[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]


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[Federal Register: July 5, 1994]


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Part III





Department of Health and Human Services





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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 LD50 
100 nanograms per kilogram body weight in organisms other 
than Escherichia coli K-12 (see Section III-B-1 and Appendices F-I and 
F-II);
    Section IV-C-3-b. Reviewing and approving certain experiments 
involving the deliberate transfer of recombinant DNA or DNA or RNA 
derived from recombinant DNA into one or more human subjects, in 
consultation with the RAC Chair and one or more RAC members, as 
necessary, that qualify for the Accelerated Review process (see Section 
III-B-2);
    Section IV-C-3-c. Reviewing and approving minor changes to human 
gene transfer protocols approved under Sections III-A-2 and III-B-2, in 
consultation with the RAC Chair and one or more RAC members, as 
necessary;
    Section IV-C-3-d. Reviewing and approving the membership of an 
institution's Institutional Biosafety Committee, and where it finds the 
Institutional Biosafety Committee meets the requirements set forth in 
Section IV-B-2 will give its approval to the Institutional Biosafety 
Committee membership;
    Section IV-C-3-e. Publishing in the Federal Register:
    Section IV-C-3-e-(1). Announcements of RAC meetings and agendas at 
least 15 days in advance (Note--If the agenda for a RAC meeting is 
modified, ORDA shall make the revised agenda available to anyone upon 
request at least 72 hours in advance of the meeting);
    Section IV-C-3-e-(2). Proposed Major Actions to the NIH Guidelines 
(see Section IV-C-1-b-(1)) at least 15 days prior to the RAC meeting;
    Section IV-C-3-f. Serve as the focal point for data management of 
NIH-approved human gene transfer protocols approved under Sections III-
A-2 and III-B-2 and registered with NIH/ORDA as required under Section 
III-C-7;
    Section IV-C-3-g. Serve as the executive secretary of the RAC; and
    Section IV-C-3-h. Maintain a list of Major and Minor Actions 
approved under Section III-A-2 and III-B-3 and a list of experiments 
registered with NIH/ORDA as described in Section III-C-7.

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