-A. Biosafety Level 1 - Animals (BL1-N)
Appendix Q-II-A-1. Standard Practices (BL1-N)
Appendix Q-II-A-1-a. Animal Facility Access (BL1-N)
Appendix Q-II-A-1-a-(1). The containment area shall be locked.
Appendix Q-II-A-1-a-(2). Access to the containment area shall be
limited or restricted when experimental animals are being held.
Appendix Q-II-A-1-a-(3). The containment area shall be patrolled or
monitored at frequent intervals.
Appendix Q-II-A-1-b. Other (BL1-N)
Appendix Q-II-A-1-b-(1). All genetically engineered neonates shall
be permanently marked within 72 hours after birth, if their size
permits. If their size does not permit marking, their containers should
be marked. In addition, transgenic animals should contain distinct and
biochemically assayable DNA sequences that allow identification of
transgenic animals from among non- transgenic animals.
Appendix Q-II-A-1-b-(2) A double barrier shall be provided to
separate male and female animals unless reproductive studies are part
of the experiment or other measures are taken to avoid reproductive
transmission. Reproductive incapacitation may be used.
Appendix Q-II-A-1-b-(3). The containment area shall be in
accordance with state and Federal laws and animal care requirements.
Appendix Q-II-A-2. Animal Facilities (BL1-N)
Appendix Q-II-A-2-(a). Animals shall be confined to securely fenced
areas or be in enclosed structures (animal rooms) to minimize the
possibility of theft or unintentional release.
Appendix Q-II-B. Biosafety Level 2 - Animals (BL2-N) (see Appendix Q-
III-A)
Appendix Q-II-B-1. Standard Practices (BL2-N)
Appendix Q-II-B-1-a. Animal Facility Access (BL2-N)
Appendix Q-II-B-1-a-(1). The containment area shall be locked.
Appendix Q-II-B-1-a-(2). The containment area shall be patrolled or
monitored at frequent intervals.
Appendix Q-II-B-1-a-(3). The containment building shall be
controlled and have a locking access.
Appendix Q-II-B-1-a-(4). The Animal Facility Director shall
establish policies and procedures whereby only persons who have been
advised of the potential hazard and who meet any specific entry
requirements (e.g., vaccination) may enter the laboratory or animal
rooms.
Appendix Q-II-B-1-a-(5). Animals of the same or different species,
which are not involved in the work being performed, shall not be
permitted in the animal area.
Appendix Q-II-B-1-b. Decontamination and Inactivation (BL2-N)
Appendix Q-II-B-1-b-(1). Contaminated materials that are
decontaminated at a site away from the laboratory shall be placed in a
closed durable leak-proof container prior to removal from the
laboratory.
Appendix Q-II-B-1-b-(2). Needles and syringes shall be promptly
placed in a puncture-resistant container and decontaminated, preferably
by autoclaving, before discard or reuse.
Appendix Q-II-B-1-c. Signs (BL2-N)
Appendix Q-II-B-1-c-(1). When the animal research requires special
provisions for entry (e.g., vaccination), a warning sign incorporating
the universal biosafety symbol shall be posted on all access doors to
the animal work area. The sign shall indicate: (i) The agent, (ii) the
animal species, (iii) the name and telephone number of the Animal
Facility Director or other responsible individual, and (iv) any special
requirements for entering the laboratory.
Appendix Q-II-B-1-d. Protective Clothing (BL2-N)
Appendix Q-II-B-1-d-(1). Laboratory coats, gowns, smocks, or
uniforms shall be worn while in the animal area or attached laboratory.
Before entering non-laboratory areas (e.g., cafeteria, library,
administrative offices), protective clothing shall be removed and kept
in the work entrance area.
Appendix Q-II-B-1-d-(2). Special care shall be taken to avoid skin
contamination with microorganisms containing recombinant DNA.
Impervious and/or protective gloves shall be worn when handling
experimental animals and when skin contact with an infectious agent is
unavoidable.
Appendix Q-II-B-1-e. Records (BL2-N)
Appendix Q-II-B-1-e-(1). Any incident involving spills and
accidents that result in environmental release or exposures of animals
or laboratory workers to organisms containing recombinant DNA molecules
shall be reported immediately to the Animal Facility Director,
Institutional Biosafety Committee, NIH/ORDA, and other appropriate
authorities (if applicable). Reports to the NIH/ORDA shall be sent to
the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
9838. Medical evaluation, surveillance, and treatment shall be provided
as appropriate and written records maintained. If necessary, the area
shall be appropriately decontaminated.
Appendix Q-II-B-1-e-(2). When appropriate and giving consideration to
the agent handled, baseline serum samples shall be collected and stored
for animal care and other at-risk personnel. Additional serum specimens
may be collected periodically depending on the agent handled and the
function of the animal facility.
Appendix Q-II-B-1-f. Transfer of Materials (BL2-N)
Appendix Q-II-B-1-f-(1). Biological materials removed from the
animal containment area in a viable or intact state shall be
transferred to a non-breakable sealed primary container and then
enclosed in a non-breakable sealed secondary container. All containers,
primary and secondary, shall be disinfected before removal from the
animal facility. Advance approval for transfer of material shall be
obtained from the Animal Facility Director. Packages containing viable
agents may only be opened in a facility having an equivalent or higher
level of physical containment unless the agent is biologically
inactivated or incapable of reproduction.
Appendix Q-II-B-1-g. Other (BL2-N)
Appendix Q-II-B-1-g-(1). All genetically engineered neonates shall
be permanently marked within 72 hours after birth, if their size
permits. If their size does not permit marking, their containers should
be marked. In addition, transgenic animals should contain distinct and
biochemically assayable DNA sequences that allow identification of
transgenic animals from among non-transgenic animals.
Appendix Q-II-B-1-g-(2). Needles and syringes shall be used only
for parenteral injection and aspiration of fluids from laboratory
animals and diaphragm bottles. Only needle-locking syringes or
disposable syringe-needle units (i.e., needle is integral to the
syringe) shall be used for the injection or aspiration of fluids
containing organisms that contain recombinant DNA. Extreme caution
shall be used when handling needles and syringes to avoid
autoinoculation and the generation of aerosols during use and disposal.
Following use, needles shall not be bent, sheared, replaced in the
needle sheath or guard, or removed from the syringe. Needles and
syringes shall be promptly placed in a puncture-resistant container and
decontaminated, preferably by autoclaving, before discard or reuse.
Appendix Q-II-B-1-g-(3). Appropriate steps should be taken to
prevent horizontal transmission or exposure of laboratory personnel. If
the agent used as a vector is known to be transmitted by a particular
route (e.g., arthropods), special attention should be given to
preventing spread by that route. In the absence of specific knowledge
of a particular route of transmission, all potential means of
horizontal transmission (e.g., arthropods, contaminated bedding, or
animal waste, etc.) should be prevented.
Appendix Q-II-B-1-g-(4). Eating, drinking, smoking, and applying
cosmetics shall not be permitted in the work area.
Appendix Q-II-B-1-g-(5). Individuals who handle materials and
animals containing recombinant DNA molecules shall be required to wash
their hands before exiting the containment area.
Appendix Q-II-B-1-g-(6). A double barrier shall be provided to
separate male and female animals unless reproductive studies are part
of the experiment or other measures are taken to avoid reproductive
transmission. Reproductive incapacitation may be used.
Appendix Q-II-B-1-g-(7). The containment area shall be in
accordance with state and Federal laws and animal care requirements.
Appendix Q-II-B-1-g-(8). A biosafety manual shall be prepared or
adopted. Personnel shall be advised of special hazards and required to
read and follow instructions on practices and procedures.
Appendix Q-II-B-2. Animal Facilities (BL2-N)
Appendix Q-II-B-2-a. Animals shall be contained within an enclosed
structure (animal room or equivalent) to minimize the possibility of
theft or unintentional release and to avoid arthropod access. The
special provision to avoid the entry or escape of arthropods from the
animal areas may be waived if the agent in use is not known to be
transmitted by arthropods.
Appendix Q-II-B-2-b. Surfaces shall be impervious to water and
resistant to acids, alkalis, organic solvents, and moderate heat.
Appendix Q-II-B-2-c. The animal containment area shall be designed
so that it can be easily cleaned.
Appendix Q-II-B-2-d. Windows that open shall be fitted with fly
screens.
Appendix Q-II-B-2-e. An autoclave shall be available for
decontamination of laboratory wastes.
Appendix Q-II-B-2-f. If arthropods are used in the experiment or
the agent under study can be transmitted by an arthropod, interior work
areas shall be appropriately screened (52 mesh). All perimeter joints
and openings shall be sealed and additional arthropod control
mechanisms used to minimize arthropod entry and propagation, including
appropriate screening of access doors or the equivalent.
Appendix Q-II-C. Biosafety Level 3--Animals (BL3-N) (See Appendix Q-
III-B)
Appendix Q-II-C-1. Standard Practices (BL3-N)
Appendix Q-II-C-1-a. Animal Facility Access (BL3-N)
Appendix Q-II-C-1-a-(1). The containment area shall be locked.
Appendix Q-II-C-1-a-(2). The containment area shall be patrolled or
monitored at frequent intervals.
Appendix Q-II-C-1-a-(3). The containment building shall be
controlled and have a locking access.
Appendix Q-II-C-1-a-(4). The Animal Facility Director shall
establish policies and procedures whereby only persons who have been
advised of the potential hazard and who meet any specific entry
requirements (e.g., vaccination) shall enter the laboratory or animal
rooms.
Appendix Q-II-C-1-a-(5). Animal room doors, gates, or other
closures shall be kept closed when experiments are in progress.
Appendix Q-II-C-1-b. Decontamination and Inactivation (BL3-N)
Appendix Q-II-C-1-b-(1). The work surfaces of containment equipment
shall be decontaminated when work with organisms containing recombinant
DNA molecules is finished. Where feasible, plastic-backed paper
toweling shall be used on nonporous work surfaces to facilitate clean-
up.
Appendix Q-II-C-1-b-(2). All animals shall be euthanized at the end
of their experimental usefulness and the carcasses decontaminated
before disposal in an approved manner.
Appendix Q-II-C-1-b-(3). Needles and syringes shall be promptly
placed in a puncture-resistant container and decontaminated, preferably
by autoclaving, before discard or reuse.
Appendix Q-II-C-1-b-(4). Special safety testing, decontamination
procedures, and Institutional Biosafety Committee approval shall be
required to transfer agents or tissue/organ specimens from a BL3-N
animal facility to a facility with a lower containment classification.
Appendix Q-II-C-1-b-(5). Liquid effluent from containment
equipment, sinks, biological safety cabinets, animal rooms, primary
barriers, floor drains, and sterilizers shall be decontaminated by heat
treatment before being released into the sanitary system. The procedure
used for heat decontamination of liquid wastes shall be monitored with
a recording thermometer. The effectiveness of the heat decontamination
process system shall be revalidated every 30 days with an indicator
organism.
Appendix Q-II-C-1-c. Signs (BL3-N)
Appendix Q-II-C-1-c-(1). When the animal research requires special
provisions for entry (e.g., vaccination), a warning sign incorporating
the universal biosafety symbol shall be posted on all access doors to
the animal work area. The sign shall indicate: (i) The agent, (ii) the
animal species, (iii) the name and telephone number of the Animal
Facility Director or other responsible individual, and (iv) any special
requirements for entering the laboratory.
Appendix Q-II-C-1-d. Protective Clothing (BL3-N)
Appendix Q-II-C-1-d-(1). Full protective clothing that protects the
individual (e.g., scrub suits, coveralls, uniforms) shall be worn in
the animal area. Clothing shall not be worn outside the animal
containment area and shall be decontaminated before laundering or
disposal. Personnel shall be required to shower before exiting the BL3-
N area and wearing of personal clothing.
Appendix Q-II-C-1-d-(2). Special care shall be taken to avoid skin
contamination with microorganisms containing recombinant DNA.
Impervious and/or protective gloves shall be worn when handling
experimental animals and when skin contact with an infectious agent is
unavoidable.
Appendix Q-II-C-1-d-(3). Appropriate respiratory protection shall
be worn in rooms containing experimental animals.
Appendix Q-II-C-1-e. Records (BL3-N)
Appendix Q-II-C-1-e-(1). Documents regarding experimental animal
use and disposal shall be maintained in a permanent record book.
Appendix Q-II-C-1-e-(2). Any incident involving spills and
accidents that result in environmental release or exposure of animals
or laboratory workers to organisms containing recombinant DNA shall be
reported immediately to the Biological Safety Office, Animal Facility
Director, Institutional Biosafety Committee, NIH/ORDA, and other
appropriate authorities (if applicable). Reports to the NIH/ORDA shall
be sent to the Office of Recombinant DNA Activities, National
Institutes of Health, Building 31, Room 4B11, Bethesda, Maryland 20892,
(301) 496-9838. Medical evaluation, surveillance, and treatment shall
be provided as appropriate and written records maintained. If
necessary, the area shall be appropriately decontaminated.
Appendix Q-II-C-1-e-(3). When appropriate and giving consideration
to the agent handled, baseline serum samples shall be collected and
stored for animal care and other at-risk personnel. Additional serum
specimens may be collected periodically depending on the agent handled
or the function of the facility.
Appendix Q-II-C-1-f. Transfer of Materials (BL3-N)
Appendix Q-II-C-1-f-(1). Biological materials removed from the
animal containment laboratory in a viable or intact state shall be
transferred to a non- breakable sealed primary container and then
enclosed in a non-breakable sealed secondary container. All containers,
primary and secondary, shall be disinfected before removal from the
animal facility. Advance approval for transfer of material shall be
obtained from the Animal Facility Director. Packages containing viable
agents may be opened only in a facility having an equivalent or higher
level of physical containment unless the agent is biologically
inactivated or incapable of reproduction.
Appendix Q-II-C-1-f-(2). Special safety testing, decontamination
procedures, and Institutional Biosafety Committee approval shall be
required to transfer agents or tissue/organ specimens from a BL3-N
animal facility to a facility with a lower containment classification.
Appendix Q-II-C-1-g. Other (BL3-N)
Appendix Q-II-C-1-g-(1). All genetically engineered neonates shall
be permanently marked within 72 hours after birth, if their size
permits. If their size does not permit marking, their containers should
be marked. In addition, transgenic animals should contain distinct and
biochemically assayable DNA sequences that allow identification of
transgenic animals from among nontransgenic animals.
Appendix Q-II-C-1-g-(2). Appropriate steps should be taken to
prevent horizontal transmission or exposure of laboratory personnel. If
the agent used as the vector is known to be transmitted by a particular
route (e.g., arthropods), special attention should be given to
preventing spread by that route. In the absence of specific knowledge
of a particular route of transmission, all potential means of
horizontal transmission (e.g., arthropods, contaminated bedding, or
animal waste) should be prevented.
Appendix Q-II-C-1-g-(3). Eating, drinking, smoking, and applying
cosmetics shall not be permitted in the work area.
Appendix Q-II-C-1-g-(4). Individuals who handle materials and
animals containing recombinant DNA molecules shall be required to wash
their hands before exiting the containment area.
Appendix Q-II-C-1-g-(5). Experiments involving other organisms that
require containment levels lower than BL3-N may be conducted in the
same area concurrently with experiments requiring BL3-N containment
provided that they are conducted in accordance with BL3-N practices.
Appendix Q-II-C-1-g-(6). Animal holding areas shall be cleaned at
least once a day and decontaminated immediately following any spill of
viable materials.
Appendix Q-II-C-1-g-(7). All procedures shall be performed
carefully to minimize the creation of aerosols.
Appendix Q-II-C-1-g-(8). A double barrier shall be provided to
separate male and female animals unless reproductive studies are part
of the experiment or other measures are taken to avoid reproductive
transmission. Reproductive incapacitation may be used.
Appendix Q-II-C-1-g-(9). The containment area shall be in
accordance with state and Federal laws and animal care requirements.
Appendix Q-II-C-1-g-(10). All animals shall be euthanized at the
end of their experimental usefulness and the carcasses decontaminated
before disposal in an approved manner.
Appendix Q-II-C-1-g-(11). Personnel shall be required to shower
before exiting the BL3-N area and wearing personal clothing.
Appendix Q-II-C-1-g-(12). Animals of the same or different species,
which are not involved in the work being performed, shall not be
permitted in the animal area.
Appendix Q-II-C-1-g-(13). Needles and syringes shall be used only
for parenteral injection and aspiration of fluids from laboratory
animals and diaphragm bottles. Only needle-locking syringes or
disposable syringe-needle units (i.e., needle is integral to the
syringe) shall be used for the injection or aspiration of fluids
containing organisms that contain recombinant DNA. Extreme caution
shall be used when handling needles and syringes to avoid
autoinoculation and the generation of aerosols during use and disposal.
Following use, needles shall not be bent, sheared, replaced in the
needle sheath or guard or removed from the syringe. The needles and
syringes shall be promptly placed in a puncture-resistant container and
decontaminated, preferably by autoclaving, before discard or reuse.
Appendix Q-II-C-1-g-(14). A biosafety manual shall be prepared or
adopted. Personnel shall be advised of special hazards and required to
read and follow instructions on practices and procedures.
Appendix Q-II-C-2. Animal Facilities (BL3-N)
Appendix Q-II-C-2-a. Animals shall be contained within an enclosed
structure (animal room or equivalent) to minimize the possibility of
theft or unintentional release and avoid arthropod access. The special
provision to avoid the entry or escape of arthropods from the animal
areas may be waived if the agent in use is not known to be transmitted
by arthropods.
Appendix Q-II-C-2-b. The interior walls, floors, and ceilings shall
be impervious to water and resistant to acids, alkalis, organic
solvents, and moderate heat, to facilitate cleaning. Penetrations in
these structures and surfaces (e.g., plumbing and utilities) shall be
sealed.
Appendix Q-II-C-2-c. Windows in the animal facility shall be
closed, sealed, and breakage resistant (e.g., double-pane tempered
glass or equivalent). The need to maintain negative pressure should be
considered when constructing or renovating the animal facility.
Appendix Q-II-C-2-d. An autoclave, incinerator, or other effective
means to decontaminate animals and waste shall be available, preferably
within the containment area. If feasible, a double-door autoclave is
preferred and should be positioned to allow removal of material from
the containment area.
Appendix Q-II-C-2-e. If arthropods are used in the experiment or
the agent under study can be transmitted by an arthropod, the interior
work area shall be appropriately screened (52 mesh). All perimeter
joints and openings shall be sealed, and additional arthropod control
mechanisms used to minimize arthropod entry and propagation, including
appropriate screening, or the equivalent of access doors.
Appendix Q-II-C-2-f. Access doors to the containment area shall be
self-closing.
Appendix Q-II-C-2-g. The animal area shall be separated from all
other areas. Passage through two sets of doors shall be the basic
requirement for entry into the animal area from access corridors or
other contiguous areas. The animal containment area shall be physically
separated from access corridors and other laboratories or areas by a
double-door clothes change room, equipped with integral showers and
airlock.
Appendix Q-II-C-2-h. Liquid effluent from containment equipment,
sinks, biological safety cabinets, animal rooms, primary barriers,
floor drains, and sterilizers shall be decontaminated by heat treatment
before being released into the sanitary system. The procedure used for
heat decontamination of liquid wastes shall be monitored with a
recording thermometer. The effectiveness of the heat decontamination
process system shall be revalidated every 30 days with an indicator
organism.
Appendix Q-II-C-2-i. An exhaust air ventilation system shall be
provided. This system shall create directional airflow that draws air
into the animal room through the entry area. The building exhaust, or
the exhaust from primary containment units, may be used for this
purpose if the exhaust air is discharged to the outside and shall be
dispersed away from occupied areas and air intakes. Personnel shall
verify that the direction of the airflow (into the animal room) is
proper.
Appendix Q-II-C-2-j. If the agent is transmitted by aerosol, then
the exhaust air shall pass through a high efficiency particulate air/
HEPA filter.
Appendix Q-II-C-2-k. Vacuum lines shall be protected with high
efficiency particulate air/HEPA filters and liquid disinfectant traps.
Appendix Q-II-C-2-l. In lieu of open housing in the special animal
room, animals held in a BL3-N area may be housed in partial-containment
caging systems (e.g., Horsfall units or gnotobiotic systems, or other
special containment primary barriers). Prudent judgment must be
exercised to implement this ventilation system (e.g., animal species)
and its discharge location.
Appendix Q-II-C-2-m. Each animal area shall contain a foot, elbow,
or automatically operated sink for hand washing. The sink shall be
located near the exit door.
Appendix Q-II-C-2-n. Restraining devices for animals may be
required to avoid damage to the integrity of the animal containment
facility.
Appendix Q-II-D. Biosafety Level 4--Animals (BL4-N) (See Appendix Q-
III-C)
Appendix Q-II-D-1. Standard Practices (BL4-N)
Appendix Q-II-D-1-a. Animal Facility Access (BL4-N)
Appendix Q-II-D-1-a-(1). Individuals under 16 years of age shall
not be permitted to enter the animal area.
Appendix Q-II-D-1-a-(2). The containment area shall be locked.
Appendix Q-II-D-1-a-(3). The containment area shall be patrolled or
monitored at frequent intervals.
Appendix Q-II-D-1-a-(4). The containment building shall be
controlled and have a locking access.
Appendix Q-II-D-1-a-(5). The Animal Facility Director shall
establish policies and procedures whereby only persons who have been
advised of the potential hazard and who meet any specific entry
requirements (e.g., vaccination) may enter the laboratory or animal
room.
Appendix Q-II-D-1-a-(6). Individuals shall enter and exit the
animal facility only through the clothing change and shower rooms.
Appendix Q-II-D-1-a-(7). Personnel shall use the airlocks to enter
or exit the laboratory only in an emergency.
Appendix Q-II-D-1-a-(8). Animal room doors, gates, and other
closures shall be kept closed when experiments are in progress.
Appendix Q-II-D-1-b. Decontamination and Inactivation (BL4-N)
Appendix Q-II-D-1-b-(1). All contaminated liquid or solid wastes
shall be decontaminated before disposal.
Appendix Q-II-D-1-b-(2). The work surfaces and containment
equipment shall be decontaminated when work with organisms containing
recombinant DNA molecules is finished. Where feasible, plastic-backed
paper toweling shall be used on nonporous work surfaces to facilitate
clean-up.
Appendix Q-II-D-1-b-(3). All wastes from animal rooms and
laboratories shall be appropriately decontaminated before disposal in
an approved manner.
Appendix Q-II-D-1-b-(4). No materials, except for biological
materials that are to remain in a viable or intact state, shall be
removed from the maximum containment laboratory unless they have been
autoclaved or decontaminated.
Equipment or material that might be damaged by high temperatures or
steam shall be decontaminated by gaseous or vapor methods in an airlock
or chamber designed for this purpose.
Appendix Q-II-D-1-b-(5). When ventilated suits are required, the
animal personnel shower entrance/exit area shall be equipped with a
chemical disinfectant shower to decontaminate the surface of the suit
before exiting the area. A neutralization or water dilution device
shall be integral with the chemical disinfectant discharge piping
before entering the heat sterilization system. Entry to this area shall
be through an airlock fitted with airtight doors.
Appendix Q-II-D-1-b-(6). Needles and syringes shall be promptly
placed in a puncture-resistant container and decontaminated, preferably
by autoclaving, before discard or reuse.
Appendix Q-II-D-1-b-(7). Supplies and materials needed in the
animal facility shall be brought in by way of the double-door
autoclave, fumigation chamber, or airlock that shall be appropriately
decontaminated between each use.
Appendix Q-II-D-1-b-(8). An autoclave, incinerator, or other
effective means to decontaminate animals and wastes shall be available,
preferably within the containment area. If feasible, a double-door
autoclave is preferred and should be positioned to allow removal of
material from the containment area.
Appendix Q-II-D-1-b-(9). Liquid effluent from containment
equipment, sinks, biological safety cabinets, animal rooms, primary
barriers, floor drains, and sterilizers shall be decontaminated by heat
treatment before being released into the sanitary system. Liquid wastes
from shower rooms and toilets shall be decontaminated with chemical
disinfectants or heat by methods demonstrated to be effective. The
procedure used for heat decontamination of liquid wastes shall be
monitored with a recording thermometer. The effectiveness of the heat
decontamination process system shall be revalidated every 30 days with
an indicator organism. Liquid wastes from the shower shall be
chemically decontaminated using an Environmental Protection Agency-
approved germicide. The efficacy of the chemical treatment process
shall be validated with an indicator organism. Chemical disinfectants
shall be neutralized or diluted before release into general effluent
waste systems.
Appendix Q-II-D-1-c. Signs (BL4-N)
Appendix Q-II-D-1-c-(1). When the animal research requires special
provisions for entry (e.g., vaccination), a warning sign incorporating
the universal biosafety symbol shall be posted on all access doors to
the animal work area. The sign shall indicate: (i) The agent, (ii) the
animal species, (iii) the name and telephone number of the Animal
Facility Director, or other responsible individual, and (iv) any
special requirements for entering the laboratory.
Appendix Q-II-D-1-d. Protective Clothing (BL4-N)
Appendix Q-II-D-1-d-(1). Individuals shall enter and exit the
animal facility only through the clothing change and shower rooms.
Street clothing shall be removed and kept in the outer clothing change
room. Complete laboratory clothing (may be disposable), including
undergarments, pants, shirts, jump suits, and shoes shall be provided
for all personnel entering the animal facility. When exiting the BL4-N
area and before proceeding into the shower area, personnel shall remove
their laboratory clothing in the inner change room. All laboratory
clothing shall be autoclaved before laundering. Personnel shall shower
each time they exit the animal facility.
Appendix Q-II-D-1-d-(2). A ventilated head-hood or a one-piece
positive pressure suit, which is ventilated by a life-support system,
shall be worn by all personnel entering rooms that contain experimental
animals when appropriate. When ventilated suits are required, the
animal personnel shower entrance/exit area shall be equipped with a
chemical disinfectant shower to decontaminate the surface of the suit
before exiting the area. A neutralization or water dilution device
shall be integral with the chemical disinfectant discharge piping
before entering the heat sterilization system. Entry to this area shall
be through an airlock fitted with airtight doors.
Appendix Q-II-D-1-d-(3). Appropriate respiratory protection shall
be worn in rooms containing experimental animals.
Appendix Q-II-D-1-e. Records (BL4-N)
Appendix Q-II-D-1-e-(1). Documents regarding experimental animal
use and disposal shall be maintained in a permanent record book.
Appendix Q-II-D-1-e-(2). A system shall be established for: (i)
Reporting laboratory accidents and exposures that are a result of overt
exposures to organisms containing recombinant DNA, (ii) employee
absenteeism, and (iii) medical surveillance of potential laboratory-
associated illnesses. Permanent records shall be prepared and
maintained. Any incident involving spills and accidents that results in
environmental release or exposures of animals or laboratory workers to
organisms containing recombinant DNA molecules shall be reported
immediately to the Biological Safety Officer, Animal Facility Director,
Institutional Biosafety Committee, NIH/ORDA, and other appropriate
authorities (if applicable). Reports to the NIH/ORDA shall be sent to
the Office of Recombinant DNA Activities, National Institutes of
Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-
9838. Medical evaluation, surveillance, and treatment shall be provided
as appropriate and written records maintained. If necessary, the area
shall be appropriately decontaminated.
Appendix Q-II-D-1-e-(3). When appropriate and giving consideration
to the agents handled, baseline serum samples shall be collected and
stored for animal care and other at-risk personnel. Additional serum
specimens may be collected periodically depending on the agents handled
or the function of the facility.
Appendix Q-II-D-1-e-(4). A permanent record book indicating the
date and time of each entry and exit shall be signed by all personnel.
Appendix Q-II-D-1-f. Transfer of Materials (BL4-N)
Appendix Q-II-D-1-f-(1). No materials, except for biological
materials that are to remain in a viable or intact state, shall be
removed from the maximum containment laboratory unless they have been
autoclaved or decontaminated. Equipment or material that might be
damaged by high temperatures or steam shall be decontaminated by
gaseous or vapor methods in an airlock or chamber designed for this
purpose.
Appendix Q-II-D-1-f-(2). Biological materials removed from the
animal maximum containment laboratory in a viable or intact state shall
be transferred to a non-breakable sealed primary container and then
enclosed in a non-breakable sealed secondary container that shall be
removed from the animal facility through a disinfectant dunk tank,
fumigation chamber, or an airlock designed for this purpose. Advance
approval for transfer of material shall be obtained from the Animal
Facility Director. Such packages containing viable agents can only be
opened in another BL4-N animal facility if the agent is biologically
inactivated or incapable of reproduction. Special safety testing,
decontamination procedures, and Institutional Biosafety Committee
approval shall be required to transfer agents or tissue/organ specimens
from a BL4-N animal facility to one with a lower containment
classification.
Appendix Q-II-D-1-f-(3). Supplies and materials needed in the
animal facility shall be brought in by way of the double-door
autoclave, fumigation chamber, or airlock that shall be appropriately
decontaminated between each use. After securing the outer doors,
personnel within the animal facility retrieve the materials by opening
the interior doors of the autoclave, fumigation chamber, or airlock.
These doors shall be secured after materials are brought into the
animal facility.
Appendix Q-II-D-1-g. Other (BL4-N)
Appendix Q-II-D-1-g-(1). All genetically engineered neonates shall
be permanently marked within 72 hours after birth, if their size
permits. If their size does not permit marking, their containers should
be marked. In addition, transgenic animals should contain distinct and
biochemically assayable DNA sequences that allow identification of
transgenic animals from among non-transgenic animals.
Appendix Q-II-D-1-g-(2). Eating, drinking, smoking, and applying
cosmetics shall not be permitted in the work area.
Appendix Q-II-D-1-g-(3). Individuals who handle materials and
animals containing recombinant DNA molecules shall be required to wash
their hands before exiting the containment area.
Appendix Q-II-D-1-g-(4). Experiments involving other organisms that
require containment levels lower than BL4-N may be conducted in the
same area concurrently with experiments requiring BL4-N containment
provided that they are conducted in accordance with BL4-N practices.
Appendix Q-II-D-1-g-(5). Animal holding areas shall be cleaned at
least once a day and decontaminated immediately following any spill of
viable materials.
Appendix Q-II-D-1-g-(6). All procedures shall be performed
carefully to minimize the creation of aerosols.
Appendix Q-II-D-1-g-(7). A double barrier shall be provided to
separate male and female animals. Animal isolation barriers shall be
sturdy and accessible for cleaning. Reproductive incapacitation may be
used.
Appendix Q-II-D-1-g-(8). The containment area shall be in
accordance with state and Federal laws and animal care requirements.
Appendix Q-II-D-1-g-(9). The life support system for the ventilated
suit or head hood is equipped with alarms and emergency back-up air
tanks. The exhaust air from the suit area shall be filtered by two sets
of high efficiency particulate air/HEPA filters installed in series or
incinerated. A duplicate filtration unit, exhaust fan, and an
automatically starting emergency power source shall be provided. The
air pressure within the suit shall be greater than that of any adjacent
area. Emergency lighting and communication systems shall be provided. A
double-door autoclave shall be provided for decontamination of waste
materials to be removed from the suit area.
Appendix Q-II-D-1-g-(10). Needles and syringes shall be used only
for parenteral injection and aspiration of fluids from laboratory
animals and diaphragm bottles. Only needle-locking syringes or
disposable syringe-needle units (i.e., needle is integral to the
syringe) shall be used for the injection or aspiration of fluids
containing organisms that contain recombinant DNA. Extreme caution
shall be used when handling needles and syringes to avoid
autoinoculation and the generation of aerosols during use and disposal.
Following use, needles shall not be bent, sheared, replaced in the
needle sheath or guard, or removed from the syringe. The needles and
syringes shall be promptly placed in a puncture-resistant container and
decontaminated, preferably by autoclaving, before discard or reuse.
Appendix Q-II-D-1-g-(11). An essential adjunct to the reporting-
surveillance system is the availability of a facility for quarantine,
isolation, and medical care of personnel with potential or known
laboratory-associated illnesses.
Appendix Q-II-D-1-g-(12). A biosafety manual shall be prepared or
adopted. Personnel shall be advised of special hazards and required to
read and follow instructions on practices and procedures.
Appendix Q-II-D-1-g-(13). Vacuum lines shall be protected with high
efficiency particulate air/HEPA filters and liquid disinfectant traps.
Appendix Q-II-D-2. Animal Facilities (BL4-N)
Appendix Q-II-D-2-a. Animals shall be contained within an enclosed
structure (animal room or equivalent) to minimize the possibility of
theft or unintentional release and avoid arthropod access.
Appendix Q-II-D-2-b. The interior walls, floors, and ceilings shall
be impervious to water and resistant to acids, alkalis, organic
solvents, and moderate heat, to facilitate cleaning. Penetrations in
these structures and surfaces (e.g., plumbing and utilities) shall be
sealed.
Appendix Q-II-D-2-c. Windows in the animal facility shall be
closed, sealed, and breakage resistant (e.g., double-pane tempered
glass or equivalent).
Appendix Q-II-D-2-d. An autoclave, incinerator, or other effective
means to decontaminate animals and wastes shall be available,
preferably within the containment area. If feasible, a double-door
autoclave is preferred and should be positioned to allow removal of
material from the containment area.
Appendix Q-II-D-2-e. Access doors to the containment area shall be
self-closing.
Appendix Q-II-D-2-f. All perimeter joints and openings shall be
sealed to form an arthropod-proof structure.
Appendix Q-II-D-2-g. The BL4-N laboratory provides a double barrier
to prevent the release of recombinant DNA containing microorganisms
into the environment. Design of the animal facility shall be such that
if the barrier of the inner facility is breached, the outer barrier
will prevent release into the environment. The animal area shall be
separated from all other areas. Passage through two sets of doors shall
be the basic requirement for entry into the animal area from access
corridors or other contiguous areas. Physical separation of the animal
containment area from access corridors or other laboratories or
activities shall be provided by a double-door clothes change room
equipped with integral showers and airlock.
Appendix Q-II-D-2-h. A necropsy room shall be provided within the
BL4-N containment area.
Appendix Q-II-D-2-i. Liquid effluent from containment equipment,
sinks, biological safety cabinets, animal rooms, primary barriers,
floor drains, and sterilizers shall be decontaminated by heat treatment
before being released into the sanitary system. Liquid wastes from
shower rooms and toilets shall be decontaminated with chemical
disinfectants or heat by methods demonstrated to be effective. The
procedure used for heat decontamination of liquid wastes shall be
monitored with a recording thermometer. The effectiveness of the heat
decontamination process system shall be revalidated every 30 days with
an indicator organism. Liquid wastes from the shower shall be
chemically decontaminated using an Environmental Protection Agency-
approved germicide.
The efficacy of the chemical treatment process shall be validated
with an indicator organism. Chemical disinfectants shall be neutralized
or diluted before release into general effluent waste systems.
Appendix Q-II-D-2-j. A ducted exhaust air ventilation system shall
be provided that creates directional airflow that draws air into the
laboratory through the entry area. The exhaust air, which is not
recirculated to any other area of the building, shall be discharged to
the outside and dispersed away from the occupied areas and air intakes.
Personnel shall verify that the direction of the airflow (into the
animal room) is proper.
Appendix Q-II-D-2-k. Exhaust air from BL4-N containment area shall
be double high efficiency particulate air/HEPA filtered or treated by
passing through a certified HEPA filter and an air incinerator before
release to the atmosphere. Double HEPA filters shall be required for
the supply air system in a BL4-N containment area.
Appendix Q-II-D-2-l. All high efficiency particulate air/HEPA
filters' frames and housings shall be certified to have no detectable
smoke [dioctylphthalate] leaks when the exit face (direction of flow)
of the filter is scanned above 0.01 percent when measured by a linear
or logarithmic photometer. The instrument must demonstrate a threshold
sensitivity of at least 1 x 10-3 micrograms per liter for 0.3
micrometer diameter dioctylphthalate particles and a challenge
concentration of 80-120 micrograms per liter. The air sampling rate
should be at least 1 cfm (28.3 liters per minute).
Appendix Q-II-D-2-m. If an air incinerator is used in lieu of the
second high efficiency particulate air/HEPA filter, it shall be
biologically challenged to prove all viable test agents are sterilized.
The biological challenge must be minimally 1 x 108 organisms per
cubic foot of airflow through the incinerator. It is universally
accepted if bacterial spores are used to challenge and verify that the
equipment is capable of killing spores, then assurance is provided that
all other known agents are inactivated by the parameters established to
operate the equipment. Test spores meeting this criterion are Bacillus
subtilis var. niger or Bacillus stearothermophilis. The operating
temperature of the incinerator shall be continuously monitored and
recorded during use.
Appendix Q-II-D-2-n. All equipment and floor drains shall be
equipped with deep traps (minimally 5 inches). Floor drains shall be
fitted with isolation plugs or fitted with automatic water fill
devices.
Appendix Q-II-D-2-o. Each animal area shall contain a foot, elbow,
or automatically operated sink for hand washing. The sink shall be
located near the exit door.
Appendix Q-II-D-2-p. Restraining devices for animals may be
required to avoid damage to the integrity of the containment animal
facility.
Appendix Q-II-D-2-q. The supply water distribution system shall be
fitted with a back-flow preventer or break tank.
Appendix Q-II-D-2-r. All utilities, liquid and gas services, shall
be protected with devices that avoid back-flow.
Appendix Q-II-D-2-s. Sewer and other atmospheric ventilation lines
shall be equipped minimally with a single high efficiency particulate/
HEPA filter. Condensate drains from these type housings shall be
appropriately connected to a contaminated or sanitary drain system. The
drain position in the housing dictates the appropriate system to be
used.
Appendix Q-III. Footnotes and References for Appendix Q
Appendix Q-III-A. If recombinant DNA is derived from a Class 2
organism requiring BL2 containment, personnel shall be required to have
specific training in handling pathogenic agents and directed by
knowledgeable scientists.
Appendix Q-III-B. Personnel who handle pathogenic and potentially
lethal agents shall be required to have specific training and be
supervised by knowledgeable scientists who are experienced in working
with these agents. BL3-N containment also minimizes escape of
recombinant DNA-containing organisms from exhaust air or waste material
from the containment area.
Appendix Q-III-C. Classes 4 and 5 microorganisms pose a high level
of individual risk for acquiring life-threatening diseases to personnel
and/or animals. To import Class 5 agents, special approval must be
obtained from U.S. Department of Agriculture, Animal and Plant Health
Inspection Service, Import-Export Products, Room 756, Federal Building,
6505 Belcrest Road, Hyattsville, Maryland 20782.
Laboratory staff shall be required to have specific and thorough
training in handling extremely hazardous infectious agents, primary and
secondary containment, standard and special practices, and laboratory
design characteristics. The laboratory staff shall be supervised by
knowledgeable scientists who are trained and experienced in working
with these agents and in the special containment facilities.
Within work areas of the animal facility, all activities shall be
confined to the specially equipped animal rooms or support areas. The
maximum animal containment area and support areas shall have special
engineering and design features to prevent the dissemination of
microorganisms into the environment via exhaust air or waste disposal.
Appendix Q-III-D. Other research with non-laboratory animals, which
may not appropriately be conducted under conditions described in
Appendix Q, may be conducted safely by applying practices routinely
used for controlled culture of these biota. In aquatic systems, for
example, BL1 equivalent conditions could be met by utilizing growth
tanks that provide adequate physical means to avoid the escape of the
aquatic species, its gametes, and introduced exogenous genetic
material. A mechanism shall be provided to ensure that neither the
organisms nor their gametes can escape into the supply or discharge
system of the rearing container (e.g., tank, aquarium, etc.) Acceptable
barriers include appropriate filtration, irradiation, heat treatment,
chemical treatment, etc. Moreover, the top of the rearing container
shall be covered to avoid escape of the organism and its gametes. In
the event of tank rupture, leakage, or overflow, the construction of
the room containing these tanks should prevent the organisms and
gametes from entering the building's drains before the organism and its
gametes have been inactivated.
Other types of non-laboratory animals (e.g., nematodes, arthropods,
and certain forms of smaller animals) may be accommodated by using the
appropriate BL1 through BL4 or BL1-P through BL4-P containment
practices and procedures as specified in Appendices G and P.
OMB's ``Mandatory Information Requirements for Federal Assistance
Program Announcements'' (45 FR 39592) requires a statement concerning
the official government programs contained in the Catalog of Federal
Domestic Assistance. Normally NIH lists in its announcements the number
and title of affected individual programs for the guidance of the
public. Because the guidance in this notice covers not only virtually
every NIH program but also essentially every Federal research program
in which DNA recombinant molecule techniques could be used, it has been
determined to be not cost effective or in the public interest to
attempt to list these programs. Such a list would likely require
several additional pages. In addition, NIH could not be certain that
every Federal program would be included as many Federal agencies, as
well as private organizations, both national and international, have
elected to follow the NIH Guidelines. In lieu of the individual program
listing, NIH invites readers to direct questions to the information
address above about whether individual programs listed in the Catalog
of Federal Domestic Assistance are affected.
Effective Date: June 24, 1994.
Harold Varmus,
Director, National Institutes of Health.
[FR Doc. 94-16199 Filed 7-1-94; 8:45 am]
BILLING CODE 4140-01-P
