Recombinant DNA Research: Actions Under the Guidelines; Notice DEPARTMENT OF HEALTH AND HUMAN SERVICES

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 50

100 nanograms per kilogram body weight in organisms other

than Escherichia coli K-12 (see Section III-B-1 and Appendices F-I and

F-II);

Section IV-C-3-b. Reviewing and approving certain experiments

involving the deliberate transfer of recombinant DNA or DNA or RNA

derived from recombinant DNA into one or more human subjects, in

consultation with the RAC Chair and one or more RAC members, as

necessary, that qualify for the Accelerated Review process (see Section

III-B-2);

Section IV-C-3-c. Reviewing and approving minor changes to human

gene transfer protocols approved under Sections III-A-2 and III-B-2, in

consultation with the RAC Chair and one or more RAC members, as

necessary;

Section IV-C-3-d. Reviewing and approving the membership of an

institution's Institutional Biosafety Committee, and where it finds the

Institutional Biosafety Committee meets the requirements set forth in

Section IV-B-2 will give its approval to the Institutional Biosafety

Committee membership;

Section IV-C-3-e. Publishing in the Federal Register:

Section IV-C-3-e-(1). Announcements of RAC meetings and agendas at

least 15 days in advance (Note--If the agenda for a RAC meeting is

modified, ORDA shall make the revised agenda available to anyone upon

request at least 72 hours in advance of the meeting);

Section IV-C-3-e-(2). Proposed Major Actions to the NIH Guidelines

(see Section IV-C-1-b-(1)) at least 15 days prior to the RAC meeting;

Section IV-C-3-f. Serve as the focal point for data management of

NIH-approved human gene transfer protocols approved under Sections III-

A-2 and III-B-2 and registered with NIH/ORDA as required under Section

III-C-7;

Section IV-C-3-g. Serve as the executive secretary of the RAC; and

Section IV-C-3-h. Maintain a list of Major and Minor Actions

approved under Section III-A-2 and III-B-3 and a list of experiments

registered with NIH/ORDA as described in Section III-C-7.

E. Amendment and Addition to Section V, Footnotes and References of

Sections I-IV of the NIH Guidelines

The amended version of Section V-U reads as follows:

Section V-U. Human studies in which the induction or enhancement of

an immune response to a vector-encoded microbial immunogen is the major

goal, such an immune response has been demonstrated in model systems,

and the persistence of the vector-encoded immunogen is not expected,

are not covered under Sections III-A-2, III-B-2, or III-B-3. Such

studies may be initiated without RAC review and NIH approval if

approved by another Federal agency.''

The following new footnote, V-W is added to Section V:

Section V-W. In accordance with accepted scientific and regulatory

practices of the discipline of plant pathology, an exotic plant

pathogen (e.g., virus, bacteria, or fungus) is one that is unknown to

occur within the U.S. (see Section V-R). Determination of whether a

pathogen has a potential for serious detrimental impact on managed

(agricultural, forest, grassland) or natural ecosystems should be made

by the Principal Investigator and the Institutional Biosafety

Committee, in consultation with scientists knowledgeable of plant

diseases, crops, and ecosystems in the geographic area of the research.

F. Addition to Appendix C-I, Recombinant DNA in Tissue Culture, of the

NIH Guidelines

The amended version of Appendix C-I reads as follows:

Appendix C-I. Recombinant DNA in Tissue Culture

Recombinant DNA molecules containing less than one-half of any

eukaryotic viral genome (all viruses from a single family (see Appendix

C-VI-D) being considered identical (see Appendix C-VI-E), that are

propagated and maintained in cells in tissue culture are exempt from

these NIH Guidelines with the exceptions listed in Appendix C-I-A.

Appendix C-I-A. Exceptions

The following categories are not exempt from the NIH Guidelines:

(i) Experiments described in Section III-A which require specific RAC

review and NIH and Institutional Biosafety Committee approval before

initiation, (ii) experiments described in Section III-B which require

NIH/ORDA and Institutional Biosafety Committee approval before

initiation, (iii) experiments involving DNA from Class 3, 4, or 5

organisms (see Appendix C-VI-A) or cells known to be infected with

these agents, (iv) experiments involving the deliberate introduction of

genes coding for the biosynthesis of molecules that are toxic for

vertebrates (see Appendix F), and (v) whole plants regenerated from

plant cells and tissue cultures are covered by the exemption provided

they remain axenic cultures even though they differentiate into

embryonic tissue and regenerate into plantlets.

G. Addition to Appendix G, Physical Containment, of the NIH Guidelines

Appendix G through G-I is amended to read as follows:

Appendix G specifies physical containment for standard laboratory

experiments and defines Biosafety Level 1 through Biosafety Level 4.

For large scale (over 10 liters) research or production, Appendix K

supersedes Appendix G. Appendix K defines Good Large Scale Practice

through Biosafety Level 3--Large Scale. For certain work with plants,

Appendix P supersedes Appendix G. Appendix P defines Biosafety Levels 1

through 4--Plants. For certain work with animals, Appendix Q supersedes

Appendix G. Appendix Q defines Biosafety Levels 1 through 4--Animals.

Appendix G-I. Standard Practices and Training

The first principle of containment is strict adherence to good

microbiological practices (see Appendices G-III-A through G-III-J).

Consequently, all personnel directly or indirectly involved in

experiments using recombinant DNA shall receive adequate instruction

(see Sections IV-B-1-e and IV-B-4-d). At a minimum, these instructions

include training in aseptic techniques and in the biology of the

organisms used in the experiments so that the potential biohazards can

be understood and appreciated.

Any research group working with agents that are known or potential

biohazards shall have an emergency plan that describes the procedures

to be followed if an accident contaminates personnel or the

environment. The Principal Investigator shall ensure that everyone in

the laboratory is familiar with both the potential hazards of the work

and the emergency plan (see Sections IV-B-4-d and IV-B-4-e). If a

research group is working with a known pathogen for which there is an

effective vaccine, the vaccine should be made available to all workers.

Serological monitoring, when clearly appropriate, will be provided (see

Section IV-B-1-f).

The Laboratory Safety Monograph (see Appendix G-III-O) and

Biosafety in Microbiological and Biomedical Laboratories (see Appendix

G-III-B) describe practices, equipment, and facilities in detail.

H. Addition of Appendix P, Physical and Biological Containment for

Recombinant DNA Research Involving Plants, to the NIH Guidelines

The following new appendix, Appendix P, reads as follows:

Appendix P specifies physical and biological containment conditions

and practices suitable to the greenhouse conduct of experiments

involving recombinant DNA-containing plants, plant-associated

microorganisms, and small animals. All provisions of the NIH Guidelines

apply to plant research activities with the following modifications:

Appendix P shall supersede Appendix G when the research plants are

of a size, number, or have growth requirements that preclude the use of

containment conditions described in Appendix G. The plants covered in

Appendix P include but are not limited to mosses, liverworts,

macroscopic algae, and vascular plants including terrestrial crops,

forest, and ornamental species.

Plant-associated microorganisms include viroids, virusoids,

viruses, bacteria, fungi, protozoans, certain small algae, and

microorganisms that have a benign or beneficial association with

plants, such as certain Rhizobium species and microorganisms known to

cause plant diseases. The appendix applies to microorganisms which are

being modified with the objective of fostering an association with

plants.

Plant-associated small animals include those arthropods that: (i)

Are in obligate association with plants, (ii) are plant pests, (iii)

are plant pollinators, or (iv) transmit plant disease agents, as well

as other small animals such as nematodes for which tests of biological

properties necessitate the use of plants. Microorganisms associated

with such small animals (e.g., pathogens or symbionts) are included.

The Institutional Biosafety Committee shall include at least one

individual with expertise in plant, plant pathogen, or plant pest

containment principles when experiments utilizing Appendix P require

prior approval by the Institutional Biosafety Committee.

Appendix P-I. General Plant Biosafety Levels

Appendix P-I-A. The principal purpose of plant containment is to

avoid the unintentional transmission of a recombinant DNA-containing

plant genome, including nuclear or organelle hereditary material or

release of recombinant DNA-derived organisms associated with plants.

Appendix P-I-B. The containment principles are based on the

recognition that the organisms that are used pose no health threat to

humans or higher animals (unless deliberately modified for that

purpose), and that the containment conditions minimize the possibility

of an unanticipated deleterious effect on organisms and ecosystems

outside of the experimental facility, e.g., the inadvertent spread of a

serious pathogen from a greenhouse to a local agricultural crop or the

unintentional introduction and establishment of an organism in a new

ecosystem.

Appendix P-I-C. Four biosafety levels, referred to as Biosafety

Level (BL) 1--Plants (P), BL2-P, BL3-P, and BL4-P, are established in

Section II. The selection of containment levels required for research

involving recombinant DNA molecules in plants or associated with plants

is specified in Section III. These biosafety levels are described in

Appendix P-II. This appendix describes greenhouse practices and special

greenhouse facilities for physical containment.

Appendix P-I-D. BL1-P through BL4-P are designed to provide

differential levels of biosafety for plants in the absence or presence

of other experimental organisms that contain recombinant DNA. These

biosafety levels, in conjunction with biological containment conditions

described in Appendix P-III, provide flexible approaches to ensure the

safe conduct of research.

Appendix P-I-E. For experiments in which plants are grown at the

BL1 through BL4 laboratory settings, containment practices shall be

followed as described in Appendix G. These containment practices

include the use of plant tissue culture rooms, growth chambers within

laboratory facilities, or experiments performed on open benches.

Additional biological containment practices should be added by the

Greenhouse Director or Institutional Biosafety Committee as necessary

(see Appendix P-III), if botanical reproductive structures are produced

that have the potential of being released.

Appendix P-II. Physical Containment Levels

Appendix P-II-A. Biosafety Level 1--Plants (BL1-P)

Appendix P-II-A-1. Standard Practices (BL1-P)

Appendix P-II-A-1-a. Greenhouse Access (BL1-P)

Appendix P-II-A-1-a-(1). Access to the greenhouse shall be limited

or restricted, at the discretion of the Greenhouse Director, when

experiments are in progress.

Appendix P-II-A-1-a-(2). Prior to entering the greenhouse,

personnel shall be required to read and follow instructions on BL1-P

greenhouse practices and procedures. All procedures shall be performed

in accordance with accepted greenhouse practices that are appropriate

to the experimental organism.

Appendix P-II-A-1-b. Records (BL1-P)

Appendix P-II-A-1-b-(1). A record shall be kept of experiments

currently in progress in the greenhouse facility.

Appendix P-II-A-1-c. Decontamination and Inactivation (BL1-P)

Appendix P-II-A-1-c-(1). Experimental organisms shall be rendered

biologically inactive by appropriate methods before disposal outside of

the greenhouse facility.

Appendix P-II-A-1-d. Control of Undesired Species and Motile

Macroorganisms (BL1-P)

Appendix P-II-A-1-d-(1). A program shall be implemented to control

undesired species (e.g., weed, rodent, or arthropod pests and

pathogens), by methods appropriate to the organisms and in accordance

with applicable state and Federal laws.

Appendix P-II-A-1-d-(2). Arthropods and other motile macroorganisms

shall be housed in appropriate cages. If macroorganisms (e.g., flying

arthropods or nematodes) are released within the greenhouse,

precautions shall be taken to minimize escape from the greenhouse

facility.

Appendix P-II-A-1-e. Concurrent Experiments Conducted in the Greenhouse

(BL1-P)

Appendix P-II-A-1-e-(1). Experiments involving other organisms that

require a containment level lower than BL1-P may be conducted in the

greenhouse concurrently with experiments that require BL1-P

containment, provided that all work is conducted in accordance with

BL1-P greenhouse practices.

Appendix P-II-A-2. Facilities (BL1-P)

Appendix P-II-A-2-a. Definitions (BL1-P)

Appendix P-II-A-2-a-(1). The term `greenhouse' refers to a

structure with walls, a roof, and a floor designed and used principally

for growing plants in a controlled and protected environment. The walls

and roof are usually constructed of transparent or translucent material

to allow passage of sunlight for plant growth.

Appendix P-II-A-2-a-(2). The term `greenhouse facility' includes

the actual greenhouse rooms or compartments for growing plants,

including all immediately contiguous hallways and head-house areas, and

is considered part of the confinement area.

Appendix P-II-A-2-b. Greenhouse Design (BL1-P)

Appendix P-II-A-2-b-(1). The greenhouse floor may be composed of

gravel or other porous material. At a minimum, impervious (e.g.,

concrete) walkways are recommended.

Appendix P-II-A-2-b-(2). Windows and other openings in the walls

and roof of the greenhouse facility may be open for ventilation as

needed for proper operation and do not require any special barrier to

contain or exclude pollen, microorganisms, or small flying animals

(e.g., arthropods and birds); however, screens are recommended.

Appendix P-II-B. Biosafety Level 2--Plants (BL2-P)

Appendix P-II-B-1. Standard Practices (BL2-P)

Appendix P-II-B-1-a. Greenhouse Access (BL2-P)

Appendix P-II-B-1-a-(1). Access to the greenhouse shall be limited

or restricted, at the discretion of the Greenhouse Director, to

individuals directly involved with the experiments when they are in

progress.

Appendix P-II-B-1-a-(2). Personnel shall be required to read and

follow instructions on BL2-P practices and procedures. All procedures

shall be conducted in accordance with accepted greenhouse practices

that are appropriate to the experimental organisms.

Appendix P-II-B-1-b. Records (BL2-P)

Appendix P-II-B-1-b-(1). A record shall be kept of experimental

plants, microorganisms, or small animals that are brought into or

removed from the greenhouse facility.

Appendix P-II-B-1-b-(2). A record shall be kept of experiments

currently in progress in the greenhouse facility.

Appendix P-II-B-1-b-(3). The Principal Investigator shall report

any greenhouse accident involving the inadvertent release or spill of

microorganisms to the Greenhouse Director, Institutional Biosafety

Committee, NIH/ORDA and other appropriate authorities immediately (if

applicable). Reports to the NIH/ORDA shall be sent to the Office of

Recombinant DNA Activities, National Institutes of Health, Building 31,

Room 4B11, Bethesda, Maryland 20892, (301) 496-9838. Documentation of

any such accident shall be prepared and maintained.

Appendix P-II-B-1-c. Decontamination and Inactivation (BL2-P)

Appendix P-II-B-1-c-(1). Experimental organisms shall be rendered

biologically inactive by appropriate methods before disposal outside of

the greenhouse facility.

Appendix P-II-B-1-c-(2). Decontamination of run-off water is not

necessarily required. If part of the greenhouse is composed of gravel

or similar material, appropriate treatments should be made periodically

to eliminate, or render inactive, any organisms potentially entrapped

by the gravel.

Appendix P-II-B-1-d. Control of Undesired Species and Motile

Macroorganisms (BL2-P)

Appendix P-II-B-1-d-(1). A program shall be implemented to control

undesired species (e.g., weed, rodent, or arthropod pests and

pathogens) by methods appropriate to the organisms and in accordance

with applicable state and Federal laws.

Appendix P-II-B-1-d-(2). Arthropods and other motile macroorganisms

shall be housed in appropriate cages. If macroorganisms (e.g., flying

arthropods or nematodes) are released within the greenhouse,

precautions shall be taken to minimize escape from the greenhouse

facility.

Appendix P-II-B-1-e. Concurrent Experiments Conducted in the Greenhouse

(BL2-P)

Appendix P-II-B-1-e-(1). Experiments involving other organisms that

require a containment level lower than BL2-P may be conducted in the

greenhouse concurrently with experiments that require BL2-P containment

provided that all work is conducted in accordance with BL2-P greenhouse

practices.

Appendix P-II-B-1-f. Signs (BL2-P)

Appendix P-II-B-1-f-(1). A sign shall be posted indicating that a

restricted experiment is in progress. The sign shall indicate the

following: (i) the name of the responsible individual, (ii) The plants

in use, and (iii) any special requirements for using the area.

Appendix P-II-B-1-f-(2). If organisms are used that have a

recognized potential for causing serious detrimental impacts on managed

or natural ecosystems, their presence shall be indicated on a sign

posted on the greenhouse access doors.

Appendix P-II-B-1-f-(3). If there is a risk to human health, a sign

shall be posted incorporating the universal biosafety symbol.

Appendix P-II-B-1-g. Transfer of Materials (BL2-P)

Appendix P-II-B-1-g-(1). Materials containing experimental

microorganisms, which are brought into or removed from the greenhouse

facility in a viable or intact state, shall be transferred in a closed

non-breakable container.

Appendix P-II-B-1-h. Greenhouse Practices Manual (BL2-P)

Appendix P-II-B-1-h-(1). A greenhouse practices manual shall be

prepared or adopted. This manual shall: (i) Advise personnel of the

potential consequences if such practices are not followed, and (ii)

outline contingency plans to be implemented in the event of the

unintentional release of organisms.

Appendix P-II-B-2. Facilities (BL2-P)

Appendix P-II-B-2-a. Definitions (BL2-P)

Appendix P-II-B-2-a-(1). The term `greenhouse' refers to a

structure with walls, a roof, and a floor designed and used principally

for growing plants in a controlled and protected environment. The walls

and roof are usually constructed of transparent or translucent material

to allow passage of sunlight for plant growth.

Appendix P-II-B-2-a-(2). The term `greenhouse facility' includes

the actual greenhouse rooms or compartments for growing plants,

including all immediately contiguous hallways and head-house areas and

is considered part of the confinement area.

Appendix P-II-B-2-b. Greenhouse Design (BL2-P)

Appendix P-II-B-2-b-(1). A greenhouse floor composed of an

impervious material. Concrete is recommended, but gravel or other

porous material under benches is acceptable unless propagules of

experimental organisms are readily disseminated through soil. Soil beds

are acceptable unless propagules of experimental organisms are readily

disseminated through soil.

Appendix P-II-B-2-b-(2). Windows and other openings in the walls

and roof of the greenhouse facility may be open for ventilation as

needed for proper operation and do not require any special barrier to

exclude pollen or microorganisms; however, screens are required to

exclude small flying animals (e.g., arthropods and birds).

Appendix P-II-B-2-c. Autoclaves (BL2-P)

Appendix P-II-B-2-c-(1). An autoclave shall be available for the

treatment of contaminated greenhouse materials.

Appendix P-II-B-2-d. Supply and Exhaust Air Ventilation Systems (BL2-P)

Appendix P-II-B-2-d-(1). If intake fans are used, measures shall be

taken to minimize the ingress of arthropods. Louvers or fans shall be

constructed such that they can only be opened when the fan is in

operation.

Appendix P-II-B-2-e. Other (BL2-P)

Appendix P-II-B-2-e-(1). BL2-P greenhouse containment requirements

may be satisfied by using a growth chamber or growth room within a

building provided that the external physical structure limits access

and escape of microorganisms and macroorganisms in a manner that

satisfies the intent of the foregoing clauses.

Appendix P-II-C. Biosafety Level 3--Plants (BL3-P)

Appendix P-II-C-1. Standard Practices (BL3-P)

Appendix P-II-C-1-a. Greenhouse Access (BL3-P)

Appendix P-II-C-1-a-(1). Authorized entry into the greenhouse shall

be restricted to individuals who are required for program or support

purposes. The Greenhouse Director shall be responsible for assessing

each circumstance and determining those individuals who are authorized

to enter the greenhouse facility.

Appendix P-II-C-1-a-(2). Prior to entering the greenhouse,

personnel shall be required to read and follow instructions on BL3-P

practices and procedures. All procedures shall be conducted in

accordance with accepted greenhouse practices that are appropriate to

the experimental organisms.

Appendix P-II-C-1-b. Records (BL3-P)

Appendix P-II-C-1-b-(1). A record shall be kept of experimental

plants, microorganisms, or small animals that are brought into or

removed from the greenhouse facility.

Appendix P-II-C-1-b-(2). A record shall be kept of experiments

currently in progress in the greenhouse facility.

Appendix P-II-C-1-b-(3). The Principal Investigator shall report

any greenhouse accident involving the inadvertent release or spill of

microorganisms to the Biological Safety Officer, Greenhouse Director,

Institutional Biosafety Committee, NIH/ORDA, and other appropriate

authorities immediately (if applicable).

Reports to the NIH/ORDA shall be sent to the Office of Recombinant

DNA Activities, National Institutes of Health, Building 31, Room 4B11,

Bethesda, Maryland 20892, (301) 496-9838. Documentation of any such

accident shall be prepared and maintained.

Appendix P-II-C-1-c. Decontamination and Inactivation (BL3-P)

Appendix P-II-C-1-c-(1). All experimental materials shall be

sterilized in an autoclave or rendered biologically inactive by

appropriate methods before disposal, except those that are to remain in

a viable or intact state for experimental purposes; including water

that comes in contact with experimental microorganisms or with material

exposed to such microorganisms, and contaminated equipment and

supplies.

Appendix P-II-C-1-d. Control of Undesired Species and Motile

Macroorganisms (BL3-P)

Appendix P-II-C-1-d-(1). A program shall be implemented to control

undesired species (e.g., weed, rodent, or arthropod pests and

pathogens) by methods appropriate to the organisms and in accordance

with applicable state and Federal laws.

Appendix P-II-C-1-d-(2). Arthropods and other motile macroorganisms

shall be housed in appropriate cages. When appropriate to the organism,

experiments shall be conducted within cages designed to contain the

motile organisms.

Appendix P-II-C-1-e. Concurrent Experiments Conducted in the Greenhouse

(BL3-P)

Appendix P-II-C-1-e-(1). Experiments involving organisms that

require a containment level lower than BL3-P may be conducted in the

greenhouse concurrently with experiments that require BL3-P containment

provided that all work is conducted in accordance with BL3-P greenhouse

practices.

Appendix P-II-C-1-f. Signs (BL3-P)

Appendix P-II-C-1-f-(1). A sign shall be posted indicating that a

restricted experiment is in progress. The sign shall indicate the

following: (i) The name of the responsible individual, (ii) the plants

in use, and (iii) any special requirements for using the area.

Appendix P-II-C-1-f-(2). If organisms are used that have a

recognized potential for causing serious detrimental impacts on managed

or natural ecosystems, their presence should be indicated on a sign

posted on the greenhouse access doors.

Appendix P-II-C-1-f-(3). If there is a risk to human health, a sign

shall be posted incorporating the universal biosafety symbol.

Appendix P-II-C-1-g. Transfer of Materials (BL3-P)

Appendix P-II-C-1-g-(1). Experimental materials that are brought

into or removed from the greenhouse facility in a viable or intact

state shall be transferred to a non-breakable sealed secondary

container. At the time of transfer, if the same plant species, host, or

vector are present within the effective dissemination distance of

propagules of the experimental organism, the surface of the secondary

container shall be decontaminated. Decontamination may be accomplished

by passage through a chemical disinfectant or fumigation chamber or by

an alternative procedure that has demonstrated effective inactivation

of the experimental organism.

Appendix P-II-C-1-h. Greenhouse Practices Manual (BL3-P)

Appendix P-II-C-1-h-(1). A greenhouse practices manual shall be

prepared or adopted. This manual shall: (i) Advise personnel of the

potential consequences if such practices are not followed, and (ii)

outline contingency plans to be implemented in the event of the

unintentional release of organisms with recognized potential for

serious detrimental impact.

Appendix P-II-C-1-i. Protective Clothing (BL3-P)

Appendix P-II-C-1-i-(1). Disposable clothing (e.g., solid front or

wrap-around gowns, scrub suits, or other appropriate clothing) shall be

worn in the greenhouse if deemed necessary by the Greenhouse Director

because of potential dissemination of the experimental microorganisms.

Appendix P-II-C-1-i-(2). Protective clothing shall be removed

before exiting the greenhouse and decontaminated prior to laundering or

disposal.

Appendix P-II-C-1-j. Other (BL3-P)

Appendix P-II-C-1-j-(1). Personnel are required to thoroughly wash

their hands upon exiting the greenhouse.

Appendix P-II-C-1-j-(2). All procedures shall be performed

carefully to minimize the creation of aerosols and excessive splashing

of potting material/soil during watering, transplanting, and all

experimental manipulations.

Appendix P-II-C-2. Facilities (BL3-P)

Appendix P-II-C-2-a. Definitions (BL3-P)

Appendix P-II-C-2-a-(1). The term 'greenhouse' refers to a

structure with walls, roof, and floor designed and used principally for

growing plants in a controlled and protected environment. The walls and

roof are usually constructed of transparent or translucent material to

allow passage of sunlight for plant growth.

Appendix P-II-C-2-a-(2). The term 'greenhouse facility' includes

the actual greenhouse rooms or compartments for growing plants,

including all immediately contiguous hallways and head-house areas, and

is considered part of the confinement area. The need to maintain

negative pressure should be considered when constructing or renovating

the greenhouse.

Appendix P-II-C-2-b. Greenhouse Design (BL3-P)

Appendix P-II-C-2-b-(1). The greenhouse floor shall be composed of

concrete or other impervious material with provision for collection and

decontamination of liquid run-off.

Appendix P-II-C-2-b-(2). Windows shall be closed and sealed. All

glazing shall be resistant to breakage (e.g., double-pane tempered

glass or equivalent).

Appendix P-II-C-2-b-(3). The greenhouse shall be a closed self-

contained structure with a continuous covering that is separated from

areas that are open to unrestricted traffic flow. The minimum

requirement for greenhouse entry shall be passage through two sets of

self-closing locking doors.

Appendix P-II-C-2-b-(4). The greenhouse facility shall be

surrounded by a security fence or protected by equivalent security

measures.

Appendix P-II-C-2-b-(5). Internal walls, ceilings, and floors shall

be resistant to penetration by liquids and chemicals to facilitate

cleaning and decontamination of the area. All penetrations into these

structures and surfaces (e.g., plumbing and utilities) shall be sealed.

Appendix P-II-C-2-b-(6). Bench tops and other work surfaces should

have seamless surfaces that are impervious to water and resistant to

acids, alkalis, organic solvents, and moderate heat.

Appendix P-II-C-2-b-(7). The greenhouse contains a foot, elbow, or

automatically operated sink, which is located near the exit door for

hand washing.

Appendix P-II-C-2-c. Autoclaves (BL3-P)

Appendix P-II-C-2-c-(1). An autoclave shall be available for

decontaminating materials within the greenhouse facility. A double-door

autoclave is recommended (not required) for the decontamination of

materials passing out of the greenhouse facility.

Appendix P-II-C-2-d. Supply and Exhaust Air Ventilation Systems (BL3-P)

Appendix P-II-C-2-d-(1). An individual supply and exhaust air

ventilation system shall be provided. The system maintains pressure

differentials and directional airflow, as required, to assure inward

(or zero) airflow from areas outside of the greenhouse.

Appendix P-II-C-2-d-(2). The exhaust air from the greenhouse

facility shall be filtered through high efficiency particulate air-HEPA

filters and discharged to the outside. The filter chambers shall be

designed to allow in situ decontamination before filters are removed

and to facilitate certification testing after they are replaced. Air

filters shall be 80-85% average efficiency by the American Society of

Heating, Refrigerating, and Air Conditioning Engineers (ASHRAE)

Standard 52- 68 test method using atmosphere dust. Air supply fans

shall be equipped with a back-flow damper that closes when the air

supply fan is off. Alternatively, a HEPA filter may be used on the air

supply system instead of the filters and damper. The supply and exhaust

airflow shall be interlocked to assure inward (or zero) airflow at all

times.

Appendix P-II-C-2-e. Other (BL3-P)

Appendix P-II-C-2-e-(1). BL3-P greenhouse containment requirements

may be satisfied using a growth chamber or growth room within a

building provided that the location, access, airflow patterns, and

provisions for decontamination of experimental materials and supplies

meet the intent of the foregoing clauses.

Appendix P-II-C-2-e-(2). Vacuum lines shall be protected with high

efficiency particulate air/HEPA or equivalent filters and liquid

disinfectant traps.

Appendix P-II-D. Biosafety Level 4--Plants (BL4-P)

Appendix P-II-D-1. Standard Practices (BL4-P)

Appendix P-II-D-1-a. Greenhouse Access (BL4-P)

Appendix P-II-D-1-a-(1). Authorized entry into the greenhouse shall

be restricted to individuals who are required for program or support

purposes. The Greenhouse Director shall be responsible for assessing

each circumstance and determining those individuals who are authorized

to enter the greenhouse facility or work in the greenhouse during

experiments.

Appendix P-II-D-1-a-(2). Access shall be managed by the Greenhouse

Director, Biological Safety Officer, or other individual responsible

for physical security of the greenhouse facility; and access limited by

means of secure, locked doors.

Appendix P-II-D-1-a-(3). Prior to entering, individuals shall be

advised of the potential environmental hazards and instructed on

appropriate safeguards for ensuring environmental safety. Individuals

authorized to enter the greenhouse facility shall comply with the

instructions and all other applicable entry/exit procedures.

Appendix P-II-D-1-a-(4). Personnel shall enter and exit the

greenhouse facility only through the clothing change and shower rooms

and shall shower each time they exit the greenhouse facility. Personnel

shall use the airlocks to enter or exit the laboratory only in an

emergency. In the event of an emergency, every reasonable effort should

be made to prevent the possible transport of viable propagules from

containment.

Appendix P-II-D-1-a-(5). Prior to entering the greenhouse,

personnel shall be required to read and follow instructions on BL4-P

practices and procedures.

Appendix P-II-D-1-b. Records (BL4-P)

Appendix P-II-D-1-b-(1). A record shall be kept of all experimental

materials brought into or removed from the greenhouse.

Appendix P-II-D-1-b-(2). A record shall be kept of experiments

currently in progress in the greenhouse facility.

Appendix P-II-D-1-b-(3). A record shall be kept of all personnel

entering and exiting the greenhouse facility, including the date and

time of each entry.

Appendix P-II-D-1-b-(4). The Principal Investigator shall report

any greenhouse accident involving the inadvertent release or spill of

microorganisms to the Biological Safety Officer, Greenhouse Director,

Institutional Biosafety Committee, NIH/ORDA, and other appropriate

authorities immediately (if applicable). Reports to the NIH/ORDA shall

be sent to the Office of Recombinant DNA Activities, National

Institutes of Health, Building 31, Room 4B11, Bethesda, Maryland 20892,

(301) 496-9838. Documentation of any such accident shall be prepared

and maintained.

Appendix P-II-D-1-c. Decontamination and Inactivation (BL4-P)

Appendix P-II-D-1-c-(1). All materials, except for those that are

to remain in a viable or intact state for experimental purposes, shall

be autoclaved prior to removal from the maximum containment greenhouse.

Equipment or material that could be damaged by high temperatures or

steam shall be decontaminated by alternative methods (e.g., gas or

vapor sterilization) in an airlock or chamber designed for this

purpose.

Appendix P-II-D-1-c-(2). Water that comes in contact with

experimental microorganisms or with material exposed to such

microorganisms (e.g., run-off from watering plants) shall be collected

and decontaminated before disposal.

Appendix P-II-D-1-c-(3). Standard microbiological procedures shall

be followed for decontamination of equipment and materials. Spray or

liquid waste or rinse water from containers used to apply the

experimental microorganisms shall be decontaminated before disposal.

Appendix P-II-D-1-d. Control of Undesired Species and Motile

Macroorganisms (BL4-P)

Appendix P-II-D-1-d-(1). A chemical control program shall be

implemented to eliminate undesired pests and pathogens in accordance

with applicable state and Federal laws.

Appendix P-II-D-1-d-(2). Arthropods and other motile macroorganisms

used in conjunction with experiments requiring BL4-P level physical

containment shall be housed in appropriate cages. When appropriate to

the organism, experiments shall be conducted within cages designed to

contain the motile organisms.

Appendix P-II-D-1-e. Concurrent Experiments Conducted in the Greenhouse

(BL4-P)

Appendix P-II-D-1-e-(1). Experiments involving organisms that

require a containment level lower than BL4-P may be conducted in the

greenhouse concurrently with experiments that require BL4-P containment

provided that all work is conducted in accordance with BL4-P greenhouse

practices. When the experimental microorganisms in use require a

containment level lower than BL4-P, greenhouse practices reflect the

level of containment required by the highest containment level

microorganisms being tested.

Appendix P-II-D-1-f. Signs (BL4-P)

Appendix P-II-D-1-f-(1). A sign shall be posted indicating that a

restricted experiment is in progress. The sign shall indicate the

following: (i) The name of the responsible individual, (ii) the plants

in use, and (iii) any special requirements for using the area.

Appendix P-II-D-1-f-(2). If organisms are used that have a

recognized potential for causing serious detrimental impacts on managed

or natural ecosystems, their presence shall be indicated by a sign

posted on the greenhouse access doors.

Appendix P-II-D-1-f-(3). If there is a risk to human health, a sign

shall be posted incorporating the universal biosafety symbol.

Appendix P-II-D-1-g. Transfer of Materials (BL4-P)

Appendix P-II-D-1-g-(1). Experimental materials that are brought

into or removed from the greenhouse in a viable or intact state shall

be transferred to a non-breakable, sealed, primary container then

enclosed in a non-breakable, sealed secondary container. These

containers shall be removed from the greenhouse facility through a

chemical disinfectant, fumigation chamber, or an airlock designed for

this purpose.

Appendix P-II-D-g-(2). Supplies and materials shall be brought into

the greenhouse facility through a double-door autoclave, fumigation

chamber, or airlock that is appropriately decontaminated between each

use. After securing the outer doors, personnel within the greenhouse

facility shall retrieve the materials by opening the interior door of

the autoclave, fumigation chamber, or airlock. These doors shall be

secured after the materials are brought into the greenhouse facility.

Appendix P-II-D-1-h. Greenhouse Practices Manual (BL4-P)

Appendix P-II-D-1-h-(1). A greenhouse practices manual shall be

prepared or adopted. This manual shall include contingency plans to be

implemented in the event of the unintentional release of experimental

organisms.

Appendix P-II-D-1-i. Protective Clothing (BL4-P)

Appendix P-II-D-1-i-(1). Street clothing shall be removed in the

outer clothing change room. Complete laboratory clothing (may be

disposable) including undergarments, pants, and shirts, jump suits,

shoes, and hats shall be provided and worn by all personnel entering

the greenhouse facility.

Appendix P-II-D-1-i-(2). Personnel shall remove laboratory clothing

when exiting the greenhouse facility and before entering the shower

area. This clothing shall be stored in a locker or hamper in the inner

change room.

Appendix P-II-D-1-i-(3). All laboratory clothing shall be

autoclaved before laundering.

Appendix P-II-D-2. Facilities (BL4-P)

Appendix P-II-D-2-a. Greenhouse Design (BL4-P)

Appendix P-II-D-2-a-(1). The maximum containment greenhouse

facility shall consist of a separate building or a clearly demarcated

and isolated area within a building. The need to maintain negative

pressure should be considered when constructing or renovating the

greenhouse facility.

Appendix P-II-D-2-a-(2). Outer and inner change rooms, separated by

a shower, shall be provided for personnel entering and exiting the

greenhouse facility.

Appendix P-II-D-2-a-(3). Windows shall be closed and sealed. All

glazing shall be resistant to breakage (e.g., double-pane tempered

glass or equivalent).

Appendix P-II-D-2-a-(4). Access doors to the greenhouse shall be

self-closing and locking.

Appendix P-II-D-2-a-(5). The greenhouse facility shall be

surrounded by a security fence or protected by equivalent security

measures.

Appendix P-II-D-2-a-(6). The walls, floors, and ceilings of the

greenhouse shall be constructed to form a sealed internal shell that

facilitates fumigation and is animal and arthropod-proof. These

internal surfaces shall be resistant to penetration and degradation by

liquids and chemicals to facilitate cleaning and decontamination of the

area. All penetrations into these structures and surfaces (e.g.,

plumbing and utilities) shall be sealed.

Appendix P-II-D-2-a-(7). Bench tops and other work surfaces shall

have seamless surfaces impervious to water and resistant to acids,

alkalis, organic solvents, and moderate heat.

Appendix P-II-D-2-a-(8). A double-door autoclave, fumigation

chamber, or ventilated airlock shall be provided for passage of all

materials, supplies, or equipment that are not brought into the

greenhouse facility through the change room.

Appendix P-II-D-2-b. Autoclaves (BL4-P)

Appendix P-II-D-2-b-(1). A double-door autoclave shall be provided

for the decontamination of materials removed from the greenhouse

facility. The autoclave door, which opens to the area external to the

greenhouse facility, shall be sealed to the outer wall and

automatically controlled so that it can only be opened upon completion

of the sterilization cycle.

Appendix P-II-D-2-c. Supply and Exhaust Air Ventilation Systems

(BL4-P)

Appendix P-II-D-2-c-(1). An individual supply and exhaust air

ventilation system shall be provided. The system shall maintain

pressure differentials and directional airflow as required to assure

inward (or zero) airflow from areas outside of the greenhouse.

Differential pressure transducers shall be used to sense pressure

levels. If a system malfunctions, the transducers shall sound an alarm.

A backup source of power should be considered. The supply and exhaust

airflow shall be interlocked to assure inward (or zero) airflow at all

times. The integrity of the greenhouse shall have an air leak rate

(decay rate) not to exceed 7 percent per minute (logarithm of pressure

against time) over a 20-minute period at 2 inches of water gauge

pressure. Nominally, this is 0.05 inches of water gauge pressure loss

in 1 minute at 2 inches water gauge pressure.

Appendix P-II-D-2-c-(2). Exhaust air from the greenhouse facility

shall be filtered through high efficiency particulate air/HEPA filters

and discharged to the outside and dispersed away from occupied

buildings and air intakes. Filter chambers shall be designed to allow

in situ decontamination before filters are removed and to facilitate

certification testing after they are replaced. HEPA filters shall be

provided to treat air supplied to the greenhouse facility. HEPA filters

shall be certified annually.

Appendix P-II-D-2-d. Other (BL4-P)

Appendix P-II-D-2-d-(1). Sewer vents and other ventilation lines

contain high efficiency particulate air/HEPA filters. HEPA filters

shall be certified annually.

Appendix P-II-D-2-d-(2). A pass-through dunk tank, fumigation

chamber, or an equivalent method of decontamination shall be provided

to ensure decontamination of materials and equipment that cannot be

decontaminated in the autoclave.

Appendix P-II-D-2-d-(3). Liquid effluent from sinks, floors, and

autoclave chambers shall be decontaminated by heat or chemical

treatment before being released from the maximum containment greenhouse

facility. Liquid wastes from shower rooms and toilets may be

decontaminated by heat or chemical treatment. Autoclave and chemical

decontamination of liquid wastes shall be evaluated by appropriate

standard procedures for autoclaved wastes. Decontamination shall be

evaluated mechanically and biologically using a recording thermometer

and an indicator microorganism with a defined heat susceptibility

pattern. If liquid wastes are decontaminated with chemical

disinfectants, the chemicals used must have demonstrated efficacy

against the target or indicator microorganisms.

Appendix P-II-D-2-d-(4). If there is a central vacuum system, it

shall not serve areas outside of the greenhouse facility. In-line high

efficiency particulate air/HEPA filters shall be placed as near as

practicable to each use point or vacuum service cock. Other liquid and

gas services to the greenhouse facility shall be protected by devices

that prevent back-flow. HEPA filters shall be certified annually.

Appendix P-III. Biological Containment Practices

Appropriate selection of the following biological containment

practices may be used to meet the containment requirements for a given

organism. The present list is not exhaustive; there may be other ways

of preventing effective dissemination that could possibly lead to the

establishment of the organism or its genetic material in the

environment resulting in deleterious consequences to managed or natural

ecosystems.

Appendix P-III-A. Biological Containment Practices (Plants)

Appendix P-III-A-1. Effective dissemination of plants by pollen or

seed can be prevented by one or more of the following procedures: (i)

Cover the reproductive structures to prevent pollen dissemination at

flowering and seed dissemination at maturity; (ii) remove reproductive

structures by employing male sterile strains, or harvest the plant

material prior to the reproductive stage; (iii) ensure that

experimental plants flower at a time of year when cross-fertile plants

are not flowering within the normal pollen dispersal range of the

experimental plant; or (iv) ensure that cross-fertile plants are not

growing within the known pollen dispersal range of the experimental

plant.

Appendix P-III-B. Biological Containment Practices (Microorganisms)

Appendix P-III-B-1. Effective dissemination of microorganisms

beyond the confines of the greenhouse can be prevented by one or more

of the following procedures: (i) Confine all operations to injections

of microorganisms or other biological procedures (including genetic

manipulation) that limit replication or reproduction of viruses and

microorganisms or sequences derived from microorganisms, and confine

these injections to internal plant parts or adherent plant surfaces;

(ii) ensure that organisms, which can serve as hosts or promote the

transmission of the virus or microorganism, are not present within the

farthest distance that the airborne virus or microorganism may be

expected to be effectively disseminated; (iii) conduct experiments at a

time of year when plants that can serve as hosts are either not growing

or are not susceptible to productive infection; (iv) use viruses and

other microorganisms or their genomes that have known arthropod or

animal vectors, in the absence of such vectors; (v) use microorganisms

that have an obligate association with the plant; or (vi) use

microorganisms that are genetically disabled to minimize survival

outside of the research facility and whose natural mode of transmission

requires injury of the target organism, or assures that inadvertent

release is unlikely to initiate productive infection of organisms

outside of the experimental facility.

Appendix P-III-C. Biological Containment Practices (Macroorganisms)

Appendix P-III-C-1. Effective dissemination of arthropods and other

small animals can be prevented by using one or more of the following

procedures: (i) Use non-flying, flight-impaired, or sterile arthropods;

(ii) use non-motile or sterile strains of small animals; (iii) conduct

experiments at a time of year that precludes the survival of escaping

organisms; (iv) use animals that have an obligate association with a

plant that is not present within the dispersal range of the organism;

or (v) prevent the escape of organisms present in run-off water by

chemical treatment or evaporation of run-off water.

I. Addition of Appendix Q, Physical and Biological Containment for

Recombinant DNA Research Involving Animals, to the NIH Guidelines

The following new appendix, Appendix Q, reads as follows:

Appendix Q specifies containment and confinement practices for

research involving whole animals, both those in which the animal's

genome has been altered by stable introduction of recombinant DNA, or

DNA derived therefrom, into the germ-line (transgenic animals) and

experiments involving viable recombinant DNA-modified microorganisms

tested on whole animals. The appendix applies to animal research

activities with the following modifications:

Appendix Q shall supersede Appendix G when research animals are of

a size or have growth requirements that preclude the use of containment

for laboratory animals. Some animals may require other types of

containment (see Appendix Q-III-D). The animals covered in Appendix Q

are those species normally categorized as animals including but not

limited to cattle, swine, sheep, goats, horses, and poultry.

The Institutional Biosafety Committee shall include at least one

scientist with expertise in animal containment principles when

experiments utilizing Appendix Q require Institutional Biosafety

Committee prior approval.

The institution shall establish and maintain a health surveillance

program for personnel engaged in animal research involving viable

recombinant DNA-containing microorganisms that require Biosafety Level

(BL) 3 or greater containment in the laboratory.

Appendix Q-I. General Considerations

Appendix Q-I-A. Containment Levels

The containment levels required for research involving recombinant

DNA associated with or in animals is based on classification of

experiments in Section III. For the purpose of animal research, four

levels of containment are established. These are referred to as BL1-

Animals (N), BL2-N, BL3-N, and BL4-N and are described in the following

sections of Appendix Q. The descriptions include: (i) standard

practices for physical and biological containment, and (ii) animal

facilities.

Appendix Q-I-B. Disposal of Animals (BL1-N through BL4-N)

Appendix Q-I-B-1. When an animal covered by Appendix Q containing

recombinant DNA or a recombinant DNA-derived organism is euthanized or

dies, the carcass shall be disposed of to avoid its use as food for

human beings or animals unless food use is specifically authorized by

an appropriate Federal agency.

Appendix Q-I-B-2. A permanent record shall be maintained of the

experimental use and disposal of each animal or group of animals.

Appendix Q-II. Physical and Biological Containment Levels

Appendix Q-II-A. Biosafety Level 1 - Animals (BL1-N)

Appendix Q-II-A-1. Standard Practices (BL1-N)

Appendix Q-II-A-1-a. Animal Facility Access (BL1-N)

Appendix Q-II-A-1-a-(1). The containment area shall be locked.

Appendix Q-II-A-1-a-(2). Access to the containment area shall be

limited or restricted when experimental animals are being held.

Appendix Q-II-A-1-a-(3). The containment area shall be patrolled or

monitored at frequent intervals.

Appendix Q-II-A-1-b. Other (BL1-N)

Appendix Q-II-A-1-b-(1). All genetically engineered neonates shall

be permanently marked within 72 hours after birth, if their size

permits. If their size does not permit marking, their containers should

be marked. In addition, transgenic animals should contain distinct and

biochemically assayable DNA sequences that allow identification of

transgenic animals from among non- transgenic animals.

Appendix Q-II-A-1-b-(2) A double barrier shall be provided to

separate male and female animals unless reproductive studies are part

of the experiment or other measures are taken to avoid reproductive

transmission. Reproductive incapacitation may be used.

Appendix Q-II-A-1-b-(3). The containment area shall be in

accordance with state and Federal laws and animal care requirements.

Appendix Q-II-A-2. Animal Facilities (BL1-N)

Appendix Q-II-A-2-(a). Animals shall be confined to securely fenced

areas or be in enclosed structures (animal rooms) to minimize the

possibility of theft or unintentional release.

Appendix Q-II-B. Biosafety Level 2 - Animals (BL2-N) (see Appendix Q-

III-A)

Appendix Q-II-B-1. Standard Practices (BL2-N)

Appendix Q-II-B-1-a. Animal Facility Access (BL2-N)

Appendix Q-II-B-1-a-(1). The containment area shall be locked.

Appendix Q-II-B-1-a-(2). The containment area shall be patrolled or

monitored at frequent intervals.

Appendix Q-II-B-1-a-(3). The containment building shall be

controlled and have a locking access.

Appendix Q-II-B-1-a-(4). The Animal Facility Director shall

establish policies and procedures whereby only persons who have been

advised of the potential hazard and who meet any specific entry

requirements (e.g., vaccination) may enter the laboratory or animal

rooms.

Appendix Q-II-B-1-a-(5). Animals of the same or different species,

which are not involved in the work being performed, shall not be

permitted in the animal area.

Appendix Q-II-B-1-b. Decontamination and Inactivation (BL2-N)

Appendix Q-II-B-1-b-(1). Contaminated materials that are

decontaminated at a site away from the laboratory shall be placed in a

closed durable leak-proof container prior to removal from the

laboratory.

Appendix Q-II-B-1-b-(2). Needles and syringes shall be promptly

placed in a puncture-resistant container and decontaminated, preferably

by autoclaving, before discard or reuse.

Appendix Q-II-B-1-c. Signs (BL2-N)

Appendix Q-II-B-1-c-(1). When the animal research requires special

provisions for entry (e.g., vaccination), a warning sign incorporating

the universal biosafety symbol shall be posted on all access doors to

the animal work area. The sign shall indicate: (i) The agent, (ii) the

animal species, (iii) the name and telephone number of the Animal

Facility Director or other responsible individual, and (iv) any special

requirements for entering the laboratory.

Appendix Q-II-B-1-d. Protective Clothing (BL2-N)

Appendix Q-II-B-1-d-(1). Laboratory coats, gowns, smocks, or

uniforms shall be worn while in the animal area or attached laboratory.

Before entering non-laboratory areas (e.g., cafeteria, library,

administrative offices), protective clothing shall be removed and kept

in the work entrance area.

Appendix Q-II-B-1-d-(2). Special care shall be taken to avoid skin

contamination with microorganisms containing recombinant DNA.

Impervious and/or protective gloves shall be worn when handling

experimental animals and when skin contact with an infectious agent is

unavoidable.

Appendix Q-II-B-1-e. Records (BL2-N)

Appendix Q-II-B-1-e-(1). Any incident involving spills and

accidents that result in environmental release or exposures of animals

or laboratory workers to organisms containing recombinant DNA molecules

shall be reported immediately to the Animal Facility Director,

Institutional Biosafety Committee, NIH/ORDA, and other appropriate

authorities (if applicable). Reports to the NIH/ORDA shall be sent to

the Office of Recombinant DNA Activities, National Institutes of

Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-

9838. Medical evaluation, surveillance, and treatment shall be provided

as appropriate and written records maintained. If necessary, the area

shall be appropriately decontaminated.

Appendix Q-II-B-1-e-(2). When appropriate and giving consideration to

the agent handled, baseline serum samples shall be collected and stored

for animal care and other at-risk personnel. Additional serum specimens

may be collected periodically depending on the agent handled and the

function of the animal facility.

Appendix Q-II-B-1-f. Transfer of Materials (BL2-N)

Appendix Q-II-B-1-f-(1). Biological materials removed from the

animal containment area in a viable or intact state shall be

transferred to a non-breakable sealed primary container and then

enclosed in a non-breakable sealed secondary container. All containers,

primary and secondary, shall be disinfected before removal from the

animal facility. Advance approval for transfer of material shall be

obtained from the Animal Facility Director. Packages containing viable

agents may only be opened in a facility having an equivalent or higher

level of physical containment unless the agent is biologically

inactivated or incapable of reproduction.

Appendix Q-II-B-1-g. Other (BL2-N)

Appendix Q-II-B-1-g-(1). All genetically engineered neonates shall

be permanently marked within 72 hours after birth, if their size

permits. If their size does not permit marking, their containers should

be marked. In addition, transgenic animals should contain distinct and

biochemically assayable DNA sequences that allow identification of

transgenic animals from among non-transgenic animals.

Appendix Q-II-B-1-g-(2). Needles and syringes shall be used only

for parenteral injection and aspiration of fluids from laboratory

animals and diaphragm bottles. Only needle-locking syringes or

disposable syringe-needle units (i.e., needle is integral to the

syringe) shall be used for the injection or aspiration of fluids

containing organisms that contain recombinant DNA. Extreme caution

shall be used when handling needles and syringes to avoid

autoinoculation and the generation of aerosols during use and disposal.

Following use, needles shall not be bent, sheared, replaced in the

needle sheath or guard, or removed from the syringe. Needles and

syringes shall be promptly placed in a puncture-resistant container and

decontaminated, preferably by autoclaving, before discard or reuse.

Appendix Q-II-B-1-g-(3). Appropriate steps should be taken to

prevent horizontal transmission or exposure of laboratory personnel. If

the agent used as a vector is known to be transmitted by a particular

route (e.g., arthropods), special attention should be given to

preventing spread by that route. In the absence of specific knowledge

of a particular route of transmission, all potential means of

horizontal transmission (e.g., arthropods, contaminated bedding, or

animal waste, etc.) should be prevented.

Appendix Q-II-B-1-g-(4). Eating, drinking, smoking, and applying

cosmetics shall not be permitted in the work area.

Appendix Q-II-B-1-g-(5). Individuals who handle materials and

animals containing recombinant DNA molecules shall be required to wash

their hands before exiting the containment area.

Appendix Q-II-B-1-g-(6). A double barrier shall be provided to

separate male and female animals unless reproductive studies are part

of the experiment or other measures are taken to avoid reproductive

transmission. Reproductive incapacitation may be used.

Appendix Q-II-B-1-g-(7). The containment area shall be in

accordance with state and Federal laws and animal care requirements.

Appendix Q-II-B-1-g-(8). A biosafety manual shall be prepared or

adopted. Personnel shall be advised of special hazards and required to

read and follow instructions on practices and procedures.

Appendix Q-II-B-2. Animal Facilities (BL2-N)

Appendix Q-II-B-2-a. Animals shall be contained within an enclosed

structure (animal room or equivalent) to minimize the possibility of

theft or unintentional release and to avoid arthropod access. The

special provision to avoid the entry or escape of arthropods from the

animal areas may be waived if the agent in use is not known to be

transmitted by arthropods.

Appendix Q-II-B-2-b. Surfaces shall be impervious to water and

resistant to acids, alkalis, organic solvents, and moderate heat.

Appendix Q-II-B-2-c. The animal containment area shall be designed

so that it can be easily cleaned.

Appendix Q-II-B-2-d. Windows that open shall be fitted with fly

screens.

Appendix Q-II-B-2-e. An autoclave shall be available for

decontamination of laboratory wastes.

Appendix Q-II-B-2-f. If arthropods are used in the experiment or

the agent under study can be transmitted by an arthropod, interior work

areas shall be appropriately screened (52 mesh). All perimeter joints

and openings shall be sealed and additional arthropod control

mechanisms used to minimize arthropod entry and propagation, including

appropriate screening of access doors or the equivalent.

Appendix Q-II-C. Biosafety Level 3--Animals (BL3-N) (See Appendix Q-

III-B)

Appendix Q-II-C-1. Standard Practices (BL3-N)

Appendix Q-II-C-1-a. Animal Facility Access (BL3-N)

Appendix Q-II-C-1-a-(1). The containment area shall be locked.

Appendix Q-II-C-1-a-(2). The containment area shall be patrolled or

monitored at frequent intervals.

Appendix Q-II-C-1-a-(3). The containment building shall be

controlled and have a locking access.

Appendix Q-II-C-1-a-(4). The Animal Facility Director shall

establish policies and procedures whereby only persons who have been

advised of the potential hazard and who meet any specific entry

requirements (e.g., vaccination) shall enter the laboratory or animal

rooms.

Appendix Q-II-C-1-a-(5). Animal room doors, gates, or other

closures shall be kept closed when experiments are in progress.

Appendix Q-II-C-1-b. Decontamination and Inactivation (BL3-N)

Appendix Q-II-C-1-b-(1). The work surfaces of containment equipment

shall be decontaminated when work with organisms containing recombinant

DNA molecules is finished. Where feasible, plastic-backed paper

toweling shall be used on nonporous work surfaces to facilitate clean-

up.

Appendix Q-II-C-1-b-(2). All animals shall be euthanized at the end

of their experimental usefulness and the carcasses decontaminated

before disposal in an approved manner.

Appendix Q-II-C-1-b-(3). Needles and syringes shall be promptly

placed in a puncture-resistant container and decontaminated, preferably

by autoclaving, before discard or reuse.

Appendix Q-II-C-1-b-(4). Special safety testing, decontamination

procedures, and Institutional Biosafety Committee approval shall be

required to transfer agents or tissue/organ specimens from a BL3-N

animal facility to a facility with a lower containment classification.

Appendix Q-II-C-1-b-(5). Liquid effluent from containment

equipment, sinks, biological safety cabinets, animal rooms, primary

barriers, floor drains, and sterilizers shall be decontaminated by heat

treatment before being released into the sanitary system. The procedure

used for heat decontamination of liquid wastes shall be monitored with

a recording thermometer. The effectiveness of the heat decontamination

process system shall be revalidated every 30 days with an indicator

organism.

Appendix Q-II-C-1-c. Signs (BL3-N)

Appendix Q-II-C-1-c-(1). When the animal research requires special

provisions for entry (e.g., vaccination), a warning sign incorporating

the universal biosafety symbol shall be posted on all access doors to

the animal work area. The sign shall indicate: (i) The agent, (ii) the

animal species, (iii) the name and telephone number of the Animal

Facility Director or other responsible individual, and (iv) any special

requirements for entering the laboratory.

Appendix Q-II-C-1-d. Protective Clothing (BL3-N)

Appendix Q-II-C-1-d-(1). Full protective clothing that protects the

individual (e.g., scrub suits, coveralls, uniforms) shall be worn in

the animal area. Clothing shall not be worn outside the animal

containment area and shall be decontaminated before laundering or

disposal. Personnel shall be required to shower before exiting the BL3-

N area and wearing of personal clothing.

Appendix Q-II-C-1-d-(2). Special care shall be taken to avoid skin

contamination with microorganisms containing recombinant DNA.

Impervious and/or protective gloves shall be worn when handling

experimental animals and when skin contact with an infectious agent is

unavoidable.

Appendix Q-II-C-1-d-(3). Appropriate respiratory protection shall

be worn in rooms containing experimental animals.

Appendix Q-II-C-1-e. Records (BL3-N)

Appendix Q-II-C-1-e-(1). Documents regarding experimental animal

use and disposal shall be maintained in a permanent record book.

Appendix Q-II-C-1-e-(2). Any incident involving spills and

accidents that result in environmental release or exposure of animals

or laboratory workers to organisms containing recombinant DNA shall be

reported immediately to the Biological Safety Office, Animal Facility

Director, Institutional Biosafety Committee, NIH/ORDA, and other

appropriate authorities (if applicable). Reports to the NIH/ORDA shall

be sent to the Office of Recombinant DNA Activities, National

Institutes of Health, Building 31, Room 4B11, Bethesda, Maryland 20892,

(301) 496-9838. Medical evaluation, surveillance, and treatment shall

be provided as appropriate and written records maintained. If

necessary, the area shall be appropriately decontaminated.

Appendix Q-II-C-1-e-(3). When appropriate and giving consideration

to the agent handled, baseline serum samples shall be collected and

stored for animal care and other at-risk personnel. Additional serum

specimens may be collected periodically depending on the agent handled

or the function of the facility.

Appendix Q-II-C-1-f. Transfer of Materials (BL3-N)

Appendix Q-II-C-1-f-(1). Biological materials removed from the

animal containment laboratory in a viable or intact state shall be

transferred to a non- breakable sealed primary container and then

enclosed in a non-breakable sealed secondary container. All containers,

primary and secondary, shall be disinfected before removal from the

animal facility. Advance approval for transfer of material shall be

obtained from the Animal Facility Director. Packages containing viable

agents may be opened only in a facility having an equivalent or higher

level of physical containment unless the agent is biologically

inactivated or incapable of reproduction.

Appendix Q-II-C-1-f-(2). Special safety testing, decontamination

procedures, and Institutional Biosafety Committee approval shall be

required to transfer agents or tissue/organ specimens from a BL3-N

animal facility to a facility with a lower containment classification.

Appendix Q-II-C-1-g. Other (BL3-N)

Appendix Q-II-C-1-g-(1). All genetically engineered neonates shall

be permanently marked within 72 hours after birth, if their size

permits. If their size does not permit marking, their containers should

be marked. In addition, transgenic animals should contain distinct and

biochemically assayable DNA sequences that allow identification of

transgenic animals from among nontransgenic animals.

Appendix Q-II-C-1-g-(2). Appropriate steps should be taken to

prevent horizontal transmission or exposure of laboratory personnel. If

the agent used as the vector is known to be transmitted by a particular

route (e.g., arthropods), special attention should be given to

preventing spread by that route. In the absence of specific knowledge

of a particular route of transmission, all potential means of

horizontal transmission (e.g., arthropods, contaminated bedding, or

animal waste) should be prevented.

Appendix Q-II-C-1-g-(3). Eating, drinking, smoking, and applying

cosmetics shall not be permitted in the work area.

Appendix Q-II-C-1-g-(4). Individuals who handle materials and

animals containing recombinant DNA molecules shall be required to wash

their hands before exiting the containment area.

Appendix Q-II-C-1-g-(5). Experiments involving other organisms that

require containment levels lower than BL3-N may be conducted in the

same area concurrently with experiments requiring BL3-N containment

provided that they are conducted in accordance with BL3-N practices.

Appendix Q-II-C-1-g-(6). Animal holding areas shall be cleaned at

least once a day and decontaminated immediately following any spill of

viable materials.

Appendix Q-II-C-1-g-(7). All procedures shall be performed

carefully to minimize the creation of aerosols.

Appendix Q-II-C-1-g-(8). A double barrier shall be provided to

separate male and female animals unless reproductive studies are part

of the experiment or other measures are taken to avoid reproductive

transmission. Reproductive incapacitation may be used.

Appendix Q-II-C-1-g-(9). The containment area shall be in

accordance with state and Federal laws and animal care requirements.

Appendix Q-II-C-1-g-(10). All animals shall be euthanized at the

end of their experimental usefulness and the carcasses decontaminated

before disposal in an approved manner.

Appendix Q-II-C-1-g-(11). Personnel shall be required to shower

before exiting the BL3-N area and wearing personal clothing.

Appendix Q-II-C-1-g-(12). Animals of the same or different species,

which are not involved in the work being performed, shall not be

permitted in the animal area.

Appendix Q-II-C-1-g-(13). Needles and syringes shall be used only

for parenteral injection and aspiration of fluids from laboratory

animals and diaphragm bottles. Only needle-locking syringes or

disposable syringe-needle units (i.e., needle is integral to the

syringe) shall be used for the injection or aspiration of fluids

containing organisms that contain recombinant DNA. Extreme caution

shall be used when handling needles and syringes to avoid

autoinoculation and the generation of aerosols during use and disposal.

Following use, needles shall not be bent, sheared, replaced in the

needle sheath or guard or removed from the syringe. The needles and

syringes shall be promptly placed in a puncture-resistant container and

decontaminated, preferably by autoclaving, before discard or reuse.

Appendix Q-II-C-1-g-(14). A biosafety manual shall be prepared or

adopted. Personnel shall be advised of special hazards and required to

read and follow instructions on practices and procedures.

Appendix Q-II-C-2. Animal Facilities (BL3-N)

Appendix Q-II-C-2-a. Animals shall be contained within an enclosed

structure (animal room or equivalent) to minimize the possibility of

theft or unintentional release and avoid arthropod access. The special

provision to avoid the entry or escape of arthropods from the animal

areas may be waived if the agent in use is not known to be transmitted

by arthropods.

Appendix Q-II-C-2-b. The interior walls, floors, and ceilings shall

be impervious to water and resistant to acids, alkalis, organic

solvents, and moderate heat, to facilitate cleaning. Penetrations in

these structures and surfaces (e.g., plumbing and utilities) shall be

sealed.

Appendix Q-II-C-2-c. Windows in the animal facility shall be

closed, sealed, and breakage resistant (e.g., double-pane tempered

glass or equivalent). The need to maintain negative pressure should be

considered when constructing or renovating the animal facility.

Appendix Q-II-C-2-d. An autoclave, incinerator, or other effective

means to decontaminate animals and waste shall be available, preferably

within the containment area. If feasible, a double-door autoclave is

preferred and should be positioned to allow removal of material from

the containment area.

Appendix Q-II-C-2-e. If arthropods are used in the experiment or

the agent under study can be transmitted by an arthropod, the interior

work area shall be appropriately screened (52 mesh). All perimeter

joints and openings shall be sealed, and additional arthropod control

mechanisms used to minimize arthropod entry and propagation, including

appropriate screening, or the equivalent of access doors.

Appendix Q-II-C-2-f. Access doors to the containment area shall be

self-closing.

Appendix Q-II-C-2-g. The animal area shall be separated from all

other areas. Passage through two sets of doors shall be the basic

requirement for entry into the animal area from access corridors or

other contiguous areas. The animal containment area shall be physically

separated from access corridors and other laboratories or areas by a

double-door clothes change room, equipped with integral showers and

airlock.

Appendix Q-II-C-2-h. Liquid effluent from containment equipment,

sinks, biological safety cabinets, animal rooms, primary barriers,

floor drains, and sterilizers shall be decontaminated by heat treatment

before being released into the sanitary system. The procedure used for

heat decontamination of liquid wastes shall be monitored with a

recording thermometer. The effectiveness of the heat decontamination

process system shall be revalidated every 30 days with an indicator

organism.

Appendix Q-II-C-2-i. An exhaust air ventilation system shall be

provided. This system shall create directional airflow that draws air

into the animal room through the entry area. The building exhaust, or

the exhaust from primary containment units, may be used for this

purpose if the exhaust air is discharged to the outside and shall be

dispersed away from occupied areas and air intakes. Personnel shall

verify that the direction of the airflow (into the animal room) is

proper.

Appendix Q-II-C-2-j. If the agent is transmitted by aerosol, then

the exhaust air shall pass through a high efficiency particulate air/

HEPA filter.

Appendix Q-II-C-2-k. Vacuum lines shall be protected with high

efficiency particulate air/HEPA filters and liquid disinfectant traps.

Appendix Q-II-C-2-l. In lieu of open housing in the special animal

room, animals held in a BL3-N area may be housed in partial-containment

caging systems (e.g., Horsfall units or gnotobiotic systems, or other

special containment primary barriers). Prudent judgment must be

exercised to implement this ventilation system (e.g., animal species)

and its discharge location.

Appendix Q-II-C-2-m. Each animal area shall contain a foot, elbow,

or automatically operated sink for hand washing. The sink shall be

located near the exit door.

Appendix Q-II-C-2-n. Restraining devices for animals may be

required to avoid damage to the integrity of the animal containment

facility.

Appendix Q-II-D. Biosafety Level 4--Animals (BL4-N) (See Appendix Q-

III-C)

Appendix Q-II-D-1. Standard Practices (BL4-N)

Appendix Q-II-D-1-a. Animal Facility Access (BL4-N)

Appendix Q-II-D-1-a-(1). Individuals under 16 years of age shall

not be permitted to enter the animal area.

Appendix Q-II-D-1-a-(2). The containment area shall be locked.

Appendix Q-II-D-1-a-(3). The containment area shall be patrolled or

monitored at frequent intervals.

Appendix Q-II-D-1-a-(4). The containment building shall be

controlled and have a locking access.

Appendix Q-II-D-1-a-(5). The Animal Facility Director shall

establish policies and procedures whereby only persons who have been

advised of the potential hazard and who meet any specific entry

requirements (e.g., vaccination) may enter the laboratory or animal

room.

Appendix Q-II-D-1-a-(6). Individuals shall enter and exit the

animal facility only through the clothing change and shower rooms.

Appendix Q-II-D-1-a-(7). Personnel shall use the airlocks to enter

or exit the laboratory only in an emergency.

Appendix Q-II-D-1-a-(8). Animal room doors, gates, and other

closures shall be kept closed when experiments are in progress.

Appendix Q-II-D-1-b. Decontamination and Inactivation (BL4-N)

Appendix Q-II-D-1-b-(1). All contaminated liquid or solid wastes

shall be decontaminated before disposal.

Appendix Q-II-D-1-b-(2). The work surfaces and containment

equipment shall be decontaminated when work with organisms containing

recombinant DNA molecules is finished. Where feasible, plastic-backed

paper toweling shall be used on nonporous work surfaces to facilitate

clean-up.

Appendix Q-II-D-1-b-(3). All wastes from animal rooms and

laboratories shall be appropriately decontaminated before disposal in

an approved manner.

Appendix Q-II-D-1-b-(4). No materials, except for biological

materials that are to remain in a viable or intact state, shall be

removed from the maximum containment laboratory unless they have been

autoclaved or decontaminated.

Equipment or material that might be damaged by high temperatures or

steam shall be decontaminated by gaseous or vapor methods in an airlock

or chamber designed for this purpose.

Appendix Q-II-D-1-b-(5). When ventilated suits are required, the

animal personnel shower entrance/exit area shall be equipped with a

chemical disinfectant shower to decontaminate the surface of the suit

before exiting the area. A neutralization or water dilution device

shall be integral with the chemical disinfectant discharge piping

before entering the heat sterilization system. Entry to this area shall

be through an airlock fitted with airtight doors.

Appendix Q-II-D-1-b-(6). Needles and syringes shall be promptly

placed in a puncture-resistant container and decontaminated, preferably

by autoclaving, before discard or reuse.

Appendix Q-II-D-1-b-(7). Supplies and materials needed in the

animal facility shall be brought in by way of the double-door

autoclave, fumigation chamber, or airlock that shall be appropriately

decontaminated between each use.

Appendix Q-II-D-1-b-(8). An autoclave, incinerator, or other

effective means to decontaminate animals and wastes shall be available,

preferably within the containment area. If feasible, a double-door

autoclave is preferred and should be positioned to allow removal of

material from the containment area.

Appendix Q-II-D-1-b-(9). Liquid effluent from containment

equipment, sinks, biological safety cabinets, animal rooms, primary

barriers, floor drains, and sterilizers shall be decontaminated by heat

treatment before being released into the sanitary system. Liquid wastes

from shower rooms and toilets shall be decontaminated with chemical

disinfectants or heat by methods demonstrated to be effective. The

procedure used for heat decontamination of liquid wastes shall be

monitored with a recording thermometer. The effectiveness of the heat

decontamination process system shall be revalidated every 30 days with

an indicator organism. Liquid wastes from the shower shall be

chemically decontaminated using an Environmental Protection Agency-

approved germicide. The efficacy of the chemical treatment process

shall be validated with an indicator organism. Chemical disinfectants

shall be neutralized or diluted before release into general effluent

waste systems.

Appendix Q-II-D-1-c. Signs (BL4-N)

Appendix Q-II-D-1-c-(1). When the animal research requires special

provisions for entry (e.g., vaccination), a warning sign incorporating

the universal biosafety symbol shall be posted on all access doors to

the animal work area. The sign shall indicate: (i) The agent, (ii) the

animal species, (iii) the name and telephone number of the Animal

Facility Director, or other responsible individual, and (iv) any

special requirements for entering the laboratory.

Appendix Q-II-D-1-d. Protective Clothing (BL4-N)

Appendix Q-II-D-1-d-(1). Individuals shall enter and exit the

animal facility only through the clothing change and shower rooms.

Street clothing shall be removed and kept in the outer clothing change

room. Complete laboratory clothing (may be disposable), including

undergarments, pants, shirts, jump suits, and shoes shall be provided

for all personnel entering the animal facility. When exiting the BL4-N

area and before proceeding into the shower area, personnel shall remove

their laboratory clothing in the inner change room. All laboratory

clothing shall be autoclaved before laundering. Personnel shall shower

each time they exit the animal facility.

Appendix Q-II-D-1-d-(2). A ventilated head-hood or a one-piece

positive pressure suit, which is ventilated by a life-support system,

shall be worn by all personnel entering rooms that contain experimental

animals when appropriate. When ventilated suits are required, the

animal personnel shower entrance/exit area shall be equipped with a

chemical disinfectant shower to decontaminate the surface of the suit

before exiting the area. A neutralization or water dilution device

shall be integral with the chemical disinfectant discharge piping

before entering the heat sterilization system. Entry to this area shall

be through an airlock fitted with airtight doors.

Appendix Q-II-D-1-d-(3). Appropriate respiratory protection shall

be worn in rooms containing experimental animals.

Appendix Q-II-D-1-e. Records (BL4-N)

Appendix Q-II-D-1-e-(1). Documents regarding experimental animal

use and disposal shall be maintained in a permanent record book.

Appendix Q-II-D-1-e-(2). A system shall be established for: (i)

Reporting laboratory accidents and exposures that are a result of overt

exposures to organisms containing recombinant DNA, (ii) employee

absenteeism, and (iii) medical surveillance of potential laboratory-

associated illnesses. Permanent records shall be prepared and

maintained. Any incident involving spills and accidents that results in

environmental release or exposures of animals or laboratory workers to

organisms containing recombinant DNA molecules shall be reported

immediately to the Biological Safety Officer, Animal Facility Director,

Institutional Biosafety Committee, NIH/ORDA, and other appropriate

authorities (if applicable). Reports to the NIH/ORDA shall be sent to

the Office of Recombinant DNA Activities, National Institutes of

Health, Building 31, Room 4B11, Bethesda, Maryland 20892, (301) 496-

9838. Medical evaluation, surveillance, and treatment shall be provided

as appropriate and written records maintained. If necessary, the area

shall be appropriately decontaminated.

Appendix Q-II-D-1-e-(3). When appropriate and giving consideration

to the agents handled, baseline serum samples shall be collected and

stored for animal care and other at-risk personnel. Additional serum

specimens may be collected periodically depending on the agents handled

or the function of the facility.

Appendix Q-II-D-1-e-(4). A permanent record book indicating the

date and time of each entry and exit shall be signed by all personnel.

Appendix Q-II-D-1-f. Transfer of Materials (BL4-N)

Appendix Q-II-D-1-f-(1). No materials, except for biological

materials that are to remain in a viable or intact state, shall be

removed from the maximum containment laboratory unless they have been

autoclaved or decontaminated. Equipment or material that might be

damaged by high temperatures or steam shall be decontaminated by

gaseous or vapor methods in an airlock or chamber designed for this

purpose.

Appendix Q-II-D-1-f-(2). Biological materials removed from the

animal maximum containment laboratory in a viable or intact state shall

be transferred to a non-breakable sealed primary container and then

enclosed in a non-breakable sealed secondary container that shall be

removed from the animal facility through a disinfectant dunk tank,

fumigation chamber, or an airlock designed for this purpose. Advance

approval for transfer of material shall be obtained from the Animal

Facility Director. Such packages containing viable agents can only be

opened in another BL4-N animal facility if the agent is biologically

inactivated or incapable of reproduction. Special safety testing,

decontamination procedures, and Institutional Biosafety Committee

approval shall be required to transfer agents or tissue/organ specimens

from a BL4-N animal facility to one with a lower containment

classification.

Appendix Q-II-D-1-f-(3). Supplies and materials needed in the

animal facility shall be brought in by way of the double-door

autoclave, fumigation chamber, or airlock that shall be appropriately

decontaminated between each use. After securing the outer doors,

personnel within the animal facility retrieve the materials by opening

the interior doors of the autoclave, fumigation chamber, or airlock.

These doors shall be secured after materials are brought into the

animal facility.

Appendix Q-II-D-1-g. Other (BL4-N)

Appendix Q-II-D-1-g-(1). All genetically engineered neonates shall

be permanently marked within 72 hours after birth, if their size

permits. If their size does not permit marking, their containers should

be marked. In addition, transgenic animals should contain distinct and

biochemically assayable DNA sequences that allow identification of

transgenic animals from among non-transgenic animals.

Appendix Q-II-D-1-g-(2). Eating, drinking, smoking, and applying

cosmetics shall not be permitted in the work area.

Appendix Q-II-D-1-g-(3). Individuals who handle materials and

animals containing recombinant DNA molecules shall be required to wash

their hands before exiting the containment area.

Appendix Q-II-D-1-g-(4). Experiments involving other organisms that

require containment levels lower than BL4-N may be conducted in the

same area concurrently with experiments requiring BL4-N containment

provided that they are conducted in accordance with BL4-N practices.

Appendix Q-II-D-1-g-(5). Animal holding areas shall be cleaned at

least once a day and decontaminated immediately following any spill of

viable materials.

Appendix Q-II-D-1-g-(6). All procedures shall be performed

carefully to minimize the creation of aerosols.

Appendix Q-II-D-1-g-(7). A double barrier shall be provided to

separate male and female animals. Animal isolation barriers shall be

sturdy and accessible for cleaning. Reproductive incapacitation may be

used.

Appendix Q-II-D-1-g-(8). The containment area shall be in

accordance with state and Federal laws and animal care requirements.

Appendix Q-II-D-1-g-(9). The life support system for the ventilated

suit or head hood is equipped with alarms and emergency back-up air

tanks. The exhaust air from the suit area shall be filtered by two sets

of high efficiency particulate air/HEPA filters installed in series or

incinerated. A duplicate filtration unit, exhaust fan, and an

automatically starting emergency power source shall be provided. The

air pressure within the suit shall be greater than that of any adjacent

area. Emergency lighting and communication systems shall be provided. A

double-door autoclave shall be provided for decontamination of waste

materials to be removed from the suit area.

Appendix Q-II-D-1-g-(10). Needles and syringes shall be used only

for parenteral injection and aspiration of fluids from laboratory

animals and diaphragm bottles. Only needle-locking syringes or

disposable syringe-needle units (i.e., needle is integral to the

syringe) shall be used for the injection or aspiration of fluids

containing organisms that contain recombinant DNA. Extreme caution

shall be used when handling needles and syringes to avoid

autoinoculation and the generation of aerosols during use and disposal.

Following use, needles shall not be bent, sheared, replaced in the

needle sheath or guard, or removed from the syringe. The needles and

syringes shall be promptly placed in a puncture-resistant container and

decontaminated, preferably by autoclaving, before discard or reuse.

Appendix Q-II-D-1-g-(11). An essential adjunct to the reporting-

surveillance system is the availability of a facility for quarantine,

isolation, and medical care of personnel with potential or known

laboratory-associated illnesses.

Appendix Q-II-D-1-g-(12). A biosafety manual shall be prepared or

adopted. Personnel shall be advised of special hazards and required to

read and follow instructions on practices and procedures.

Appendix Q-II-D-1-g-(13). Vacuum lines shall be protected with high

efficiency particulate air/HEPA filters and liquid disinfectant traps.

Appendix Q-II-D-2. Animal Facilities (BL4-N)

Appendix Q-II-D-2-a. Animals shall be contained within an enclosed

structure (animal room or equivalent) to minimize the possibility of

theft or unintentional release and avoid arthropod access.

Appendix Q-II-D-2-b. The interior walls, floors, and ceilings shall

be impervious to water and resistant to acids, alkalis, organic

solvents, and moderate heat, to facilitate cleaning. Penetrations in

these structures and surfaces (e.g., plumbing and utilities) shall be

sealed.

Appendix Q-II-D-2-c. Windows in the animal facility shall be

closed, sealed, and breakage resistant (e.g., double-pane tempered

glass or equivalent).

Appendix Q-II-D-2-d. An autoclave, incinerator, or other effective

means to decontaminate animals and wastes shall be available,

preferably within the containment area. If feasible, a double-door

autoclave is preferred and should be positioned to allow removal of

material from the containment area.

Appendix Q-II-D-2-e. Access doors to the containment area shall be

self-closing.

Appendix Q-II-D-2-f. All perimeter joints and openings shall be

sealed to form an arthropod-proof structure.

Appendix Q-II-D-2-g. The BL4-N laboratory provides a double barrier

to prevent the release of recombinant DNA containing microorganisms

into the environment. Design of the animal facility shall be such that

if the barrier of the inner facility is breached, the outer barrier

will prevent release into the environment. The animal area shall be

separated from all other areas. Passage through two sets of doors shall

be the basic requirement for entry into the animal area from access

corridors or other contiguous areas. Physical separation of the animal

containment area from access corridors or other laboratories or

activities shall be provided by a double-door clothes change room

equipped with integral showers and airlock.

Appendix Q-II-D-2-h. A necropsy room shall be provided within the

BL4-N containment area.

Appendix Q-II-D-2-i. Liquid effluent from containment equipment,

sinks, biological safety cabinets, animal rooms, primary barriers,

floor drains, and sterilizers shall be decontaminated by heat treatment

before being released into the sanitary system. Liquid wastes from

shower rooms and toilets shall be decontaminated with chemical

disinfectants or heat by methods demonstrated to be effective. The

procedure used for heat decontamination of liquid wastes shall be

monitored with a recording thermometer. The effectiveness of the heat

decontamination process system shall be revalidated every 30 days with

an indicator organism. Liquid wastes from the shower shall be

chemically decontaminated using an Environmental Protection Agency-

approved germicide.

The efficacy of the chemical treatment process shall be validated

with an indicator organism. Chemical disinfectants shall be neutralized

or diluted before release into general effluent waste systems.

Appendix Q-II-D-2-j. A ducted exhaust air ventilation system shall

be provided that creates directional airflow that draws air into the

laboratory through the entry area. The exhaust air, which is not

recirculated to any other area of the building, shall be discharged to

the outside and dispersed away from the occupied areas and air intakes.

Personnel shall verify that the direction of the airflow (into the

animal room) is proper.

Appendix Q-II-D-2-k. Exhaust air from BL4-N containment area shall

be double high efficiency particulate air/HEPA filtered or treated by

passing through a certified HEPA filter and an air incinerator before

release to the atmosphere. Double HEPA filters shall be required for

the supply air system in a BL4-N containment area.

Appendix Q-II-D-2-l. All high efficiency particulate air/HEPA

filters' frames and housings shall be certified to have no detectable

smoke [dioctylphthalate] leaks when the exit face (direction of flow)

of the filter is scanned above 0.01 percent when measured by a linear

or logarithmic photometer. The instrument must demonstrate a threshold

sensitivity of at least 1 x 10-3 micrograms per liter for 0.3

micrometer diameter dioctylphthalate particles and a challenge

concentration of 80-120 micrograms per liter. The air sampling rate

should be at least 1 cfm (28.3 liters per minute).

Appendix Q-II-D-2-m. If an air incinerator is used in lieu of the

second high efficiency particulate air/HEPA filter, it shall be

biologically challenged to prove all viable test agents are sterilized.

The biological challenge must be minimally 1 x 108 organisms per

cubic foot of airflow through the incinerator. It is universally

accepted if bacterial spores are used to challenge and verify that the

equipment is capable of killing spores, then assurance is provided that

all other known agents are inactivated by the parameters established to

operate the equipment. Test spores meeting this criterion are Bacillus

subtilis var. niger or Bacillus stearothermophilis. The operating

temperature of the incinerator shall be continuously monitored and

recorded during use.

Appendix Q-II-D-2-n. All equipment and floor drains shall be

equipped with deep traps (minimally 5 inches). Floor drains shall be

fitted with isolation plugs or fitted with automatic water fill

devices.

Appendix Q-II-D-2-o. Each animal area shall contain a foot, elbow,

or automatically operated sink for hand washing. The sink shall be

located near the exit door.

Appendix Q-II-D-2-p. Restraining devices for animals may be

required to avoid damage to the integrity of the containment animal

facility.

Appendix Q-II-D-2-q. The supply water distribution system shall be

fitted with a back-flow preventer or break tank.

Appendix Q-II-D-2-r. All utilities, liquid and gas services, shall

be protected with devices that avoid back-flow.

Appendix Q-II-D-2-s. Sewer and other atmospheric ventilation lines

shall be equipped minimally with a single high efficiency particulate/

HEPA filter. Condensate drains from these type housings shall be

appropriately connected to a contaminated or sanitary drain system. The

drain position in the housing dictates the appropriate system to be

used.

Appendix Q-III. Footnotes and References for Appendix Q

Appendix Q-III-A. If recombinant DNA is derived from a Class 2

organism requiring BL2 containment, personnel shall be required to have

specific training in handling pathogenic agents and directed by

knowledgeable scientists.

Appendix Q-III-B. Personnel who handle pathogenic and potentially

lethal agents shall be required to have specific training and be

supervised by knowledgeable scientists who are experienced in working

with these agents. BL3-N containment also minimizes escape of

recombinant DNA-containing organisms from exhaust air or waste material

from the containment area.

Appendix Q-III-C. Classes 4 and 5 microorganisms pose a high level

of individual risk for acquiring life-threatening diseases to personnel

and/or animals. To import Class 5 agents, special approval must be

obtained from U.S. Department of Agriculture, Animal and Plant Health

Inspection Service, Import-Export Products, Room 756, Federal Building,

6505 Belcrest Road, Hyattsville, Maryland 20782.

Laboratory staff shall be required to have specific and thorough

training in handling extremely hazardous infectious agents, primary and

secondary containment, standard and special practices, and laboratory

design characteristics. The laboratory staff shall be supervised by

knowledgeable scientists who are trained and experienced in working

with these agents and in the special containment facilities.

Within work areas of the animal facility, all activities shall be

confined to the specially equipped animal rooms or support areas. The

maximum animal containment area and support areas shall have special

engineering and design features to prevent the dissemination of

microorganisms into the environment via exhaust air or waste disposal.

Appendix Q-III-D. Other research with non-laboratory animals, which

may not appropriately be conducted under conditions described in

Appendix Q, may be conducted safely by applying practices routinely

used for controlled culture of these biota. In aquatic systems, for

example, BL1 equivalent conditions could be met by utilizing growth

tanks that provide adequate physical means to avoid the escape of the

aquatic species, its gametes, and introduced exogenous genetic

material. A mechanism shall be provided to ensure that neither the

organisms nor their gametes can escape into the supply or discharge

system of the rearing container (e.g., tank, aquarium, etc.) Acceptable

barriers include appropriate filtration, irradiation, heat treatment,

chemical treatment, etc. Moreover, the top of the rearing container

shall be covered to avoid escape of the organism and its gametes. In

the event of tank rupture, leakage, or overflow, the construction of

the room containing these tanks should prevent the organisms and

gametes from entering the building's drains before the organism and its

gametes have been inactivated.

Other types of non-laboratory animals (e.g., nematodes, arthropods,

and certain forms of smaller animals) may be accommodated by using the

appropriate BL1 through BL4 or BL1-P through BL4-P containment

practices and procedures as specified in Appendices G and P.

OMB's ``Mandatory Information Requirements for Federal Assistance

Program Announcements'' (45 FR 39592) requires a statement concerning

the official government programs contained in the Catalog of Federal

Domestic Assistance. Normally NIH lists in its announcements the number

and title of affected individual programs for the guidance of the

public. Because the guidance in this notice covers not only virtually

every NIH program but also essentially every Federal research program

in which DNA recombinant molecule techniques could be used, it has been

determined to be not cost effective or in the public interest to

attempt to list these programs. Such a list would likely require

several additional pages. In addition, NIH could not be certain that

every Federal program would be included as many Federal agencies, as

well as private organizations, both national and international, have

elected to follow the NIH Guidelines. In lieu of the individual program

listing, NIH invites readers to direct questions to the information

address above about whether individual programs listed in the Catalog

of Federal Domestic Assistance are affected.

Effective Date: June 24, 1994.

Harold Varmus,

Director, National Institutes of Health.

[FR Doc. 94-16199 Filed 7-1-94; 8:45 am]

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