THURSDAY, SEPTEMBER 9, 1976

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PART Il:

DEPARTMENT OF
HEALTH,
EDUCATION, AND
WELFARE

National Institutes of Health

RECOMBINANT DNA
RESEARCH GUIDELINES

Draft Environmental Impact Statement
38 126

DEPARTMENT OF HEALTH,
EDUCATION, AND WELFARE

National Institutes of Health

RECOMBINANT DNA RESEARCH
GUIDELINES

Draft Environmental! Impact Statement

On Wednesday, June 23, 1976, the
Director of the National Institutes of
Health, with the concurrence of the Sec-
retary of Health, Fducation, and Welfare
and the Assistant Secretary for Health,
issued Guidelines that will govern the
conduct of NIH-supported researcn on
recombinant DNA molecules.

The decision by the NIH Director to
release the Guidelines was reached after
extensive scientific and public airing of
the issues. The issues were discussed at
public meetings of the Recombinant DNA
Molecule Program Advisory Committee
(Recombinant Advisory Committee) and
the Advisory Committee to the NIH
Director. The Recombinant Advisory
Committee debated three different ver-
sions of the Guidelines during this period,
and made detailed recommendations to
the NIH Director on how this line of re-
search could proceed effectively with
maximum protection of workers and the
environment against possible hazards.

The Advisory Committee to the NIH
Director, augmented with consultants
representing law, ethics, consumer af-
fairs, and the environment, was asked to
advise on whether the proposed Guide-
lines balanced responsibility to protect
the public with the potential benefits
through the pursuit of new knowledge.
The many points of view expressed at an
open meeting of the Committee on Feb-
ruary 9 and 10, 1976, and in subsequent
correspondence, were taken into con-
sideration in the Director’s decision.

A number of public commentators
urged NIH to consider preparing an en-
vironmental impact statement on re-
combinant DNA research activity. They
evoked the possibility that organisms
containing recombinant DNA molecules
might escape and affect the environment
in potentially harmful ways. It should be
noted that the development of the guide-
lines was in large part tantamount to
conducting an environmental impact
assessment. For example, the objectives
of recombinant DNA research were con-
sidered and the potential hazards and
risks analyzed. Possible alternative ap-
proaches to the cbjectives were thor-
oughly explored, to maximize safety and
minimize potential risks. And an elab-
orate review structure to ensure safety
has been created.

She Guidelines are premised on physi-
cal and biological containment to pre-
vent the release or propagation of DNA
recombinants outside the laboratory.
Deliberate release of organisms into the
environment is prohibited. The stipulated
physical and biological containment en-
sures that this research will proceed with
a high degree of safety and precaution.

With a view to promoting public un-
derstanding of its issuance of the Guide-
lines, NIH conducted an environmental
impact assessment and prepared the

NOTICES

present draft environmental impact
statement in accordance with the Na-
tional Environmental Policy Act of 1969.
Notice of the availability of this docu-
ment appeared in the FEDERAL REGISTER
of September 2.

In order to extend the opportunity for
public comment and consideration, the
present draft environmental impact
statement is offered for general comment.
Please address any comments on this
draft statement to the Director, National
Institutes of Health. 9000 Rockville Pike,
Bethesda, Maryland 20014. All comments
should be submitted by October 18, 1976.

Additional copies of this draft are
available from Dr. Rudolf G. Wanner,
Associate Director for Environmental
Health and Safety, Building 12A, Room
4051, National Institutes of Health, 9000
Rockville Pike, Bethesda, Maryland
20014,

Dated: August 26, 1976.

Dona.p 8S, FREDRICKSON,
Director,
National Institutes of Health.

DraFrTt ENVIRONMENTAL IMPACT STATEMENT

GUIDELINES FOR RESEARCH INVOLVING RE-
COMBINANT DNA MOLECULES

NATIONAL INSTITUTES OF HEALTH
BETHESDA, MARYLAND
August 19, 1976

GUIDELINES FOR RESEARCH INVOLVING
RECOMBINANT DNA MOLECULES

National Institutes of Health, Public Health
Service, DHEW, Bethesda, Maryland

(X)} Draft (
Impact Statement.

) Final Environmental

Name of Action

(XX) Administrative (
Action.

) Legislative

Additional Information

Additional information on the proposed
action, including technical documents perti-
nent to this statement may be obtained
from:

Dr. Donald S. Fredrickson, Director. Na-
tional Institutes of Health, 9000 Rockville
Pike, Bethesda, Maryland 20014, Tele-
phone: (301) 496-2433.

A copy of the “Guidelines for Research
Involving Recombinant DNA Molecules’ is
attached. (Appendix D)

COMMENTS

The Department, in issuing this draft, is
requesting comments on the accuracy of the
factual information (including the absence
of relevant material) and projections con-
tained therein. Comments shall be submitted
by October 18, 1976, the Council on Environ-
mental Quality weekly notice in the FEpERAL
Recister. Address comments to Dr. Donald
S. Fredrickson.

CONTENTS
I. Foreword.
II. Authority.
III. Objective of the NIH Action.
IV. Background.
A. Description of the recombinant DNA
experimental process.
B. Events leading to the development
of guidelines.
C. Description of issues raised by re-
combinant DNA research.
1. Possible hazardous situations.

2. Expected benefits of DNA recom-
binant research.

8. Long-range implications,

4. Possible deliberate misuse.

V. Description of the proposed action.
VI. Description of alternatives.
A. No action.
B. NIH prohibition of funding of all
experiments with recombinant
DNA.
C,. Development of different guidelines.
D. No guidelines but NIH consideration
of each proposed project on an in-
dividual basis before funding.
E. General Federal regulation of ali
such research.
VII. Environmental impact of the guidelines.
A. Impact of issuance of NIH guide-
lines.

1. Impact on the safety of labora-
tory personnel and on the
spread of possibly hazardous
agents by infected laboratory
personnel.

2. Impact on
spread of possibly
agents.

3. Cost impact.

4. Secondary impacts.

the environmental
hazardous

B. Impact of experiments conducted
under the guidelines.
1. Possible undesirable impacts.
2. Beneficial impacts of DNA recom-
binant research.
APPENDICES
A. Glossary.
B. Suggested references for additional
reading.
Cc. Documents describing the imple-
mentation of the guidelines.
D. “Recombinant DNA Research” con-

taining ‘‘Decision of the Director,
National Institutes of Health to
Release Guidelines for Research
on Recombinant DNA Molecules”
and “Guidelines for Research In-
volving Recombinant DNA Mole-
cules” as published in the FEDERAL
REGISTER, Part II, July 7, 1976.

ForREWoORD

Recent developments in molecular
genetics, particularly in the last 4 years,
open avenues to science that were previ-
ously inaccessible. In the “recombinant

DNA” experiments considered here.
genes—deoxyribonucleic acid (DNA)
molecules—from virtually any living

organism can be transferred to cells of
certain completely unrelated organisms.
For example, the genes from one species
of bacteria have been transferred to
bacteria of another species. And genes
from toads and from fruit flies have
been introduced into the bacterium
Escherichia coli.

If the recipient bacterium is then
allowed to multiply. it will propagate
these newly acquired genes as part of its
own genetic complement. It appears
likelv that any kind of gene from any
kind of organism could be introduced
into EF. coli and certain other organisms.

This ability to join together genetic
material from two different sources and
to propagate these hybrid elements in
bacterial and animal cells has resulted
in a profound and qualitative change in
the field of genetics. Now, for the first
time, there is a methodology for crossing
very large evolutionary boundaries, and
for moving genes between organisms that
are believed to have previously had little
genetic contact.

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
The promise of recombinant DNA
research for better understanding and
improved treatment of human disease is
great. There is also a possible risk that
microorganisms with foreign genes might
cause disease or alter the environment
should they escape from the laboratory
and infect human beings, animals, or
plants. However, in the absence of fur-
ther experimental data neither the bene-
fits nor the risks can be precisely identi-
fied or assessed. .

On June 23, 1976, the Director of the
National Institutes of Health released
Guidelines governing the conduct of
NIH-supported research on recombinant
DNA molecules (See Appendix D). Pro-
mulgation of these Guidelines followed 2
years of intensive discussion and debate
within the scientific community and NIH
itself, with public participation, concern-
ing the possible hazards of such research
and the best means for averting them, al-
though the possible hazards remain
speculative. The Guidelines prohibit cer-
tain kinds of recombinant DNA experi-
ments and, for those experiments that
are permitted, they specify safety pre-
cautions and conditions designed to pro-
tect the health of laboratory workers, the
general public, and the environment
should the putative hazards prove real.

The issuance of Guidelines establish-
ing conditions and precautions with re-
spect to such experiments is viewed by
NIH as a Federal action that may
significantly. affect the quality of the
human environment, and NIH Director
Dr. Donald 8. Fredrickson ordered the
preparation of this statement pursuant
to the National Environmental Policy
Act.

Although NEPA assumes that such
Federal actions will not be taken until
the NEPA procedures are completed, the
Director of NIH concluded that the pub-
lic interest required immediate issuance
of the Guidelines, rather than deferral
for the months that would be required
for completion of the NEPA process. This
was because the escape of potentially
hazardous organisms was more likely in
the absence of NIH action. Further,
prompt issuance of the Guidelines was
believed necessary in order to promote
their acceptance by scientists in the
United States and abroad who do not
come under the purview of NIH.

Issuance of and compliance with the
Guidelines is, in itself, expected to de-
crease the chance of any detrimental
environmental impact. However, since
there has been little actual experience to
date with recombinant DNA experiments,
the indicated confidence in the Guide-
lines rests essentially upon the judgment
of scientists. Their confidence is based on
two premises. First, it is believed that
the containment measures specified in
the Guidelines make the escape of poten-
tially harmful recombinant organisms
into the environment highly improbable.
Second, it is believed that, even if an
experiment performed in accordance
with the Guidelines does result in acci-
dental release of recombinant organisms,
adverse effects will either not occur or

not be serious.

NOTICES

In the absence of an adequate base of
data derived from either experiments or
experience, it must be recognized that
future events may not conform to these
judgments. There is some statistical
probability that recombinant organisms
will find their way into the environment
either from experiments under NIH
auspices or from the activities of others.
It is not difficult to construct scenarios in
which injury could result. Although the
possibility of significant environmental
consequences is entirely speculative, the
chance of an event that could cause
severe injury, however low the probabil-
ity, must be treated as an environmental
impact.

The NIH Guidelines, in addition to en-
suring the safety of NIH-supported re-
searchers, the general public and the
environment, are serving as a model for
other laboratories throughout the world,
thereby promoting environmental pro-
tection beyond that achievable through
other actions available to the Federal
Government. And the experiments them-
selves may be expected ultimately to lead
to an increase of knowledge and the ad-
vancement of medicine and other
sciences.

Although the action in question—that
is, issuance of the Guidelines—has al-
ready been taken, the Director of NIH
believes that the NEPA review will fur-
ther enlighten the public and focus at-
tention on the important issues involved,
in the interest of gaining the under-
standing and views of the broadest pos-
sible segment of the American people. In
issuing the Guidelines, the NIH Director
pointed out that they will be subject to
continuous review and modification in
the light of changing circumstances.
Constructive modification could result
from information received during the
NEPA process.

II. AUTHORITY

The Federal action discussed in this
document is taken under the authority of
Title III of the Public Health Service
Act—General Powers and Duties of
Public Health Service; Part A—Re-
search and Investigation; sections 301
and 307 (42 U.S.C. 241 and 242]).

III. OBJECTIVE OF THE NIH AcTION

The objective of the proposed action—
release of the NIH Guidelines—is the
protection of laboratory workers, the
general public, and the environment
from infection by possibly hazardous
agents that may result from re-
combinant DNA research. The Guide-
lines are meant to ensure that experi-
ments involving recombinant DNA
molecules and which are supported by
NIH, are carried out under conditions
and safeguards that minimize the possi-
bility of the harmful exposure of any
human being or other component of the
environment to these possibly hazardous
agents.

It is NIH policy that all work sup-
ported by NIH, either in its own labora-
tories or through grants or contracts to
various organizations, must be carried
out according to the Guidelines. As part

38427

of this objective, the Guidelines describe
procedures that will be used to ensure
implementation. A further objective of
establishing the Guidelines is to in-
fluence, to the extent possible, other
Federal, non-Federal, and foreign or-
ganizations in their efforts to assure that
recombinant DNA experiments will be
carried out with minimal risk to labora-
tory workers, the general public, and the’
environment.

IV. Backcrounp

A. DESCRIPTION OF THE RECOMBINANT DNA
EXPERIMENTAL PROCESS

All living things, from subcellular
particles to higher organisms, require
specific information for their reproduc-
tion and functions. The basic source of
this information is deoxyribonucleic acid
(DNA), which is the principal substance
of the genes, the units of heredity (1).
Each cell of an organism is composed of
various organized structures, several of
which contain DNA. Figure IV-1 il-
lustrates a typical cell.

 

FIGURE IV-12

DNA plays two roles: (1). Provides in-
formation for the reproduction, growth,
and functions of the cell, and (2) pre-
serves and directs replication of this in-
formation and transfers it to the off-
spring. These two roles of DNA are com-
mon to animals, plants, single-cell or-
ganisms, and many viruses. The DNA of
cells is mainly found in organized struc-
tures called chromosomes.

IntraceNular DNA also occurs cutside
of the chromosomes as separately rep-
licating molecules. Such DNA molecules
include the plasmids, found in bacteria;
the DNA of chloroplasts, common to
green plants; and the DNA of mito-
chondria, the energy-preducing units of
the cells of complex organisms. These
DNAs, while not strictly part of the in-
herent genetic make-up of a cell, help
define the cell's functional capability.
Another type of DNA commonly found in
cells is the DNA of infecting viruses.

In the past 30 years the structure of
the DNA molecule has been studied in-

FEDERAL REGISTER, VOL. 41, NO. 176-—THURSDAY, SEPTEMBER 9, 1976
38428

tensively, and it can now be described in
much detail. The molecule may be com-
pared to a very long, but twisted step-
ladder with thousands to millions of
rungs (shown in Figure IV—2). The sides
of the ladder are formed of sugar mole-
cules (deoxyribose) attached end to end
through phosphate groups. At right
angles to each sugar molecule is one of
four possible bases—adenine, guanine,
thymine, and cytosine. The precise se-
quence of these bases, the rungs of the
ladder, codes the information content.
The “reading” of the code contained in
the sequence of bases results in the for-
mation of proteins which in turn permit
the essential functions of the cell.

A gene is a portion of the DNA mole-
cule which codes for the manufacture
of a single protein. In higher organisms,
much of the DNA may not serve as genes
in this sense, but may regulate the
activity of nearby genes. It is possible to
break open cells and isolate DNA, free of
other cellular constituents.

 

Ficure IV-2

In recombinant DNA _ experiments,
DNA is first isolated from two different
cell types. Each DNA is then broken into
segments. Each segment may contain one
or more genes, or it may contain a por-
tion of the DNA that lacks functional
genes. The breaking is‘accomplished by
means of bacterial enzymes (restriction
endonucleases), which cut the DNA in
such a way that the chemical structure
at the ends of the segments permits in-
terchangeable rejoining when the two
different DNAs are mixed. In this way
single ‘DNA molecules containing por-
tions of the two different DNAs are con-
structed. The DNA recombined in these
experiments can be derived from widely
divergent sources. The DNA from one of
the sources serves as a carrier, or vector,
for the insertion of the recombined DNA
into a cell, or host. The vector may be
DNA from a virus or a plasmid, usually
derived from the same species as will
serve as the host of the recombinant
DNA. From a growth culture of the host

cells, those containing the DNA frag-
ment of particular interest are selected

NOTICES

and allowed to multiply. The resulting
population of identical cells is called a
“clone.” In some experiments the DNA
will be extracted from the cells for
study; in others, the properties of the
celis themselves will be investigated.

In the experiments discussed in the
Guidelines, the host cells are generally
single-cell microorganisms such as bac-
teria, or animal or plant cells that were
originally obtained from living tissue but
are grown as single cells under special
laboratory conditions.

The process of producing recombinant
DNA molecules and introducing them
into cells is illustrated in Figure IV-3.

> ts eS
ce =
O =
wince ~om.. -—,."
Pacorinnt OMA,

Ficure I[V-3

The cell represented at the upper left con-
tains chromosomal DNA and several sep-
arately replicating DNA molecules. The non-
chromosomal DNA molecules can be isolated
from the cell and manipulated to serve as
vectors (carriers) for DNA from a foreign
cell. Most DNA molecules used as vectors are
circular. They can be cleaved, as shown, by
enzymes (restriction endonucleases) to yleld
Hnear molecules with rejoinable ends.

At the upper right is another cell, repre-
sented here as a rectangle. It serves as the
source of the foreign DNA to be inserted in
the vector. This DNA can also be cleaved by
enzymes. The rectangular cell could be de-
rived from any living species, and the foreign
DNA might contain chromosomal or non-
chromosomal DNA, or both.

In the next steps, the foreign DNA frag-
ment is mixed and combined with the vector
DNA, and the recombinant DNA Is reinserted
into a host cell. In most experiments this
host cell will be of the same species as the
source of the vector. The recipient cells are
then placed under conditions where they
grow and multiply by division. Each new
cell will contain recombinant DNA,

B. EVENTS LEADING TO DEVELOPMENT OF
GUIDELINES

On June 23, 1976, the Director, NIH,
released “National Institutes of Health
Guidelines for Research Involving Re-
combinant DNA Molecules” (see Appen-
dix D). This action was approved by the
Secretary of Health, Education, and Wel-
fare and the Assistant Secretary for
Health. The Guidelines established care-
fully controlled conditions for the con-
duct of experiments involving the inser-
tion of recombinatn genes into orga-
nisms, such as bacteria. The chronology
leading to the present Guidelines and
the decision to release them are out-
lined below.

It was some of the scientists engaged
in recombinant DNA research who called
for a moratorium on certain kinds of ex-
periments in order to assess the risks
and devise appropriate guidelines. The
capability to perform DNA recombina-

tions, and the potential hazards, had be-
come apparent at the Gordon Research
Conference on Nucleic Acids in July 1973.
Those in attendance voted to send an
open letter to Dr. Philip Handler, Presi-
dent of the National" Academy of
Sciences, and to Dr. John R. Hogness,
President of the Institute of Medicine,
NAS. The letter, appearing in “Science”
(2), suggested that the Academy “estab-
lish a study committee to consider this
problem and to recommend specific ac-
tions or guidelines, should that seem
appropriate.”

In response, NAS formed a committee,
and its members published another letter
in “Science” in July of 1974 (3). Under
the title “Potential Biohazards of Re-
combinant DNA Molecules,” the letter
proposed:

First, and most important, that until the
potential hazards of such recombinant DNA
molecules have been better evaluated or until
adequate methods are developed for prevent-
ing their spread, scientists throughout the
world join with the members of this com-
mittee in voluntarily deferring * * * [cer-
tain] experiments * * *.

Second, plans to link fragments of ani-
meal DNAs to bacterial plasmid DNA or
bacteriophage DNA should be carefully
weighed * * *,

Third, the Director of the National In-
stitutes of Health is requested to give im-
mediate consideration to establishing an ad-
visory committee charged with (1) oversee-
ing an experimental program to evaluate
the potential biological and ecological haz-
ards of the above types of recombinant DNA
molecules; (ii) developing procedures which
will minimize the spread of such molecules
within human and other populations; and
(fli) devising guidelines to be followed by in-
vestigators working with potentially hazard-
ous recombinant DNA molecules.

Fourth, an international meeting of in-
volved scientists from all over the world
should be convened early in the coming year
to review scientific progress in this area and
to further discuss appropriate ways to deal
with the potential biohazards of recom-
binant DNA molecules.

On October 7, 1974, the NIH Recom-
binant DNA Molecule Program Advisory
Committee (hereafter “Recombinant
Advisory Committee’) was established to
advise the Secretary of HEW, the As-
sistant Secretary for Health, and the Bi-
rector of NIH” concerning a program
for developing procedures which will
minimize the spread of such molecules
within human and other populations,
and for devising guidelines to be followed
by investigators working with potentially
hazardous recombinants.”

The international meeting proposed
in the “Science” article (2) was held in
February 1975 at the Asilomar Confer-
ence Center, Pacific Grove, California. It
was sponsored by the National Academy
of Sciences and supported by the Na-
tional Institutes of Health and the Na-
tional Science Foundation. One hundred
and fifty people attended, including 52
foreign scientists from 15 countries, 16
representatives of the press, and 4
attorneys. ,

The conference reviewed progress in
research on recombinant DNA molecules
and discussed ways to deal with the po-
tential biohazards of the work. Partic-
ipants felt that experiments on con-

FEDERAL REGISTER, VOL. 41, NO. 176-—-THURSDAY, SEPTEMBER 9, 1976
struction of recombinant DNA moile-
cules should proceed: Provided, that ap-
propriate containment is utilized. The
conference made recommendations for
matching levels of containment with
levels of possible hazard for various types
of experiments. Certain experiments
were judged to pose such serious poten-
tial dangers that the conference recom-
mended against their being conducted at
the present time.

A report on the conference was sub-
mitted to the Assembly of Life Sciences,
National Research Council, NAS, and
approved by its Executive Committee on
May 20, 1975. A summary statement of
the report (4) was published in “Science,
Nature,” and the “Proceedings of the
National Academy of Sciences.” The re-
port noted that “in many countries steps
are already being taken by national
bodies to formulate codes of practice
for the conduct of experiments: with
known or potential bichazards. Until
these are established, we urge individual
scientists to use the proposals in this
document as a guide.”

The NIH Recombinant Advisory Com-
mittee held its first meeting in San Fran-
cisco immediately after the Asilomar
conference. It proposed that NIH use the
recommendations of the Asilomar con-
ference as guidelines for research until
the committee had an opportunity to
elaborate more specific guidelines, and
that NIH establish a newsletter for in-
formal distribution of information. NIH
accepted these recommendations.

At the second meeting, held on May
12-13, 1975, in Bethesda, Maryland, the
committee received a report on biohaz-
ard-containment facilities in the United
States and reviewed a proposed NIH
contract program for the construction
and testing of microorganisms that would
have very limited ability to survive in
natural environments and would thereby
limit any possible hazards. A subcom-
mittee chaired by Dr. David Hogness was
appointed to draft guidelines for research
involving recombinant DNA molecules,
to be discussed at the next meeting.

The NIH committee, beginning with
the draft guidelines prepared by the Hog-
ness subcommittee, prepared proposed
guidelines for research with recombinant
DNA molecules at its third meeting, held
on July 18-19, 1975, in Woods Hole,
Massachusetts.

Following this meeting, many letters
were received which were critical of the
guidelines. The majority of critics felt
that they were too lax, others that they
were too strict. The committee reviewed
all letters, and a new subcommittee,
chaired by Dr. Elizabeth Kutter, was ap-
pointed to revise the guidelines.

A fourth committee meeting was held
on December 4-5, 1975, in La Jolla, Cali-
fornia. For this meeting a ‘‘variorum edi-
tion” had been prepared, comparing line-
for-line the Hogness, Woods Hole, and
Kutter guidelines. The committee re-
viewed these, voting item-by-item for
their preference among the three varia-
tions and, in many cases, adding new
material. The result was the “Proposed
Guidelines for Research Involving Re-

NOTICES

combinant DNA Molecules,” which were
referred to the Director, NIH, for a final
decision in December 1975.

The Director of the National Institutes
of Health called a special meeting of the
Advisory Committee to the Director to
review these proposed guidelines. The
meeting was held at NIH, Bethesda, on
February 9-10, 1976. The Advisory Com-
mittee is charged to advise the Director,
NIH, on matters relating to the broad
setting—scientific, technological, and
socioeconomic—in which the continuing
development of the biomedical sciences,
education for the health professions, and
biomedical communications must take
place, and to advise on their implica-
tions for NIH policy, program develop-
ment, resource allocation, and admin-
istration. The members of the committee
are knowledgeable in the fields of basic
and clinical biomedical sciences, the so-
cial sciences, physical sciences, research,
education, and communications. In addi-
tion to current members of the commit-
tee, the Director, NIH, invited a number
of former committee members as well as
other scientific and public representa-
tives to participate in the special Feb-
ruary session.

The purpose of the meeting was to seek
the committee’s advice on the guidelines
proposed by the Recombinant Advisory
Committee. The Advisory Committee to
the Director was asked whether, in their
judgment, the guidelines balanced
scientific responsibility to the public with
scientific freedom to pursue new knowl-
edge.

Public responsibility weighs heavily in
this genetic research area. The scientific
community must have the public’s con-
fidence that the goals of this profoundly
important research accord respect to im-
portant ethical, legal, and social values
of our society. A key element in achiev-
ing and maintaining this public trust is
for the scientific community to ensure an
openness and candor in its proceedings.
Representatives of the international
press were invited to the Asilomar con-
ference, and the proceedings received ex-
tensive coverage. The meetings of the Di-
rector’s Advisory Committee and the
Recombinant Advisory Committee have
also reflected the intent of science to be
an open community in considering the
conduct of recombinant DNA experi-
ments. Notification of all the meetings
was published in the FEDERAL REGISTER
and all the meetings were attended and
reported by representatives of the press.
At the Director’s Advisory Committee
meeting, there was ample opportunity
for comment and an airing of the issues,
not only by the committee members but
by public witnesses as well. All major
points of view were broadly represented.

The guidelines were reviewed in light
of the comments and suggestions made
by participants at that meeting, as well
as the written comments received after-
ward. As part of that review the Recom-
binant Advisory Committee was asked
to consider at its meeting of April 1-2,
1976, a number of selected issues ralsed
by the commentators. Those issues and

the response of the Recombinant Ad-

38429

visory Committee were taken into ac-
count in arriving at the final decision
on the Guidelines.

The history of the events and discus-
sions leading to the development of the
Guidelines are described in greater de-
tail in the “Decision of the Director,
NIH,” published as a preamble to the
Guidelines in the FepERAL REGISTER, Part
II, July 7, 1976 (See Appendix D).

C. DESCRIPTION OF ISSUES RAISED BY
RECOMBINANT DNA RESEARCH

1. Possible hazardous situations. The
stable insertion of DNA derived from a
different species into a cell or virus (and
therefore the progeny thereof) may
change certain properties of the host.
The changes may be advantageous, detri-
mental, or neutral with regard to (a) the
survival of the recipient species, (b)
other forms of life that come in contact
with the recipient and (c) aspects of the
nonliving environment. Current knowl-
edge does not permit accurate assess- —
ment of whether such changes will be
advantageous, detrimental or neutral,
and to what degree, when considering a
particular recombinant DNA experiment.
At present it is only possible to speculate
on ways in which the presence of recom-
binant DNA in a cell or virus could bring
about these effects. It should be empha-
sized that there is no known instance
in which a hazardous agent has been
created by recombinant DNA technology.
The following discussion is speculative
and consider ways in which hazardous
agents might be produced.

a. The effect of foreign DNA on the
survival of recipient species (host cells
or viruses). The effect of foreign DNA
on the survival of recipient species is im-
portant to the discussion of possible haz-
ards of recombinant DNA experiments
because although a recipient species may
acquire a potential for harmful effects
as a result of the foreign DNA, the possi-
bility that the harmful effect will occur
will depend on the survival of the recipi-
ent and its ability to multiply. If acqui-
sition of foreign DNA increases the prob-
ability of survival and multiplication the

‘possibility of harmful effects will in-

crease. Similarly, if acquisition of for-
eign DNA decreases the probability of
survival or multiplication, the possibility
of harmful effects will decrease. It is
important to recognize, in evaluating the
potential for harmful effects, that sig-
nificant infections of animals and plants
by bacteria or viruses May require con-
tact with either a large or small number
of the infectious agent. derending on
the agent. .

There are various indications that bac-
teria and viruses containing inserted for-
eign DNA are less likely to survive and
multiply than are the original organisms.
Natural evolution results in the survival
of well-balanced and efficient organisms.
Essential functions are carefully con-
trolled, and can be switched on and off
as needed. It is unlikely that uncon-
trolled, nonessential properties such as
might be introduced by foreign genes
would result in any advantage to the
survival and multiplication of an other-

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
38430

wise well-balanced organisms. It is more
likely that the new properties accom-
panying insertion of foreign genes will
confer some relative disability to the
recipient organisms. Therefore it is likely
that bacterial cells containing inserted
foreign DNA will multiply more slowly
than the same cells without foreign DNA.
Thus, in a natural competitive environ-
ment, bacteria containing recombinant
DNA would generally be expected to dis-
appear. The rate of disappearance will
depend on the relative rate of growth
compared to other, competing bacteria.
The following calculation demonstrates
this point.

Assume that a new organism constitutes
90 percent of a population, but grows 10 per-
cent less rapidly than its natural counter-
part. The new organism will drop from a con-
centration of 90 percent to a concentration
of 0.0001 percent (1 part in 1,000,000) in 207
generations. If the generation time of the
natural organism is one hour, this amounts
to about 8 days.

One example of a situation in which
the capability of recipient bacterial host
cells to survive may be significantly in-
creased as the result of the presence of a
foreign DNA is the case of resistance to
antibiotics and drugs. It is well known
that such resistance is often genetically
determined and genes specifying resist-
ance have been described. Furthermore
it is well known that such genes may be
transferred, by natural DNA recombina-
tion, from one species of microorganism
to another. Such natural events are in
fact responsible for the rapid and wide
spread of resistance to clinically im-
portant drugs that has been observed
during the last 20 years.

The ability of recipient bacterial host
cells to survive and multiply might also
be enhanced by acauisiton and expres-
sion of a foreign gene conferring the
ability to metabolize particular nu-
trients. In an environmental niche con-
taining the metabolite, such a recombi-
nant might compete succesfully against
organisms native to the niche. This
could result in destruction of an environ-
mental component—that is, the metabo-
lite. Also, if the native organisms were
performing beneficial functions, those
functions could be lost upon the success-
ful establishment of the recombinant in
the niche.

b. The effect of bacteria and viruses
containing recombined DNA on other
forms of life. The analysis leading to the
Guidelines centered on the possibility of
deleterious effects, since the concern was
the health and safety of living orga-
nisms, including humans, and the en-
vironment. Agents constructed by re-
combinant DNA technology could prove
hazardous to other forms of Hfe by be-
coming pathogenic (disease-producing)
or toxigenic (toxin-producing), or by be-
coming more pathogenic or toxigenic
than the original agent.

There are two basic mechanisms by
which a recipient microorganism might
be altered with regard to its patho-
genicity or toxicity as a result of a resi-
dent recombinant:

(1) The recombinant DNA may result
in formation of a protein that has un-

NOTICES

desirable effects. The case in which bac-
terial cells are used as carriers of foreign
DNA is discussed first. A foreign protein,
specified by the foreign DNA, might act
after being liberated from the micro-
organism, or it could function within the
microorganism and alter, secondarily,
normal microbial cell function in such a
way that the cell is rendered harmful to
other living things. Either means depends
on the expression of the foreign genes;
that is, the information in the foreign
genes must be used by the recipient bac-
terium to produce a foreign protein.
Examples of protein that might prove
harmful to other organisms are hor-
mones, enzymes and toxins.

The weight of present evidence sug-
gests that foreign DNA from bacteria of
one species, when inserted into bacteria
of another species, may be expressed in
the recipient. For example, if the donor
of the foreign DNA produces a toxic sub-
stance, then the recipient call may pro-
duce such a substance if the gene for
the toxic substance is present in the re-
combinant. The recipient may or may
not be more hazardous than the original
donor organism, depending on the rela-
tive ability of the two organisms to grow
and infect an animal or plant species at
risk.

The évidence available at present is in-
sufficient to predict whether or not for-
eign genes derived from a complex orga-
nism (animals, plants, yeasts, and fungi)
will be expressed in a bacterium.in any
particular instance. It may be that spe-
cific manipulations will be required to
permit bacteria to express information of
a foreign DNA efficiently. Faithful ex-
pression of a gene requires accurate func-
tioning of the complex bacterial machin-
ery involved in protein synthesis. At each
step, specific signals originating in the
foreign gene must be recognized by the
bacterial machinery. Evolutionar, diver-
gence has resulted in different signals in
bacteria and complex organisms.

Attempts to translate animal virus and
animal cell genes into portein, using cell-
free systems containing the protein-
synthesizing machinery isolated from
bacteria such as E. coli yield some pro-
tein-like products. The protein products
characterized to date were not faithful
products of the information in the genes.

In a few cases, intact bacteria contain-
ing recombined genes from complex or-
ganisms have been tested for evidence of
expression of the inserted gene. By and
large, accurate expression of the genes
has not yet been demonstrated, although
it may occur at a low frequency. In some
instances, a new protein has been found,
replacing one encoded by a bacterial
gene. This result is expected if a bacterial
gene is interrupted by insertion of the
new DNA sequence within it, and does
not necessarily indicate expression of the
foreign gene. DNA fragments from yeast
have been inserted into a strain of the
bacterium EF. coli which cannot manu-
facture the amino acid histidine (5).
(Histidine is a component of most pro-
teins and therefore is required for the
growth of all organisms.) After insertion,
some cells no longer required histidine;

thus, the presence of the yeast DNA over-

came the requirement for histidine. This
is the first suggestion that a foreign gene
from an organism more complex than
bacteria may actually function in a
bacterial cell. (Although yeast is a single-
cell organism, it contains an organized
nucleus like cells of higher organisms.)
However, the detailed mechanism ex-
plaining this observation is unknown.

Analogous issues must be considered
for the case in which animal viruses are
the carriers of foreign DNA. Many viruses
are simply described as DNA molecules
enclosed and protected by coats of pro-
tein molecules. The protein coat protects
the DNA from environmental effects,
thus increasing the ability of the viral
DNA to infect a cell. If viral DNAs are re-
combined with foreign DNAs in such a
way that necessary viral genes remain
intact, then the recombinant DNA may in
turn be able to produce, and be packaged
in, the coat of the virus. Inadvertent dis-
persal of such a viral particle outside of
the laboratory might then result in entry
of the recombinant DNA into cells of
living organisms. The foreign genes may
be expressed, resulting in the formation
of a protein foreign to the infected cell,
or the uncontrolled synthesis of a normal
protein. The likelihood of expression of
the foreign genes will probably depend on
the degree of relatedness between its
source and the infected organism as well
as its location in the viral DNA used as
vector. Currently, few if any relevant ex-
perimental data are available so that
estimates of the probability of expression
are, in these instances, impossible.

(2) The recombined DNA may itself
cause pathogenic or toxic effects. Foreign
DNA inserted in a bacterial gene, might
so alter the microbial cell’s properties
that it becomes harmful to other orga-
nisms. This might happen, for example,
through a change in the growth rate and
competitive advantage of the recipient
microbial cell, resulting in increased
virulence of a mildly pathogenic bacteria.
In general, one would expect the inserted
DNA to result in a reduced growth rate
and a selective disadvantage to the orga-
nism, as discussed in “a” above. Similar
issues arise where animal viruses serve as
carriers of foreign DNA.

It is also necessary to consider situa-
tioris in which DNA molecules themselves
may escape from the laboratory or from
the experimental host cell and enter cells
of living organisms with which they come
in contact. Although free DNA molecules
are themselves relatively fragile (and the
probability that they would survive, in a
significant form or for a significant time,
in alr, water, or any other medium, is
considered remote), they can be pro-
tected in nature in a variety of ways and
be released either into, or close to, a living
cell.

When a cell or virus dies, or comes
close to or invades the tissue of another
living organism, the recombinant DNA
may effectively enter a new cell. A haz-
ardous situation similar to that described
above might ensue if foreign proteins
were manufactured in this “secondary”

‘recipient. The recombinant DNA might

survive as an independent cellular com-
ponent, or it could recombine by natural

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
process with the DNA of the secondary
recipient. Various possible deleterious
consequences of such a recombination
may be considered.

If the secondary recipient is another
microorganism, the same considerations
described in IV-C-l-a apply. If the sec-
ondary recipient is one of the cells of an
animal or plant, different considerations
apply. The latter include alterations of
normal cellular control mechanisms, syn-
thesis of a foreign protein (such as a hor-
mone), and insertion of genes involved
in cancer production (if, for example, the
foreign DNA were derived from a cancer-
producing virus).

It should be pointed out that the like-
lihood of causing inheritable changes in
the offspring of complex organisms by
such a mechanism is extremely low in
animals because of the protection
afforded germ-line cells (eggs and
sperm) by their location. Thus, the pos-
sibility that recombined foreign DNA
would reach germ line cells at a time in
the life of such cells when secondary re-
combination can occur is extremely re-
mote. With one-celled organisms, plants,
or simple multicellular organisms, the
probability of causing heritable change
by secondary recombination may be
higher.

What is the probability of secondary
recombination between prokaryotes and
eukaryotes in nature? It is generally held
that recombination in nature is more
likely if similar or identical sequences of
bases (rungs in the DNA ladder) occur
In the two recombining DNAs. The
greater the degree of similar sequences,
the more likely is recombination. In gen-
eral, the more closely two species are re-
lated, the more likely it is that similar
sequences will be found in their DNAs.
Thus, DNA from primates has more DNA
sequences in common with human DNA
than does DNA from mice, or fish, or
plants. Recombination may also occur
between DNAs not sharing sequences but
at lower frequencies. .

It is possible that the capacity for
interspecies recombination between dis-
tantly related species exists in nature.
For example, bacteria in animal intes-
tines are constantly exposed to fragments
of animal DNA released from dead intes-
tinal cells. Significant recombination re-
quires the uptake of intact segments of
animal DNA and their subsequent incor-
poration into the’ bacterial DNA. The
frequency of such events is unknown.

There are very few available data per-
mitting assessment of the reverse proc-
ess—namely, the incorporation of bac-
terial DNA into the cells, or DNA, of
more complex organisms. Although there
are reports of experiments in which bac-
terial DNA was inserted into animal and
plant species and production of the
bacterial protein followed, the process is
very inefficient and many investigators
have been unable to repeat these experi-
ments (6-8).

There are certain well-documented in-
stances in which the DNAs of different
living things become more or less per-
manently recombined in nature. These
instances involve recombination between

the DNAs of nonchromosomal genes, such

NOTICES

as those of viruses or plasmids, or re-
combination between the DNAs of viruses
or plasmids and chromosomal genes.
The former instance, for example, is the
mechanism behind the rapid spread of
resistance to antibiotics among different
bacterial species (9, 10). This spread ac-
companied the prevalent use of antibi-
otics in medicine and agriculture. Some
viral DNAs recombine into and persist
in chromosomal DNA of cells of recep-
tive organisms (11, 12). Some viral DNAs
acquire, in stable form, DNA sequences
derived from their host cells (13, 14).
There is also strong evidence for re-
combination of the DNA form of RNA
tumor virus genes with chromosomal
genes (15-17).

2. Expected benefits of DNA recombi-
nant research, Benefits may be divided
into two broad categories: An increased
understanding of basic biological proc-
esses, and practical applications for med-
icine, agriculture, and industry.

At this time the practical applications
are, of course, speculative. It is impor-
tant to stress that the most significant
results of this work, as with any truly
innovative endeavor, are likely to arise
in unexpected ways and will almost cer-
tainly not follow a predictable path.

a. Increased understanding of basic
biological processes. There are many im-
portant fundamental biomedical ques-
tions that can be answered or approached
by DNA recombinant research. In order
to advance against diseases in inherit-
ance, we need to understand the struc-
ture of genes and how they work. The
DNA recombinant methodology provides
a simple and inexepensive way to prepare
large quantities of specific genetic in-
formation in pure form. This should per-
mit elucidation of the organization and
function of the genetic information in
higher organisms. For example, current
estimates of the fraction of this informa-
tion that codes for proteins are simply
educated guesses. There are almost no
clues about the function of the portions
of DNA that do not code for proteins,
although these DNA sequences are sus-
pected of being involved in the regula-
tion of gene expression.

The existing state of ignorance is
largely attributable to our previous in-
ability to isolate discrete segments of the
DNA in a form that permits detailed
molecular analysis. Recombinant DNA
methodology remove this barrier. Fur-
thermore, ancillary techniques have been
developed whereby pure DNA segments
that contain particular sequences of in-
terest can be identified and selected. Of
particular interest is the isolation of pure
DNA segments that contain the genes
for the variable and constant portions of
the immunoglobin proteins. The analyses
of such segments obtained from both
germline and somatic cells should be of
inestimable value in determining the
mechanism of immunologic diversity.

A major problem in understanding the
mechanism by which certain viruses
cause cancer is how and where the in-
fecting or endogenous viral genomes are
integrated into the cell’s chromosome.
This bears on the question of how the
expression of the integrated viral genes

Qf
vetod

affects cellular regulation, thus leading
to the abnormal growth characteristics
of cancer cells. With the recombinant
DNA techniques for isolation and purifi-
cation of specific genes, this research
problem is reduced to manageable pro-
portions. It is possible to isolate the de-
sired DNA segment in pure form, Large
quantities can be obtained for detailed
study by simply extracting a culture of
the bacteria carrying the viral DNA seg-
ment in a plasmid.

b. Potential practical applications for
medicine, agriculture and industry. Cer-
tain of the potential applications will
only be realized if the reproduction of the
recombined foreign DNA in a recipient
host cell is followed by expression of the
genetic information contained in the
DNA in the form of synthesis of pro-
teins. Since the efficient translation of
eukaryote genes in bacterial (prokary-
ote) hosts has yet to be proved, these po-
tential applications are speculative at
this time. Applications that depend on
the expression of foreign prokaryotic
genes in prokaryotic recipient cells are
presently more certain.

(1) Synthesis of medically important
proteins and other substances. It has
been suggested that genes coding for
medically important substances be at-
tached to bacterial vectors, and that the
bacteria then be used to produce large
quantities of the desired material. A
number of costly and/or rare substances
would be prime candidates for such syn-
thesis:

Human insulin (a future shortage of cur-
rently used animal tnsulin appears to be
likely);

Human growth hormone (presently avail-
able only from human cadavers and in short
supply);

Clothing factor VIII
hemophilia).

Specific antibodies and antigens (for pre-
venting and treating infectious, allergic, and
autoimmune disease, and perhaps even can-
cer);

Certain enzymes, such as fibrinolysin and
urokinase (promising agents 1n the treat-
ment of embolism) and lysosomal enzymes.

(2) Endowment of plants with new
synthesis capabilities. Whole plants may
be generated from a single cell, and thus
insertion of recombinant DNA into such
cells might make it possible to endow
plant species with the capability of—

Improved photosynthetic fixation of car-
bon dioxide;

Nitrogen fixation by presently inept species
(thereby reducing the need for costly chem-
ical fertilizers that cause pollution—e.g., eu-
trophication) ;

Producing a higher quality cr quantity of
food protein.

(3) Some industrial applications. A
number of industrial processes utilize
microorganisms containing enzymes
(which are proteins) to produce impor-
tant chemicals (e.g., steroid hormones or
other drugs, vitamins) or foodstuffs (e.g.,
cheese). Such processes could be im-
proved through innovations effected by
DNA recombinant research. Completely
new biosynthetic reactions may thereby
become available, permitting the synthe-
sis of large amounts of complex and

‘for treatment of

FEDERAL REGISTER, VOL. 41, NO. 176—-THURSDAY, SEPTEMBER 9, 1976
38432

valuable compounds with ease and at
low cost. :

Some highly speculative applications
relate to the area of energy production
and neutralization of pollutants—e.g., as
in oil spills. Genetic modification through
DNA recombination might it possible to
devise microorganisms tailor-made for
such important purposes.

3. Long-range implications. The exper-
imental situations treated in the Guide-
lines are those that appear feasible either
currently or in the near future. The ex-
periments primarily involve insertion of
recombined DNA into bacteria or into
single cells derived from more complex
organisms and maintained under special
laboratory conditions. It is only in the
case of plants that the Guidelines cover
experiments involving insertion of DNA
into cells capable of developing into com-
plex, multicellular organisms. The Guide-
lines and the discussions leading to their
development have focused on problems of
safety.

It is possible that techniques similar to
or derived from current recombinant
DNA methodology may, in the future,
be applicable to the deliberate modifica-
tion of complex animals, including hu-
mans. Such modification might have as
its aim correction of an inherited defect
in an individual, or alteration of herit-

‘able characteristics in the offspring of
individuals of a given species. The latter
type of alteration has been successfully
achieved in agriculture for centuries, by
classical breeding techniques. It may be
that recombinant DNA methods, should
they develop in appropriate ways, may
offer new opportunities for specificity and
accuracy in animal breeding.

The deliberate application of such
methods for the correction of individual
genetic defects or the alteration of herit-
able characteristics in man raises com-
plex and difficult problems. In addition to
philosophical, moral, and ethical ques-
tions of concern to individuals, serious
societal issues are involved. Broad dis-
cussion of these problems in a variety of
forums will be required to inform both
private and public decision-making.

4. Possible deliberate misuse. In the
event that recombinant DNA technology
can yield hazardous agents, such agents
might be considered for deliberate per-
petration of harm to animals (including
humans), plants or the environment. The
possibilities include biological warfare or
sabotage. Because it is not known
whether recombinant DNA technology
can yield such agents, discussion of these
problems such as theft by saboteurs is
hypothetical and difficult. With regard
to biological warfare, a July 3, 1975 let-
ter to Dr. David Baltimore from James
L. Malone, General Counsel of the United
States Arms Control and Disarmament
Agency says, “you raise the question as to
whether the Biological Weapons Conven-
tion prohibits production of recombinant
DNA molecules for purposes of construct-

ing biological weapons. In our opinion
the answer is in the affirmative. The use
of recombinant DNA molecules for such
purposes clearly falls within the scope

of the Convention’s provisions.”

NOTICES

REFERENCES

(1) Handler, Philip, (ed.) (1970). Biology
and the Future of Man. Oxford University
Press, New York, N.Y.

(2) Singer, M. F. and D. Soll (1973). Guide-
lines for DNA Hybrid Molecules. Science
181:1114.

(3). Berg, P., D. Baltimore, H. W. Boyer,
8S. N. Cohen, R. W. Davis, D. S. Hogness, D.
Nathans, R. O. Roblin, J. D. Watson, S. Weiss-
man, and N. D. Zinder (1974). Potential Bio-
hazards of Recombinant DNA Molecules.
Science 185:303. :

(4) Berg, P.; D. Baltimore, S. Brenner, R.
O. Roblin, and M. F. Singer (1975). Summary
Statement of the Asilomar Conference on
Recombinant DNA Molecules. Science
188:991; Nature 225:442; Proc. Nat'l. Acad.
Sci. 7271981.

(5) Struhl, K., J. R. Cameron, and R. W.
Davis (1976). Functional Expression of
Eukaryotic DNA in Escherichia Coli, Proc.
Nat'l, Acad. Sci. U.S.A. 73:1471-1475.

(6) Goebel, W. and W. Schiess (1975). The
Fate. of a Bacterial Plasmid in Mammalian
Cells. Mol. Gen. Genet. 138:213-223,

(7) Merril, C. R., M. R. Geier and J. C.
Petricciani (1971). Bacterial Virus Gene Ex-
pression in Human Cells. Nature 233:398-400.

(8) Doy, C. H., P. M. Gresshoff and B. G.
Rolfe (1973). Biological and Molecular Evi-
dence for the Transgenesis of Genes from
Bacteria to Plant Cells. Proc. Nat'l. Acad. Sci.
U.S.A. 70:3: 28-726.

(9) Novick, R. P. and S. I. Morse (1967).
In Vivo Transmission of Drug Resistance
Factors Between Strains of Staphylococcus
Aureus. J. Exp. Med. 125:45-59.

(10) Anderson, J. D., W. A. Gillespie and
M. H. Richmond (1974). Chemotherapy and
Antibiotic Resistance Transfer Between En-
terobacteria in the Human Gastrointestinal
Tract. J. Med. Microbial. 6:461-473,

(11) Hays, W. (1968). Genetics of Bacteria
and Their Viruses. John Wiley and Sons, Inc.,
Second Edition, New York, N.Y.

(12) Tooze, J. (1973). Molecular Biology
of Tumour Viruses. Cold Spring Harbor Lab-
oratory, Cold Spring Harbor, N.Y.

(13) Lavi, S., and E. Winocour (1972).
Acquisition of Sequence Homologous to Host
Deozyribonucleic Acid by Closed Circular
Simian Virus 40 Deoxyribonucleic Acid. J. of
Virology 9:309-316.

(14) Brockman, W., T. N. H. Lee and
D. Nathans (1973). The Evolution of New
Species of Viral DNA During Serial Passage
of Simian Virus 40 at High Multiplicity.
Virology 54:384-397.

(15) Gillespie, D., W. C. Saxinger and R. C.
Gallo (1976). Information Transfer in Cells
Infected by RNA Tumor Viruses and Exten-
sion to Human Neoplasia. Prog. Nuc. Ac. Res.
and Mol. Biol. 15:1-108. .

(16) Markham, P. D. and M. A. Baluda
(1973). Integrated State of Oncornavirus
DNA in Normal Chicken Cells and in Celis
Transformed by Avian Myeloblastosts Virus.
J. Virol. 12:721.

(17) Hill, M. and H. Hillova (1972). Virus
Recovery in Chicken Cells Tested with Rous
Sarcoma Cell DNA. Nature New Biology
237335.

V. DESCRIPTION OF THE PROPOSED ACTION

The Director, National Institutes of
Health, has issued Guidelines that will
govern the conduct of NIH-supported re-
search on recombinant DNA molecules.
The Guidelines will apply to all NIH-
supported research on such molecules—
that is, molecules which are made by
combining segments of DNA from differ-
ent organisms in a cell free-system and
which can be inserted into some living
cell, there to replicate. The objective of

the Guidelines is the protection of the
laboratory worker, the general public,
and the environment from infection by

- possibly hazardous agents that may re-

sult from this research. The complete
text of the Guidelines is found in the
FEDERAL REGISTER, Part II, for Wednes-
day, July 7, 1976. As an integral part of
this Draft Environmental Impact State-
ment the Guidelines are found in Appen-
dix D.

The mechanisms by which the NIH will
implement the application of the Guide-
lines are outlined in the Guidelines them-
selves and are specified in greater detail
in Appendix C. Noncompliance with the
Guidelines will result in termination of
funding of research grants and contracts.

The Guidelines describe (1) safeguards
that protect the laboratory worker, the
general public, and the environment, (2)
the criteria for assessing the possible
dangers from experiments involving re-
combinant DNA molecules, (3) the cri-
teria for matching the assessed possible
dangers of individual experiments with
the appropriate safeguards, and (4) the
roles and responsibilities of principal in-
vestigators, their institutions, and NIH
for ensuring the implementation of the
requirements specified in these Guide-
lines. The emphasis on protection of lab-
oratory workers from infection reflects
the fact that laboratory workers are the
persons at the greatest risk of infection
and that the most likely route of escape
of possibly hazardous agents from the
laboratory is the laboratory worker.

The physical safeguards have been
grouped into four levels providing in-
creasing capability for containment.
The four levels approximate those rec-
ommended by the Center for Diseasé
Control for the control of known in-
fectious agents that have been deter-
mined to be of (1) no or minimal, (2)
ordinary, (3) special, or (4) extreme
hazard to man and other living things.
These correspond to the terms Minimal,
Low, Moderate, and High risk, respec-
tively, as used in the NIH Guidelines.
The safeguards include usual and spe-
cial microbiologicfil safety practices,
primary physical barriers that isolate
the experiment from the laboratory
worker, and facility installations that
either markedly reduce or eliminate the
potential for accidental dissemination of
recombinant DNA molecules to the en-
vironment. The four levels, designated
Pl to P4, provide increasing protection
against. contact with or accidental re-
lease of microorganisms containing re-
combinant DNA molecules.

Additional safeguards are provided
by the use of host cells and vectors with
demonstrably limited ability to survive
in other than specially designed labora-
tory environments. This concept is called
“biological containment” in the Guide-
lines. In the case of bacterial host cells
and vectors, this means that particu-
lar strains of cells and vectors with
genetically determined and fastidious
survival requirements must be used. For
those experiments judged to be of poten-
tially moderate or high risk, the proper-
ties of the bacterial strains to be used

FEDERAL REGISTER, VOL. 41, NO. 176—-THURSDAY, SEPTEMBER 9, 1976
must be certified by the NIH Recom-
binant Advisory Committee prior to in-
itiation of experiments. In the case of a
vector derived from an animal virus, the
virus itself must be a low risk agent
(CDC or National] Cancer Institute), and
a strain of the virus that is defective in
infection must serve as the source of the
vector DNA. :

The selection of containment (safe-
guard) levels is dependent on the
assessed possible dangers of the experi-
ment. The Guidelines provide standards
for evaluating the conceivable dangers
of particular experiments involving re-
combinant DNA molecules. In the ab-
sence of evidence of any hazard actually
occurring, these standards are based on
relevant current knowledge. Permis-
sible experiments are placed into four
elasses of increasing possible danger
which correspond to the four levels of in-
creasing containment capability (safe-
guards). Certain experiments, judged to
have the potential for extreme hazard,
should they prove dangerous, are pro-
hibited.

The possibility for danger depends
on—

(1) The biohazard associated with the
DNA of the cell or microorganism that serves
as the DNA source (e.g., genes for toxin pro-
duction),

(2) The degree to which the DNA seg-
ment has been purified away from other
genes and shown to be free of harmful char-
acteristics,

(3) The biohazard associated with the vec-
tor that serves to transmit the source DNA
to a recipient host cell,

(4) The ability of the vector to survive in
natural environments or habitats,

(5) The kinds and number of different
organisms that are susceptible to infection
by the recipient or vector,

(6) The biohazard of the recipient host
cell that serves to replicate the recom-
binant DNA molecule,

(7) The ability of the recipient cell to
survive tn natural environments or habitats,

(8) The ability of the recipient cell to
transmit the recombinant DNA molecule to
other cells capable of surviving in natural
environments or habitats,

(9) The potential of the reciplent cell to
obtain the source DNA by natural means, and

- (10) ‘The evolutionary relatedness of the
DNA source to humans.

- The Guidelines prohibit a number of
types of experiments, including those in
which an organism contributing DNA
is itself a biohazard of greater than low
risk as determined by conventional
methods of risk assessment (low risk cor-
responds to class 2 agents as defined by
the Center for Disease Control). The
host cells and vectors are required to be
of no or minimal risk. The potential
dangers are considered to increase as the
organism providing the source DNA ap-
proaches humans phylogenetically.
Thus, source DNA from primate cells is
considered to have greater potential
dangers than source DNA from lower
_ eukaryotes. In general, greater possible
dangers are assigned to recombinants
than are present in the most hazardous
component used to construct the DNA.
The risk-assessment standards are
specified in detail for one prokaryote

"NOTICES

host-vector system employing a variant
of KE. coli called strain K12, which is, by
itself, of no or minimal risk. Eukaryote
host-vector systems using defective viral
vectors are also described. The descrip-
tions of these systems provide principles
by which the potential dangers of recom-
binant DNA experiments with other
host-vector systems can be assessed.

The Guidelines also establish an ad-
ministrative framework for assigning the
responsibility for ensuring safety in rec-
combinant DNA research supported by
NIH. This responsibility is shared among
the principal investigators, their institu-
tions, and NIH. The principal investiga-
tors have the primary responsibility for
hazard assessment and for implemen-
tation of appropriate safeguards. The in-
stitutions are responsible for ensuring
that the principal investigators have the
capabilities for meeting the requirements
stipulated in the Guidelines. NIH is re-
sponsible for securing an independent as-
sessment of the potential dangers of this
research and for ensuring that no re-
search is supported unless it conforms to
the requirements stipulated in the Guide-
lines.

The Guidelines require that the insti-
tutions establish biohazard committees to
earry out the institutional responsibility,
and stipulate the qualifications and ex-
pertise of the committee membership.
NIH responsibilities are detailed in the
Guidelines and are divided among (1)
NIH Initial Review Groups, (2) thé NIH
Recombinant DNA Molecule Program
Advisory Committee, and (3) the NIH
staff.

Physical containment requirements

The safeguards in the Guidelines re-
quire the use of procedures and physical
containment systems to protect labora-
tory workers and the environment from
exposure to potentially harmful orga-
nisms. The requirements include pro-
cedures and equipment in which work is
to be done and special laboratory room
and building features, as well as appro-
priate training of workers. The systems
are grouped into four levels of contain-
ment—P1, P2, P3, and P4—each provid-
ing a level of containment greater than
the one preceding it. The level of con-
tainment that must be provided by a lab-
oratory in which an experiment is to be
done is based on an assessment of the
degree of hazard involved.

The following description of the physi-
cal containment levels is presented to
outline these requirements. A complete
description may be found in the Guide-
lines (Appendix B).

Pi Level (Minimal). A laboratory suit-
able for experiments involving recom-
binant DNA molecules requiring physical
containment at the P1 level is shown in
Figure V-—1. Such a laboratory possesses
no special engineering design features.
Work in this laboratory is generally con-
ducted on open bench tops. Special con-
tainment equipment is neither required
nor generally available. The laboratory
is not separated from the general traffic
patterns of the building, and public ac-
cess is permitted. Control of biohazards

pe
OMA

is provided by standard microblologica}
practices.

P2 Level (Low). A laboratory suitable
for experiments involving recombinant
DNA molecules requiring physical con-
tainment at the P2 level (see Figure V—2)
is similar in construction and design to
the P1 laboratory. The P2 laboratory
must have access to an autoclave within
the building, and it may have a biological
safety cabinet. Work that does not pro-
duce a considerable aerosol is conducted
on the open bench. However, when exces-
sive aerosols may be produced, low-risk
experiments must be conducted in special
cabinets (biological safety cabinets)
that provide physical barriers against
possible release of organisms. Although
this laboratory is not separated from the
general traffic patterns of the building,
access to it is limited when experiments
requiring P2-level physical containment.
are being conducted.

 

PA Laboratory

Ficure V-2

P3 Level (Moderate). As shown in Fig-
ure V-3, a laboratory suitable for experi-
ments involving recombinant DNA mole-
cules requiring physical containment at
the P3 level has special engineering
design features and physical contain-
ment equipment. The laboratory is sepa-
rated from areas that are open to the
general public. Separation is generally
achieved by controlled access corridors
and air locks, locker rooms, or other dou-
ble-doored facilities not available for use
by the general public. Access to the labo-
ratory is controlled. Biological safety
cabinets are available within the con-
trolled laboratory area. An autoclave
shall be available within the building and
preferably within the controlled labo-
ratory area. Environmental protection is
provided by waste sterilization tech-
niques. The surfaces of walls, floors,

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
“BRt2d

bench tops, and ceilings are easily clean-
able to facilitate housekeeping and space
decontamination. The laboratory venti-
lation system is balanced to provide for
an inflow of supply air from the access
corridor into the laboratory. No work in
open vessels is conducted on the open
bench; all such procedures are confined
to biological safety cabinets.

P4 Level (High). As shown in Figure
V-4, experiments involving recombinant
DNA molecules requiring physical con-
tainment at the P4 level shall be con-
fined to work areas in a maximum-secu-
rity facility of the type designed to con-
tain mieroorganisms that are extremely
hazardous to man or may cause serious
epidemic disease. The facility is either a
separate building or a controlled interior
area completely isolated from all other
areas of a building. Access to the facility
is under strict control. Class II biolog-
ical safety cabinets are available.

 

 

 

Ficurm V-4

A P4 facility has engineering fea-
tures, shown in Figure V-5, designed to
prevent the escape of microorganisms
to the environment (1-4). The special
features in a P4 facility include:

Monolithic walls, floors, and ceilings in
which all penetrations such as for air ducts,
electrical conduits, and utility pipes are
sealed to ensure the physical isolation of
the work area and to facilitate housekeep-
ing and space decontamination.

Air locks through which supplies and
materials can be brought safely into the
facility.

Contiguous clothing change and shower
rooms through which personnel enter
into and exit from the facility.

Double-door autoclaves to sterilize and
safely remove wastes and other materials
from the facility. .

A biowaste treatment system to sterilize

liquid effluents if facility drains are in-
stalled.

NOTICES

 

NN Lior Site
Secondary Saniery

PicurE V-5

A separate ventilation system that matn-
tains negative air pressures and directional
airflow within the facility.

A treatment system to decontaminate ex-
haust alr before it is dispersed to the at-
mosphere. A central yacuum utility system
is not encouraged; if one is installed,
each branch line leading to a laboratory
shall be protected by a high-efficiency par-
ticulate air filter.

REFERENCES

1. Design Criteria for Viral Oncology Re-
search Facilities. US. Department of
Health, Education and Welfare, Publie
Health Service, National Institutes of
Health; DHEW Publication No. (NIH) 75-
891, 1975.

2. Kuehne, R. W. (1973). Biological Con-
tainment Factitty for Studying Infectious
Disease. Appl. Microbiol. 26:239-248.

3. Runkle, R. S. and G. B. Phillips (1969).
Microbial Containment Control Facilities.
Van Nostrand Reinhold, New York.

4. Chatigny, M. A. and D. IL. Clinger (1969).
Contamination Control in Aerobiology. In_
R. L. Dimmick and A. B. Akers (eds.). AD
Introduction to Experimental Aerobtology.
John Wiley & Sons, New York, pp. 194-
263. .

VI. DESCRIPTION OF ALTERNATIVES

The following general classes of action
have been considered as alternatives to,
or in addition to, the proposed action.
The impact of each is described briefly,
and reference is made to other portions
of this document which have a more
complete discussion of the particular im-
pact in question.

A. NO ACTION

This alternative would perpetuate the
situation existing prior to June 23, 1976.
At that time the only restrictions on
recombinant DNA research stemmed
from voluntary compliance of the re-
search community with the guidelines
developed at the International Confer-
ence on Recombinant DNA Molecules,
held at Asilomar, California, in Febru-
ary of 1975, which were published in
scientific journals. The Asilomar guide-
lines differ in substance from the NIH
Guidelines, and are considerably less
stringent and less detailed in their re-
quirements for containment of poten-
tially hazardous organisms. For example,
experiments that may be carried out with
minimal containment according to the
specific language of the Asilomar guide-
lines (e.g., the construction of an E. coli
Plasmid containing the noncancer-pro-
ducing DNA segment of SV40) require
P3 or P4 according to the NIH Guide-

lines. In addition, while the Asilomar
guidelines recommend that certain ex-
periments be deferred, the list of experi-
ments to be deferred is expanded in the
NIH Guidelines. Furthermore, disregard
of the Asilomar guidelines carries no
sanctions on investigators, and it could
be expected that the currently high level
of voluntary compliance would be eroded
with time.

The “no action” alternative would
greatly increase the probability that pos-
sibly hazardous organisms would be re-
leased into the environment. In addi-
tion, public concern would be increased
in the absence of any Federal action. It
is concluded that the “no action” alter-
native would not afford adequate pro-
tection of laboratory workers, the gen-
eral public, and the environment from
the possible hazards described in sec-
tion IV-C-1.

The alternative of “no action” would
essentially remove from the conduct of
research the restrictions inherent in the
NIH Guidelines. Experiments concerning
basic biological processes, and the devel-
opment of technology applicable to medi-
cal, agricultural, and industrial prob-
lems, would proceed at a faster rate.
Moreover, the immediate cost of con-
ducting research would be markedly de-
creased with the “no action” alternative,
since the need for costly physical con-
tainment would be less.

B. NIH PROHIBITION OF FUNDING OF ALL
EXPERIMENTS WITH RECOMBINANT DNA

NIH could refuse to fund any any re-
combinant DNA experiments. This would
not necessarily result in the cessation of
such research, since it may still be sup-
ported by non-NIH funds both in this
country and abroad. Therefore a reduc-
tion of risks but not elimination of risks
might be achieved by total NIH prohibi-
tion. Because the NIH funds a large pro-
portion of the total biomedical research
effort, a significant delay might be ex-
pected in the achievement of the goals
and missions of programs designed to
elucidate basic biological processes and,
in turn, the mechanisms underlying vari-
ous disease states. It is widely antici-
pated that a variety of research—im-
pacting on health and other areas of hu-
man concern—will benefit from recom-
binant DNA technology (see Section
IV-C-2).

American scientists have played a
leading role in bringing the potential
hazards of recombinant DNA research to
the attention of scientists, governments,
and international organizations. As a re-
sult, there is an effort to adopt safety
procedures for the conduct of this re-
seearch in many countries. Although na-
tions differ in their perceptions of the
need to adopt safety measures, and of
what the exact measures should be, the
NIH Guidelines are being used as a
model. NIH. prohibition of the work
would undermine American leadership
in the establishment of worldwide stand-
ards for safety.

Finally, prohibition would be likely to
have important impacts on American
science, both in research and in develop-
ment of technology. The leadership of

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
the United States in biological research
would be threatened. Further, historical
precedents indicate that measures which
interfere with free inquiry in one area
of interest, often inhibit the vitality of
other aspects of society.

C. DEVELOPMENT OF DIFFERENT GUIDELINES

Each of the stipulations in the NIH
Guidelines was made after assessment of
the possible hazards associated with par-
ticular experiments. The available data,
however, were limited, and different con-
clusions could have been reached. Some
issues addressed in the preparation of
the Guidelines which could have led to
different specifications. are as follows:

1. Levels of physical containment. For
certain experiments in which the poten-
tial risk is controversial, the physical
containment level could have been high-
er or lower. Examples of controversial
issues are the recommendations with re-
spect to containment levels for recombi-
nant experiments involving bacterial
cells and DNA derived from cold-blooded
animals, and for-experiments involving
the use of DNA from animal viruses.

2. Establishment of a few national P3
facilities openly available to all investi-
gators, with the requirement that all ex-
periments requiring P3 containment be
conducted therein. In effect, this will be
the situation with respect to P4 facilities
under the Guidelines. There are several
advantages to working in regional cen~
ters:

a. It would be less expensive to construct
and staff a few such regional centers than
many such facilities.

b. Training would be centralized.

ce. P38 facilities would be more uniformly
accessible to qualified investigators from a
variety of institutions.

a. There would be greater assurance that
the facilities meet the specified require-
ments,

e. Banks of cells containing recombinant
DNA could be maintained, with a view to
decreasing the number of times the actual
recombination process would be performed
(such banks can also be maintained in the
absence of centralized P3 facilities).

f. The sites could be placed away from
population centers.

The disadvantages of establishing re-
gional centers include:

a. Long-range planning would be neces-
sary.
b. Scheduling would be a problem.
ce. The investigator's independence would
be diminished,
a. Competition for access might favor es-
tablished investigators or established ideas.
e. The nature of the process, which might
require only brief access of P3 facilities in a
given day but over a lengthy period of time.
f. Access problems might unnecessarily dis-
ecurage valuable research.

3. All permissible recombinant DNA
experiments be conducted in P4 facili-
ties. This alternative implies no distinc-
tion among experiments. It does not rec-
ognize that certain recombinant DNA
experiments are widely agreed to pose
little, if any, possible hazard. It is equi-
valent to a total prohibition on much
recombinant DNA research because of
the limited number of P4 facilities that
are available and the high cost of con-

NOTICES

struction. Because of access problems,
interesting and important research of
low or moderate possible hazard would
be discouraged.

4. Experiments prohibited at this time.
Certain types of experiments are pro-
hibited by the Guidelines. Their selec-
tion was a matter of judgment, and de-
pended on the assessment of the seri-
ousness of the possible hazard. Alterna-
tive assessments would result in either an
expansion or a contraction of the list of
prohibited experiments and consequent
decrease or increase in the possible
risks. Some of the controversial recom-
mendations are—

a. The prohibition of experiments in-
volving more than 10 liters of culture
fluid containing recombinant DNAs
known to make harmful products with~
out the express approval of the NIH Re-
combinant Advisory Committee. Contro-
versy over this recommendation relates
+o the fact that some investigators and
laboratories contend that larger volumes
of culture fluid can be safety contained
by special procedures and facilities. The
recommendation places responsibility for
evaluating the containment on the NIH
Recombinant Advisory Committee.

b. Sanction of the use of the bacterium
Escherichia coli as a recipient for recom-
binant DNA molecules. This organism
has been studied extensively and is well
suited to recombinant DNA research. It
has been argued, however, that E. coli
should not be used at the present time.
This is because many E. coli strains are
intimately associated with humans and
other living things, and because they
readily exchange DNA (genes) with cer-
tain other bacteria in nature.

Theoretically, the most desirable bac-
terial recipient of recombinant DNA
would be a species uniquely adapted to
carefully controlled laboratory environ-
ments and unable to survive or transmit
DNA to other organisms in any natural
environment. This means that the bac-
teria should be unable to survive in nor-
mal ecological niches, either in the lab-
oratory or neighboring areas. It should
be unable to colonize or survive in or on
other living things, or in soil or waiter.
In addition, these properties should not
be significantly altered by the insertion
into the bacterium of the recombined
DNA. The bacteria must also be able to
be manipulated for successful execution
of the proposed experiment.

No bacteria is known to meet all these
requirements. The guidelines permit the
use of various forms of a particular strain
of E. coli called K12. (The forms are
called EK1, EK2 and EK3 in the Guide-
lines where they are discussed in detail.)
Some of these forms already exist, others
need to be constructed. Although related
to other E. coli strains that do not in any
way meet the definition of the ideal or-
ganism, these permissible strains of £.
coli partially fulfill many of the criteria
in the definition of the ideal strain. At
present, no other bacterial species 1s
known to approximate the definition as
closely as E. coli K-12 and its derivatives.
In the future, other bacteria, closer to
the ideal, may become known, or the

38435

properties of already known species may
be shown to approach the ideal more
closely than E, coli strain K12 and tts de-
rivatives, as defined in the Guidelines.

c. Sanction of the use of Simian Virus
40 (SV 40) as a carrier of a foreign DNA
fragment. It has been argued that SV40
should not be permitted, since it is
known to cause cancer in laboratory ani-
mals. There is little evidence that SV40
results in disease in humans. However,
SvV40 infects humans, and demonstrable
antibodies to SV40 indicate that infec-
tion has occurred in some members of the
general population. Some of the infection
may have resulted from the inadvertent
inoculation of millions of individuals dur-
ing. the initial mass program of immuni-
zation aganist polio virus before SV40 was
identified as a contaminant in the vac-
cine. The antibodies may have been
formed against SV40-like viruses known
to exist naturally in humans (1). It is
possible that a recombined DNA carried
by SV40 could infect humans and sig-
nificantly affect their health (2). The
Guidelines restrict the use of SV40 DNA
to DNA from strains of the virus that are
defective in the infection process. In
addition, stringent physical containment
is required.

d. Sanction of experiments involving
the transfer of uncharacterized mixtures
of DNA segments derived from warm-~
biooded animals into bacteria. Such ex-
periments are believed to present a
greater possible risk than others because
they involve a conglomeration of un-
defined genes that might include DNA
capable of causing disease.

e. Sanction of the use of oncogenic
viruses. It has been argued that the
introduction into E. coli of the whole
DNA cor any purified segment of the DNA
of any virus oncogenic in any species
should not be permitted.

D. No guidelines but NIH consideration
of each proposed project on an individual
oasis before funding. With this alterna-
tive, individual investigators requesting
NIH funds for projects involving recom-
binant DNA research would bring plans
for proposed experiments to an NIH
cominittee that would, without the use of
formal guidelines, recommend suitable
containment measures, Depending on the
criteria used by the committee, this might
result in lower or higher containment
levels than are currently imposed by the
Guidelines. The advantages of such a
procedure would include constant re-
evaluation of potential hazards and con-
tainment measures, and up-to-date in-
formation for investigators. The dis-
advantages include the enormous time
and resources required for review, given
the size of the biological research enter-
prise in the United States, the problem
of finding knowledgeable individuals to
serve on such a committee—-essentially a
full-time occupation—the opportunity
for arbitrary decisions, and the bypass-
ing of local input in assessment of
hazards.

It should be pointed out that under the
present NIH Guidelines, loc:l institu-
tional biohazards committees must con-
sider proposed research projects on an
individuals basis and may impose more

FEDERAL REGISTER, VOL. 43, NO. 176—THURSDAY, SEPTEMBER 9, 1976
1 BR86

stringent safeguards than those required
by the Guidelines. The judgments. of the
investigator and his local committee will
be reevaluated by the NIH Study Section
reviewing the scientific merit of the
proposal.

E. GENERAL FEDERAL REGULATION OF ALL
SUCH RESEARCH

The NIH Guidelines control only re-
combinant DNA research supported by
NIH. Nevertheless, NIH has assumed a
real responsibility to work toward the
promulgation of safety measures for all
such research. Nationally, NIH has con-

ducted and is continuing to conduct

meetings with representatives of other
Federal agencies and of private industry.
In the case of the Federal Government,
consideration is being given to the im-
position of the Guidelines either by indi-
vidual agency adoption or through an
Executive Order. Non-Federal groups
have indicated that they will voluntarily
comply with reasonable gujdelines de-
signed to be applicable to their specific
needs.

From the international standpoint, the
NIH has been in communication with
relevant national bodies, the World
Health Organization, the European Mo-
lecular Biology Organization, and the In-
ternational Council of Scientific Unions,
among others, to encourage the widest
possible application of the Guidelines.

A variety of administrative mecha-
nisms could be employed to regulate re-
combinant DNA _ research. Relevant
agencies are the Center for Disease Con-
trol (CDC), including the National In-
stitute for Occupational Safety and
Health (NIOSH), or the Occupational
Safety and Health Administration, De-
partment of Labor (OSHA). For example
NIH could petition OSHA to enforce and
monitor such research through its stand-
ard procedures. If OSHA concurred, the
adopted guidelines could be extended to
all facilities under OSHA’s responsibility.

Legislation could be passed to impose
procedures and specify containment for
recombinant DNA experiments. Specific
guidelines, as well as appropriate en-
forcement mechanisms and penalties,
could be established as statute. The ad-
vantages of this approach would include
uniformity in coverage and process. The
disadvantages include the need for estab-
lishment of a new administrative mech-
anism and consequent costs, the long
time generally required for enactment of
legislation, and the relative inflexibility
of law. Flexibility is desirable because
presently recommended containment pro-
cedures will surely require timely revision
as knowledge and experience are accu-
mulated. :

A body like the National Commission
on the Protection of Human Subjects of
Biomedical and Behavioral Research
could be legislatively established. It
should be noted that a bill (S. 2515) cur-
rently under consideration in the Con-
gress would assign responsibility for con-
sideration of recombinant DNA experi-
ments to a permanent President’s Com-
mission for the Protection of Human Sub-

jects of Biomedical and Behavioral Re-

INQTICES

search. A real concern would be the in-

ability of a group with such a broad man-

date to deal effectively with the highly

specialized subject of recombinant DN.

research. ;
REFERENCES

(1) Millarkey, M. F., J. F. Hruska and K. K.
Takemoto (1974). Comparison of Human
Papova Viruses With Simian Virus 40. J. Virol.
13;1014-1019.

(2) Shah, K. and N. Nathanson (1975).
Human Exnosure to SV40: Review and Com-
ment, A resource document for the meeting
on Recombinant DNA molecules, Asilomar
Conference Center, February 24-26, 1975.

VIT. ENVIRONMENTAL IMPACT OF THE
GUIDELINES

A, IMPACT OF ISSUANCE OF NIH GUIDELINES

The primary impact of issuance of the
Guidelines is to provide a mechanism for
the protection of the laboratory worker,
the general public, and the environment
from the possible hazards that might re-
sult from recombinant DNA molecule re-
search. These hazards are purely specu-
lative at present; the speculations may
prove to be wrong. Nevertheless the
Guidelines take cognizance of the possi-
bility of dangers to the laboratory work-
er, other persons, and the environment
posed by the emergency research tech-
nology involving recombinant DNA mole-
cules, and call for a number of measures
aimed at reducing or eliminating human
and environmental exposure {o materials
containing recombinant DNA molecules,
in case they should prove hazardous. The
Guidelines govern only work supported
by the NIH, including NIH supported re-
search at various institutions (grants and
contracts) and research carried out with-
in NIH intramural laboratories.

With regard to the anticipated but
speculative benefits of recombinant DNA
research, adherence to the Guidelines
may postpone their realization. Certain
experiments are prohibited; many per-
missible experiments will be delayed
pending availability of suitable contain-
ment facilities and certification of ap-
propriate hosts and vectors.

1. Impact on the safety of laboratory
personnel and on the spread of possibly
hazardous agents by infected laboratory
personnel. The NIH Guidelines are di-
rectly concerned with reducing and elim-
inating exposures of laboratory person-
nel and all other persons to host cells and
microorganisms containing recombinant
DNA molecules. Because laboratory per-
sonnel would be the chief source of in-
fection of other people, protection of
personnel is of primary importance. Lack
of knowledge about the real risks of such
molecules makes it impossible to deter-
mine either the nature of the hazards or
the extent to which laboratory personnel
are endangered by exposures to the ma-
terials. Nevertheless present understand-
ing of biology permits a ranking of the
possible risks that may be associated
with a given experiment.

Four levels of possible risk have been
established: minimal, low, moderate, and
high. Protection of personnel from min-

imal risk materials is provided by or-
dinary microbiological techniques. Since

these procedures are generally per-
formed on the open bench, exposures
may occur. The avoidance of harmful
effects depends more on the exceedingly
low potential of these materials to cause
a harmful infection than of the elimina-
tion of potential exposures. Potential
harmful effects would require exposure
to large numbers of organisms, e.g., due
to accidental ingestion by poor pipetting
techniques or self-inoculation by needle
and syringe). Such exposures should be
prevented by adherence to practices rec-
ommended for this risk level.

The safety, of personnel handling ma-
terials of minimal risk in the prescribed
manner is supported by the absence of
any documented laboratory-acquired
bacterial or viral infections involving
known human etiologic agents that are
customarily handled in the same fash- -
jion—i.e., CDC class 1 agents (see Glos-
sary).

The protection of personnel from po-
tential dangers associated with-low- and
moderate-risk materials is provided by a
greater reliance on physical barriers sep-
arating the laboratory personnel from
the experimental process as well as on
safe microbiological practices. Acci-
dental exposure by ingestion would be
prevented by the adherence to the re-
quired use of mechanical pipetting for
low- and moderate-risk materials. Po-
tential exposure to low-risk materials
through aerosols is reduced by the re-
quirement that all processes that pro-
duce significant aerosols are to be con-
fined to biological safety cabinets. Po-
tential exposure to moderate-risk ma-
terials through aerosols is further re-
duced by the requirement to contain all
processes that produce any aerosol. The
use of Class I and Class II biological
safety cabinets that comply with the
standards specified in the Guidelines can
reduce the potential exposure by a factor
of 10,000 (1). Potential exposures of
laboratory personnel not involved in
these experiments are further controlled
by the specified laboratory access pro-
cedures. These measures do not provide
absolute protection from exposures, and
the required primary barriers can be
compromised by lack of attention to
technique, poor placement of equipment,
and human error. Experience demon-
strates that the use of these measures
reduces but does not prevent the poten-
tial for laboratory-acquired infections
with relatively infectious agents such as
class 2 and class 3 agents.

The nature of the harmful effects from
exposures to low- and moderate-risk re-
combinant DNA materials cannot be de-
termined. However, the ability for these
materials to cause disease or injury,
should they be hazardous can be esti-
mated by comparison of their infectivity
with that of known class 2 and class 3
agents. The requirement that recipient
bacterial cells be class 1 agents (no or
minimal risk) and that animal virus vec-
tors be similarly low risk agents (in the
absence of recombined DNA) reduces the
likelihood that they will have the infec-
tious properties of class 2 or 3 agents
upon insertion of foreign DNA.

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
Recombinant DNA experiments as-
sessed to have high-risk potential require
special precautions designed to prevent
exposures, as specified in the Guidelines.
All such experimental procedures are re-
quired to be surrounded by absolute pri-
mary barriers that are gas-tight. These
are barriers that physically isolate the
experimental process from the laboratory
worker. Research is conducted within
these barriers through attached gloves.
Materials are not removed from the bar-
riers until they have been sterilized or
put into hermetically sealed containers,
which are then surface sterilized.

Experience with class 3 and 4 human
etiologic agents demonstrates that the
absolute primary barriers can be oper-
ated without exposure of the operators
under standardized procedures, employ-
ing stable, well trained and well-disci-
plined personnel (2). This conclusion is
based on those data in referenee 2 that
refer to the experience of recent years;
the earlier experience is less relevant be-
cause of important recent developments
in the design and availability of contain-
ment equipment. The procedures for
combining segments of DNA and insert-
ing them into recipient cells can be
standardized, and the Guidelines require
that research personnel be well trained
and proficient in the necessary opera-
tional practices. Inspection and certifica-
tion of all high-risk research facilities by
NIH personnel provide additional assur-
ances that these requirements will be
met.

Thus, potentially harmful effects from
research with high risk recombinant
DNA molecules should be extremely un-
likely given strict adherence to the NIH
Guidelines.

Insofar as research sponsored by NIH
is concerned, potentially harmful effects
from experiments judged to present the
possibility of very severe hazard should
be prevented completely since those ex-
periments are prohibited.

2. Impact on the environmental spread
of possibly hazardous agents. The NIH
guidelines are directly concerned with
preventing the release of cells and micro-
organisms containing recombinant DNA
molecules, or the release of recombinant
DNA molecules themselves, into the en-
vironment, thus preventing potential ex-
posures of humans, other animals and
plant communities.

The Guidelines require decontamina-
tion of all Hquid and solid wastes gen-
erated by low-, moderate-, or high-risk
experiments. As the potential risk of
these materials increases (low —» high),
further measures are required to in-
crease the certainty of containment. The
Guidelines recommend the decontamina-
tion of no- or minimal-risk materials
before their disposal to the environment.
This is standard microbiological practice.

The Guidelines prohibit the release of
contaminated air under ordinary condi-
tions. Procedures involving low- and
moderate-risk materials that may pro-
duce aerosols are confined to primary
barriers. Contaminants in the exhaust
air from these barriers are removed by
filtration.

NOTICES

The potential for accidental release of
recombinant DNA materials into the
atmosphere, however, increases with de-
creasing containment requirements
(moderate —+ minimal). Harmful sec-
ondary effects from such accidental re-
lease of minimal-, low-, or moderate-risk
materials are exceedingly remote. An
analysis of 36 reported laboratory-ac-
quired micro-epidemics in the period
1925-1975 involving over 1,000 infections
with class 2, class 3, and class 4 human
etiologic agents demonstrated no infec-
tions among persons who were never in

the laboratory building or who were not

associated in some way with the labora-
tory (2). Almost all of these outbreaks
occurred in the absence of genuine efforts
to control contaminated air, Nquid
wastes, refuse, and laundry.

Any potential release of high-risk ma-
terials to the environment should be pre-
vented by adherence to the NIH Guide-
lines. All high-risk materials are required
to be isolated in physically contained, ab-
solute primary barriers. All effluents from
these barriers are sterilized. The bar-
riers themselves are located in maxi-
mum-security facilities, which are pro-
vided with additional barriers to prevent
any accidental release. Air locks, nega-
tive air pressure, clothes-change rooms,
filtration and incineration of all air ex-
hausted from the facility, and the sec-
ondary sterilization of all liquid and solid
wastes, provide additional protection to
the environment.

The NIH Guidelines also define re-
quirements for protecting the environ-
ment from potential dangers that may be
associated with the shipment of recom-
binant DNA materials. Federal packag-
ing standards appropriate for the ship-
ment of class 4 human etiologic agents
are required for the shipment of all re-
combinant materials.

3. Cost impact. The direct cost impact
of the NIH guidelines is the cost of com-
plying with their provisions. The costs
will vary according to the level of poten-
tial risk of the research. There are no
special facility. requirements for work
with minimal- and low-risk recombinant
DNA materials (P1 and P2). There are
equipment requirements for work involv-
ing low-risk recombinant DNA materials
that will involve little cost impact. Low-
risk research requires a biological safety
cabinet for procedures that may produce
significant aerosols and an autoclave for
sterilizing waste materials. These items
of equipment, however, are generally
available within the existing facilities
where such research is being conducted.
The cost impact of the NIH guidelines on
minimal- and low-risk research is there-
fore not significant.

Special equipment and facility require-
ments are specified for moderate-risk re-
combinant DNA research (P3). All work
at this level of potential risk is to be
conducted within biological safety cabi-
nets (Class I or IT). This requirement
will necessitate the acquisition of many
additional cabinets, the number being
dependent on the scope of the research
effort. It is estimated that one cabinet
will be required for every three persons

38437

involved in the research. The cost of each
cabinet is approximately $5,000.

Directional air flow, single-pass ven-
tilation, and provisions for ensuring re-
stricted access are facility requirements
specified for moderate risk (P3) recom-
binant DNA research. While many new
facilities (those constructed in the last
decade) have been constructed with this
capability, few older facilities can provide
this capability without extensive renova-
tion. Creating adequate access control by
construction of architectural barriers
(e.g., air locks, double-door alcove, etc.)
is not expensive. However, th cost of ren-
ovation of air-handling systems to pro-
vide for single-pass, directional air flow
may prevent some institutions from con-
ducting moderate-risk research. It has
been estimated that installation of air-
handling systems that comply with the
NIH Guidelines would cost approximate-
ly $200 per square foot of space serviced
by the system. .

The NIH Guidelines require that high-
risk «P4) research involving recombinant
DNA materials be conducted only in class
Tif biological safety cabinets (glove
boxes} that are installed in maximum
security facilities. Fewer than 30 facil-
ities within the United States have the
potential for meeting the requirements
specified in the Guidelines for such facil-
ities. A smaller number may actually be
available for this research. It is esti-
mated that approximately $750,000 would
be required to construct and equip a
maximum-security facility having two
10-foat by 20-foot laboratory modules
with class III cabinetry. This great cost
is due to sophisticated mechanical sup-
port sxstems (e.g., negative pressure, ex-
haust sir filtration, air waste treatment
plant) and architectural barrier: (e.g..
elothes-change rooms, air locks, waste-
staging areas, and monolithic walls.
floors, and ceilings). The cost of class III
cabinetry installed is approximately
$3000 per linear foot. In addition, the
cabinetry line and the facility each re-
quire a double-door autoclave, costing a
minimum of $15,000 and $65,000 respec-
tively.

4. Secondary impacts. There are three
secondary impacts which further pro-
vide for environmental protection—i.e.,
reduce the potential risk to the environ-
ment from recombinant DNA research:

a. Limited maximum-security contain-
ment capability. The small number of
facilities available to support high-risk
research greatly restricts the number of
such experiments that can be conducted.
The reduction in the number of experi-
ments minimizes the probability of acci-
dentai exposure of laboratory workers
and subsequent secondary environmental
impacts.

b. Safety awareness. The safe perform-
ance of biomedical research is dependent
on an awareness of the risks and the
safeguards required to control the risks.
Issuance of the NIH Guidelines should
strengthen safety performance in gen-
eral by providing safety information and
increasing the awareness of the labora-
tory worker to the potential hazards as-
sociated with biomedical research.

 

FEDERAL REGISTER, VOL. 41, NO. 176-——THURSDAY, SEPTEMBER %, 1976
38438

ct. Early recognition of potential haz-
ards. The Guidelines require that the
principal investigator notify NIH of any
serious or extended illness or accident
that may result in serlous exposure to
man or to the environment. This moni-
toring procedure will provide an early
warning of possible unforeseen hazard.
For example, if a laboratory infection
from exposure to a recombinant DNA
molecule is confirmed, indicating a real
hazard, an increase in safeguards or ces-
sation of experiments can be required to
minimize the hazard to other investiga-
tors. conducing similar studies. This up-
grading will also reduce any potential] for
environmental effects.

B. IMPACT OF EXPERIMENTS CONDUCTED
UNDER THE GUIDELINES

1. Possible undesirable impact-—a.
Dispersion of potentially hazardous
agents. The hypothetical mechanisms by
which insertion of foreign genes into
cells or viruses might result in the for-
mation of hazardous agents are de-
scribed in Section IV-C. There is, as
stated before, no known instance in which
a hazardous agent has been created by
recombinant DNA technology. Current
knowledge permits no more than specu-
lation that such agents may be produced
and an equally speculative assessment of
the nature and extent of hazards that
may follow upon a particulr recombinant
DNA experiment. This is the underlying
reason that the thrust of the Guidelines
is to minimize contact of organisms con-
taining recombinant DNA with other or-
ganisms or the environment. Therefore
the following analysis of possible un-
desirable impacts due to dispersion of
potentially hazardous agents emphasizes
the likelihood of significant dispersion
rather than the nature of the hazard it-
self. The analysis given does not apply
in detail to all the possible situations,
but can serve as a model for analyzing
different situations.

In order that any potential hazard be
realized, it is necessary that each of a
number of sequential events occur. Each
event in the sequence is possible only if
the earlier events have occurred. The or-
ganism must—

(a) Contain foreign genes,

(b) Escape from the experimenial situa-
tion,

(c) Survive after escape,

‘(d) Become established in an environment
permitting its growth and multiplication,

(e) Contact other living organisms in a
significant manner, including contact by a
sufficient number of organisms to ensure sur-
vival and growth and to cause infection.
(Note that the environment in (d) may be
& living organism itself) .

In those cases where the detrimental
effect results from the formation of a
harmful protein, the organism contain-
ing the recombinant DNA must—

(f) Contain a gene for a potentially harm-
ful protein,

(g) Be able to express the foreign gene—
that is, synthesize the foreign protein,

(h) Synthesize the protein in suffictent
quantity to be deleterious to the infected
organism.

NOTICES

In those cases where the foreign DNA
itself may be the cause of undesirable
effects, another set of events must be
«considered. In the case where the foreign
DNA increases the pathogenicity of the
initial host cell or virus, the inserted
DNA must—

(i) Impart a selective advantage for growth
to the carrier of the recombinant DNA as
compared with the original cell or virus,

(Jj) Alter the metabolism of the carrier so
that it becomes disease producing.

In the case where the foreign DNA
causes undesirable effects by virtue of its
transfer out of the original recipient and
reinsertion into cells of another species,
the DNA must—

(k) Leave the original recipient without
being destroyed,

(1) Survive transfer to another cell,

(m) Become associated with the other cell
in a stable manner, either as an independ-
ent element or by natural recombination.

For example, in a hypothetical experi-
ment classified as low-risk and carried
out according to the requirements of the
Guidelines, events (a) through (h) might
be required to yield a hazardous situa-
tion. Available data might permit assign-
ment of probabilities of : 1 for (a): of
10“ (1 in 100) for (b); of 10~ (1 in 10,-
000) for (c); and of 10* (1 in a mil-
lion) for (d@. Lack of any pertinent
knowledge concerning events (e) through
(h) would make assignment of probabili-
ties impossible. Even assuming a proba-
bility of one for each event (e) through
(h), the overall probability of a deleteri-
ous effect on a member of a species at
risk in this hypothetical situation would
then be the product of all probabilities
(a) through (h), namely 10-7 (one in a
trillion). This probability then needs to
be compared with the number of orga-
nisms grown for the experiment. Typi-
cally, bacteria are grown in liquid mix-
tures to a concentration of between 10°
and 10” organisms per ml. The probabil-
ity will also need to be corrected for the
length of time over which the experiment
is to be conducted. In reality, it may fre-
quently be difficult to assess the relevant
probabilities.

It is currently impossible to assign
specific probabilities for many experi-
ments, although crude estimates can
often be made from current knowledge
of laboratory-acquired infections, from
prototype experiments set up to measure
bacterlal or viral escape (4), and from
knowledge concerning the stability of or-
ganisms and DNA. NIH jis currently sup-
porting research designed to improve the
ability to evaluate certain of these prob-
abilities.

b. Other considerations. The foregoing
descriptions of the kinds of possibly
hazardous situations that might arise
from organisms obtained through recom-
binant DNA experiments must be con-
sidered in the light of certain more gen-
eral issues.

(1) Monitoring for release of orga-
nisms containing recombined DNA. Con-
trol of the spread of any agent outside of
an experimental situation to laboratory
workers or the outside environment ts
greatly assisted by adequate means for

monitoring the agent in question. A per-
tinent example is the monitoring for spil-
lage and spread of radioisotopes. The
presence of radioisotopes is readily meas-
ured, and the exposure of laboratory per-
sonnel or the environment to radiation
can be quantified. The situation is funda-
mentally different in the case of orga-
nisms or viruses containing recombined
DNA. No simple general procedure exists
for identifying an organism released
from the laboratory against the large
background level of related and un-
Telated organisms occurring naturally.

It is possible, however, to devise special
pertinent procedures for detection of
some of the agents used in recombinant
DNA experiments. For example, develop-
ment of bacterial strains, phages, or
Plasmids carrying readily detectable
genetic traits would enable the monitor-
ing of laboratory personnel, people work-
ing in the area, and their families for the
presence of those agents. This would be
analogous to the examination of drink-
ing water, lakes, etc., for fecal contami-
nation with enteric organisms. Detection
in such instances could be at levels as
low as 10" (1 part in 10,000,000). The
adequacy of such screening is not pres-
ently Known.

Given the nature of the series of events
that might characterize a hazardous
situation, the time factors involved in
those events become relevant. Certain
possible types of organisms containing
recombinant DNA might, if they escaped
and if they were hazardous; be immedi-
ately perceived as such—e.g., production
of toxic foreign proteins. We might
therefore be aware of the potential prob-
lem soon after dispersal of the organism,
and reasonable means for minimizing
further dispersal could be undertaken.
In other instances—e.g., a cancer-pro-~
ducing DNA fragment—evidence of
harmful effects might not be apparent
for many years. The connection between
the causative organisms and the ob-
served harmful effects could be difficult
to establish. Further, dispersal of the
hazardous agent might then be so wide-
spread as to make control difficult or
impossible.

(2) Natural occurrence of DNA re-
combination between unrelated orga-
nisms. Concern over the potential for
hazard in organisms containing re-
combined DNA develops from the central
idea that such recombinants will be
unique types of organisms, not normally
arising in nature, and that their prop-
erties will therefore be unknown and
unpredictable. Natural environments
provide many opportunities for recombi-

nation of DNA between unrelated species, .

as for example, in the intestines of ani-
mals. Whether, or at what frequency,
such recombinations may occur is not
known at present, but it is probably low
given the very low extent of shared base
sequences that can be detected in DNAs
derived from distantly related organisms.
It would appear that naturally occurring
interspecies recombinants, if they occur
in nature, may have been selected against
in evolution. However tests for shared
base sequences are of Hmited sensitivity.

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
X3) Relative irreversibility of spread of
organisms. Should organisms containing
recombined DNA be dispersed into the
environment, they might, depending on
their fitness relative to naturally occur-
ing organisms, find a suitable ecological
niche for their own reproduction, and a
potentially dangerous organism could
then multiply and possibly spread. Sub-
sequent cessation of experiments would
not stop the diffusion of the hazardous
agent. While means to eradicate the or-
ganism might be found, as in the case
of smallpox, it is also possible that such
means will not be available, or that they
will be available too late to prevent or
stop untoward events. .

As described earlier, the Hkelihood ts
that newly constructed organisms will be
less fit than those occurring naturally
and therefore will disappear over time.

2. Beneficial impacts of recombinant
DNA research, Section TV-C-2 describes
the various anticipated benefits of re-
combinant DNA research. As with the
possible hazards, many of the proposed
benefits are speculative. Assessment of
the likelihood that they will be realized
will depend on information acquired
from future experimentation. For ex-
ample, assessment of the category of
anticipated benefits that depends on the
synthesis of eukaryote proteins in
prokaryote cells (see IV-C-1-b) awaits
additional data on the expression of the
foreign genes. Should these benefits be
realized, it may be expected that the cost
of manufacturing certain clinically im-
portant proteins can be markedly de~
creased. Other clinically important pro-
teins that are either in short supply (e.g.
human growth hormone) or unobtain-
able by existing techniques may be made
readily available. Innovative approaches
to immunization against infectious dis-
eases can also be expected.

Some of the indicted benefits appear
certain. These are the benefits to be
derived from an increased understanding
of both basic biological processes and the
mechanisms underlying a variety of dis-
ease states.

Application of the restrictions imposed
by the Guidelines will retard progress
toward the redlization of the possible
benefits. In addition to the prohibitions
on certain experiments, there are many
permissible experiments which will need
to be postponed until the requirements
in the Guidelines can be met. The ac-
quisition and installation of P3 facilities
requires adequate funds, extensive plan-
ning and installation. P4 facilities are
limited in number. Experiments that re-
quire hosts and vectors with demonstra-
bly limited ability to survive in natural
environments must await development of
appropriate hosts and vectors, their test-
ing, and finally their certification by the
NIH Recombinant Advisory Committee.
Time will also be required for the various
review processes that are required.

REFERENCES

{1) Chatigny, M. A, W. EB Barkley and W.
Vogl (1974). Aerosol Biohazard in Microbio-
logicai Laboratories and How It Is Affected
by Air Conditioning Systems. Am. Soc. Heat.
Ref. Aircond. Engr. &0:Part 1.

FEDERAL REGISTER, VOL. 47, NO. 176—THURSDAY, SEPTEMBER &

NOTICES

(2) Wedum, A. G. (1976). The Detrick Ex-
perience As a Guide to the Probable Efficacy
of P4 Microbiological Containment Facilities
for Studies on Microbial Recombinant DNA
Facilities. Unpublished Report to the Na-
tional Cancer Institute.

(3) Falkow, S. (1975). Unpublished experi-
ments quoted in: Appendix D of the Report
of the Organizing Committee of the Asilomar
Conference on Recombinant DNA Molecules
(P. Berg, D. Baltimore, S. Brenner, R. O. Rob-
lin and M, Singer, eds.). Submitted to the
National Academy of Sciences.

(4) Dimick, R. L., W. Vogl and Chatigny,
M. A. (1973). Potential for Accidental Micro-~-
bial Aerosol Transmission in the Biological
Laboratory (in) Biohazards in Biological Re-
search, Helman, A., M. N. Oxman and R. Pol-
lack, eds. Cold Spring Harbor Laboratery,
N.Y.

APPENDIX A

GLOSSARY

1. Aerosol; A colloid of liquid or solid par-
ticles suspended in a gas, usually air.

2, Antibody: A protein which 1s formed in
the body as a result of the inoculation of an
antigen.

3. Antigen: A substance which when in-
jected into an animal causes the formation
of antibodies.

4. Autoclave: An apparatus for effecting
sterilization by steam under pressure. It is
fitted with a gauge that automatically regu-
lates the pressure, and therefore the degree
of heat to which the contents are subjected.

5. Bacteriophage: A virus that infects only
bacteria.

6. Bid: Bureaus, Institutes, and Divisions
of NIH.

7. Biohazard: A contraction of the words
biological hazard; infectious agents present-
ing a risk or potential risk to the well-being
of man, or other animals, either directly
through infection or indirectly through dis-
ruption of the environment.

8. Biohazardous Agent: Any microbial unit
capable or potentially capable of presenting
a biohazard.

9. Biohazard Area: Any area (2 complete
operating complex, a single facility, a single
room within a facility, etc.) in which work
has been, or is being performed with biohaz-
ardous agents or matertfals.

10. Biohazard Control: Any set of equip-
ment and procedures utilized to prevent or
minimize the exposure of man and his en-
vironment to biohazardous agents or mate-
rials.

11. Biohazardous Material: Any substance
which contains or potentially contains bio-
hazardous agente.

12. Blowaste: Liquid wastes from biological
research procedures.

13. CDC: Center for Disease Control,
United States Public Health Service, Atlanta,
Georgia.

14. CDC Classification of etdologic agenta
on the basis of hazard: A system for evaluat-
ing the hazards associated with various
etiologic agents, and definition of minimal
safety conditions for their management tn
microbiological investigations. The basis for
Agent Classification is as follows:

Class 1: Agents or no or minimal hazard
unger ordinary conditions of handling.

Class 2: Agents of ordinary ‘potential
hazard. This class includes agents which may
produce disease of varying degrees of severity
from accidental inoculation or injection or
other means of cutaneous penetration but
which are contained by ordinary laboratory
techniques.

Class 3: Agents involving special hazard
or agents derived,4rom outside the United
States which uire a federal permit for
importation unless they are specified for
higher classification. This class includes

38439

pathogens which require special conditicns
ior containment.

Class 4: Agents that require the most
stringent conditions for their containment
because they are extremely hazardous to
laboratory personnel or may cause serious
epidemic disease. This class includes Class 3
agents from outside the United States when
they are employed in entomological experi-
ments or when other entomological experl-
menis are conducted in the same laboratory
are

  

lass 6: Forelgn animal pathogens that are
schided from the United States by law or
whose entry is restricted by USDA adminic-
trative policy.

Nort: Federally licensed vaccines contain-
ing live bacteria or viruses are not subject
tO these classifications. These classifications
wre applicable, however, to cultures of the
atvains used for vaccine production, or fur-
‘her passages of the vaccine strains,

15. Class I biological safety cabinet: A
ventilated cabinet for personnel protection
only, having an open front with inward fiow
of air away from the operator. The cabinet
exhaust air is filtered through a high effi-
ciency particulate air (HEPA) filter before
being discharged to the outside atmosphere.
This cabinet can be used for work with low-
to moderate-hazard risk agents where no
product protection is required.

18. Class II biological safety cabinet: An
cpen-front cabinet for personnel and product
protection with mass recirculated airflow
with HEPA filtered exhaust and HEPA filtered
recirculated air. This cabinet can be used for
wors with low- to moderate-hazard risk
agents It is not suitable for use with ex-
plosive and flammable substances, toxie
agents, or radioactive materials.

17. Class III biological safety cabinet: A
gas-tight cabinet providing total isolation for
personnel and product protection with a
HEPA-fiitered air supply and a HEPA-filtered
exhaust. The cabinet is fitted with gloves and
is maintained under negative air pressure.
This cabinet provides the highest contain-
ment reliability and should be utilized for ali
activities involving high-hazard risk agents.

18. Clone: A population of cells derived, by
asexual reproduction, from a single cell, Every
cell in the population is presumed to be
genetically identical. In recombinant DNA re-
search, every cell in a clone contains the
fame recombinant DNA species.

19. Coding sequence: The orderly array of
codons which are subunits of a gene.

20. Chromosome: One or more smali rod-
shaped body(s) in the nucleus of a cell that
contains genetic information for that cell. A
collection of genes.

21, Deoxyribonucleic acid, or DNA: A com-
plex substance of which genes are composed.

22. Effuent: A Hquid or gas flowing from
a@ process.

23. Endogenous: Developing or originating
within the organism, or arising from causes
within the organism.

24. Escherichia coll: A bacterium com-
monly found in the intestinal tract of
animals.

25. Eticlogic agent: A viable microorgs-
nism or its toxin which causes, or may cause,
human disease. .

26, Eukaryotic cell: A cell that contains a
nucleus with a nuclear membrane surround-
ing muitiple chromosomes; also contains ex-
tranuclear organelles.

27. Gene: The smallest portion of a chrom-
csome that contains the hereditary informa-
tion for the production of a protein.

28. Genetic engineering: Directed inter-
vention with the content and/or organization
of an organism’s genetic complement.

29. Genome: The complete set of hereditary
information in a cell as the chromosomes in

VO?76
38440

a eukaryote or the single chromosome in a
prokaryote,

30. HEPA filter: (High Efficiency Particu-
late Air Filter) A disposable, extended medi-
um, dry type filter with a particle removal
efficiency of no less than 99.97% for 0.3um
particles,

31. Infectious: Capable of invading a sus-
ceptible host, replicating, and causing an al-
tered host reaction commonly referred to as
a disease.

32. Laminar alr flow: Air flow in which the
entire body of air within a designated space
moves with uniform velocity in one direction
along parallel flow lines.

33. Laboratory acquired infection: Any in-
fection resulting from exposure to biohazard=
ous materials in a laboratory environment.
Exposure may be the result of a specific acci-
dent or inadequate bichazard control proce-
dure or equipment.

34. Messenger ribonuclele acid (MRNA):
A complex molecule that transmits the in-
formation from the gene to a template on
which a protein is formed.

35. Mitochondrion: A DNA-containing
structure present in all aerobic eukaryotic
cells. The mitochrondia produce energy for
the cell and divide by fission after cell divi-
sion has occurred,

36. Nucleotide: A basic unit of the poly-
meric structure of DNA. Each unit contains a
sugar (deoxyribose), phosphoric acid, and one
of the following organic substances: adenine,
guanine, thymine or cytosine.

37. Oncogenesis: The process of tumor for-
mation.

38. Organelle; An independent structural
body existing within cells, generally related
to a particular cellular function, and contain-
ing a special group of genes within an extra-
chromosomal DNA molecule (e.g., mitochron-
dria and chloroplasts).

39. Pathogenic: Producing or capable of
producing disease.

40. Phenotype: The visible traits of an
organism as determined by the genome or
genotype.

41. Plasmid: A genetic element outside of
the cromosome that ls capable of replicating
independently of the chromosome.

42. Polymer: A large molecule composed
of simpler repeating units. DNA is a polymer
composed of nucleotides, while starch and
cellulose are polymers composed of sugars.

43. Prokaryotic organism or Prokaryote:
Celis of bacteria or blue-green algae which
ere characterized as being rather small,
having a single chromosome that is not en~
closed by a nuclear membrane, and lacking
organelles.

44. Restriction endonuclease: An enzyme
capable of breaking DNA at specific sites.
The action of the enzyme is unique in that
“sticky” ends are formed which can join with
other fragments of DNA to form a recombi-
nant DNA molecule. In nature, these bac-
terial enzymes restrict invasion of foreign
DNA, :

45. Reverse transcriptase: An enzyme
found in certain viruses which reverses the
normal synthesis of RNA from DNA. DNA is
formed for the replication of viral RNA.

46. R plasmid: A plasmid that carries
genetic information for resistance to anti-
biotics and/or other antibacterial drugs.

47. Shotgun experiment: An experiment in
which all the DNA fragments cleaved by a8
restriction endonuclease are inserted into a
vector DNA, which is then put into a cell.
This is in contrast to other recombinant
DNA experiments where only selected frag-
ments of DNA are inserted into a vector
DNA.

48. Sterilize: Any act which results in the
absence of all life on or in an object.

49. Vector: A carrier of a recombinant
DNA molecule; usually a plasmid or bacte-
riophage,

NOTICES

50. Viable: Literally, “capable of life.”
Generally refers to the ability of microbial
cells to grow and multiply as evidenced by,
for example, formation of colonies on an agar
culture medium. Frequently ozxganisms may
be viable under one set of culture conditions
and not under another set, making it ex-
tremely important to define precisely the
conditions used for determining viability.

APPENDIX B
SUGGESTED REFERENCES FOR ADDITIONAL READING

Alderson, T. (1967). Induction of Geneti-
cally Recombinant Chromosomes in the Ab-
sence of Induced Mutation. Nature 215:1281-
3

Basu, S. K, et al. (1975). Factor Which Af-
fects the Mode of Genetic Recombination
in E. Coli. Nature 253:138-40.

Berg, P. et al. (1974). Potential Hazards of
Recombinant DNA Molecules (letter.). Sci-
ence 185-308.

Berg, P. et al. (1975). Asilomar Conference
on Recombinant DNA Molecules. Science
188 :991-4.

Berg, P. et al. (1975). Summary Statement
of the Asilomar Conference on Recombinant
DNA Molecules. Proc, Nat’. Aca. Sci. USA
72:1981-4.

Berg, P. (1976). Genetic Engineering:
Chalienge and Responsibility ASM News 42;
273-277.

Capaldo, FP. N. (1974). Analysis of the
Growth of Recombination-Deficient Strains
of E. Coli K-12. J. Bact. 118:242-9.

Clark, A. J. (1967). The Beginning of a
Genetic Analysis of Recombination Profici-
ency. J. Cell. Physiol. 70:Suppl.:165-80.

Clark, A. J. (1971). Toward a Metabolic In-
terpretation of Genetic Recombination of E.
Colt and Its Phages. Ann, Rev. Microbiol. 25:
437-64,

Clark, A. J. (1974). Progress Toward a
Metabolic Interpretation of Genetic Recom~
dination of E. Colt and Bacteriophage Lamb-
da. Genetics 78 :269-71.

Cohen, S. N. (1975). Manipulation of Genes.
Sei. Amer. 233 724-33.

Curtiss, R. II (1968). Ultraviolet-Induced
Genetic Recombination in a Partially Diploid
Strain of E. Coli. Genetics 58 :9-64.

Felsenstein, J. (1974). The Evolutionary
Advantage of Recombination. Genetics 78:
737-56.

Fowler, J. V. (1974). Molecular Biologists
Call for Temporary Ban on DNA Experiments.
Bio. Science 24;533.

Green, M. M. (1968). Some Genetic Prop-
erties of Intrachromosomal Recombination.
Molec, Gen. Genet. 103:209-17.

Harvey, C. L. (1973). Lambda Phage DNA:
Joining of a Chemically Synthesized Cohesive
End. Science 179:291-3.

Helling, R. B. (1969). Recognition of Al-
tered DNA in Recombination. J. Bact. 100:
224-30.

Hotchkiss, R. D. (1971). Toward a General
Theory of Genetic Recombination in DNA,
Adv. Genet. 16:325-48.

Hotchkiss, R. D. (1974). Models of Genetic
Recombination, Ann. Rev. of Microbiol. 28
(0) :445-68.

Hotchkiss, R. D. (1974). Molecular Basis for
Genetic Recombination, Genetics 78 :247~57.

Hubacek, J. et al. (1967). Formation of
Recombinants in E. Coli and a Gene Control-
ling the Expression of a Donor Marker. Folia
Microbiol. (Praha) 12:422-31.

Kilbourne, E. D. (1969). Future Influenza
Vaccines and the Use of Genetic Recombi-
nants. Bull. W.H.O, 41:643-5.

Kushner, S. R. et al. (1971). Genetic Re-
combination in E. Coli: The Role of Exonu-
clease. Proc. Nat'l Acad. Sci. USA 68-924—-7.

Lederberg, J, (1975). DNA Splicing: Wiil
Fear Rob Us of Its Benefits? Prism, Nov. 1975,

p. 33) -

Marx, J. L. (1973). Restriction Enzymes:
New Tools for Studying DNA. Science 180:
482-5.

McCahon, D. et al. (1973). Use of Recom-
Gination in the Production of Influenza Vac-
cine Strains. Postgrad, Med. J. 49:195-9.

Meselson, M. 8. et al. (1975). A General
Model for Genetie Recombination. Proc. Nat'l.
Aca. Sci. USA 72;358-61.

Nash, H. A. (1975). Integrative Recombina-
tion in Bacteriophage Lambda: Analysis of
Recombinant DNA. J. Mol. Biol. 91:501~14.

Norkin, L. C. (1970). Marker Specifie Effects
in Genetic Recombination. J. Mot. Biol. 52:
491-9.

Putrament, A. (1971). Recombination and
Chromosome Structure in Eukaryotes. Genet.
Res. 18:85-95.

Potential Hazards of Recombinant DNA
Molecules. Proc. Nat'l. Acad. Scl. USA 71:
2593-4,

Russo, V. E. (1973). On the Physical
Structure of Lambda Recombinant DNA.
Mol. Gen. Genet. 222:353-66.

Signer, E. et al. (1968). The General Re-
combination System of Bacteriophage
Lambda. Sympos. Quant. Biol. 33:11-4.

Stmchen, G. et al. (1969). Fine and Coarse
Controls of Genetic Recombination. Nature
(London) 222:329-32.

Sinsheimer, R. (1975). Troubled Dawn
for Genetic Engineering. New Scientist,
Oct. 16, 1975.

Smith, J. M. (1974). Recombination and
the Rate of Evolution. Genetics 78:299-304.

Smithies, O. (1974). Letter: Recombinant
NDA Molecules. Genetics 77:819-20.

Strauss, B. S. (1968). DNA Repair Mech-
anisms and Thetr Relation to Mutation and
Recombination. Curr. Top. Microbiol. Im-
munol, 4471-85.

Tomizawa, J. I (1967). Molecular Mecha-
nizms of Genetic Recombination in Bac-
tertophage: Joint Molecules and Their Con-
version to Recombinant Molecules. J. Cell.
Physiol. 70:Suppl. 201-13.

Wade, N. (1976). Recombinant DNA: The
Last Look Before the Leap. Science 192:236—
8

Walmsley, R. H. (1969). The General The-
ory of Mapping Functions for Random Ge-
netic Recombination. Biophys. J. 9:421-$1.

“Whitehouse, H. L. (1970). The Mechanism
of Genetic Recombination. Biol. Rev. 45:-265-
315.

Wood, T. H. (1967). Genetic Recombina-
tion in E. Coli: Clone Hetrogenity and the
Kinetics of Segregation. Science 157:319-21,

Appendix C

DOCUMENTS DESCRIBING THE IMPLEMENTATION

OF THE GUIDELINES
DEPARTMENT OF HEALTH, EDUCATION,

AND WELFARE, PUBLIC HEALTH
SERVICE, NATIONAL INSTITUTES
OF HEALTH
June 18, 1976.
To: Director, NIH, Through: Director,
NIGMS, NIH.
From: Executive Secretary, Recombinant
DNA Molecule Program Advisory
Committee.

Subject: Operation of the Office of Recom-
binant DNA Activities (ORDA).

The proposed structure and responsibill-
ties of an Office of Recombinant DNA Ac-
tivities (ORDA) were described in Dr.
Kirschstein’s memorandum to you of
April 28, 1976. The purpose of this docu-
ment is to present our views as to how such
an office can function effectively at the
NIH. Consequently, the following relation-
ships and activities are discussed:

I. Office of the Director, NIH (OD, NIH).
The Office of Recombinant DNA Activities
(ORDA) will be responsible for keeping the
Office of the Director, NIH, (OD. NIH} and

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
particularly the Deputy Director for Sci-
ence informed concerning the activities of
ORDA, the status of BID extramural pro-
grams and intramural] research involving re-
combinant DNA molecules, and on scien-
tific and public developments which may
affect NIH policy decisions and procedures re-
garding recombinant DNA technology, ORDA
will interact with the Deputy Director for
Science and the Associate Director for Col-
laborative Research, NIH in their capacities
as chairman and vice-chairman, respectively,
of the Recombinant DNA Molecule Program
Advisory Committee (Recombinant Advisory
Committee). *

OD, NIH, in turn, should involve ORDA
in all major planning activities leading to
formation of NIH policies and procedures.
As is current practice, #he Executive Secre-
tariat, NIH will provide ORDA with infor-
mation copies of all correspondence relat-
ing to recombinant DNA matters. OD, NIH
should provide ORDA with copies of out-
going correspondence, responses to Con-
gressional inquiries, etc., to ensure that the
NIH maintains uniform positions on the
yarious matters relating to this technology.

An Executive Recombinant DNA Commit-
tee (Executive Committee) should be estab-
HMshed in the Office of the Director, NIH.
Members of the Committee would be: Deputy
Director for Science (Chairman); Associate
Director for Extramural Research and Train-
ing; Associate Director for Collaborative Re-
search; Associate Director for Program Plan-
ning and Evaluation; Director, Office of Re-
search Safety, NCI; and Associate Director for
Environmental Health and Safety, DRS. The
Special Assistant for Intramural Affairs will
serve as Executive Secretary of the Executive
Committee. There will be representation on
this committee of one or more of the BIDS
most involved in the support of research
utilizing this technology. Problems (such as
suitability of institutional biohazards com-
mittees, adequacy of review of applications,
appropriateness of proposed new initiatives
by BIDs, etc.) which cannot be resolved at
the level of ORDA will be referred to the Ex-
ecutive Committee. Problems which can not
be resolved at the level of the Executive Com-
mittee will be referred to the Recombinant
Advisory Committee or subcommittees there-
of by telephone, mail or presentation at the
next meeting. The Deputy Director for
Science will have the ultimate responsibillty
for decisions and can make urgent decisions
without consultation with the committees.
(OD, NIH may wish to propose an alternative
approach other than the establishment of an
Executive Committee) .

The Deputy Director for Science, NIH, will
have the responsibility for assigning to the
individual BIDs various projects developed
by the Executive Committee or Recombinant
Advisory Committee or solicited by ORDA.

The Office of the Director, NIH will be
responsible for promulgation and enforce-
ment of regulations, and for accountability
to congressional committees, DHEW, and the
public. ORDA will keep the Deputy Director
for Science, NIH informed of any potential
problems which may lead to regulatory ac-
tivities and/or need for accountability and
may make appropriate recommendations in
these circumstances.

II. Dissemination of information. The Office
of the Director, NIH will be responsible for
the promulgation of “Guidelines for Research
Involving Recombinant DNA Molecules”
(Guidelines), and should undertake the ini-
tial mass distribution of Guidelines to in-
stitutions and research laboratories through
its mailing keys and use of the appropriate
instrument, such as the “NIH Guide for
Grants and Contracts” (NIH Guide), NIH
Manual Issuance (NIH Manual), etc. There-
after, ORDA will be responsible for distribut-
ing Guidelines and responding to requests by

NOTICES

institutions and investigators regarding NIH
policies and procedures. Dissemination of
major changes in the Guidelines should also
be handled as above.

The NIAID wiil have responsibility for pub-
lication of the “Nucleic Acid Recombinant
Scientific Memoranda” (NARSM). Major an-
nouncements in NARSM, such as distribution
of Guidelines, statements of NIH policies and
procedures, announcements of certified host-
vector systems, training courses, workshops,
conferences, etc. will be coordinated with
ORDA.

IIL. Division of research grants. ORDA will
work with the Associate Director for Scien-
tific Review, DRG on procedures for the re-
view of grant applications involving recom-
binant DNA technology. On request, ORDA
will brief executive secretaries and study sec-
tions on NIH policies and procedures,

Involvement of DRG in the processing of
grant applications is discussed in Appendix A.

IV. Institutes and divisions. Institutes and
Divisions will be required to report to ORDA
on all activities involving recombinant DNA
technology, including: intramural research
projects, extramural grants and contracts,
workshops, training courses, conferences, etc.
BIDs will provide ORDA with copies of all
incoming and outgoing correspondence deal-
ing with recombinant DNA activities. Award-
ing components must consult with ORDA
prior issuing Requests for Proposals (RFPs)
and Requests for Applications (RFAs) likely
to result in projects utilizing recombinant
DNA technology. Procedures for obtaining in-
formation on extramural research, and for
the processing of applications are proposed
in Appendix A. Procedures for monitoring
intramural research are proposed in Appendix
B.

ORDA will be a source of information for
the Institutes and Divisions, and their initial
review groups, regarding current NIH policies
and procedures. ORDA may establish an
inter-BID Recombinant DNA Coordinating
Committee to facilitate interchange of infor-
mation. Because of the procedures proposed,
NIH awarding components may wish to con-
sider naming specific staff for Haison wit!
ORDA. :

V. Special NIH relationships. ORDA will
maintain close working relationships with
the Director, Office of Research Safety, NCT,
the Associate Director for Environmental
Health and Safety, DRS and the NIH Bio-
hazards Committee, and witl coordinate their
activities on matters relating to recombinant
DNA technology. The Office of Research
Safety, NCI will continue to have primary
responsibility for matters relating to physical
containment of recombinant DNA materials,
and will continue to maintain a register of
high containment facilities in this country
and abroad. However, plans for site inspec-
tions of P4 physical containment facilities
currently or envisaged to be engaged in re-
combinant DNA research, and of other fa-
cilities as deemed necessary, will be coordi-
nated through ORDA, and copies of site visit
reports will be filled with ORDA.

VI. Federal relationships. ORDA will de-
velop interrelationships with other federal
agencies concerned with recombinant DNA
technology, including but not limited to the
following: National Academy of Sciences, Na-
tional Science Foundation, Energy Research
and Development Administration, Depart-
ment of Agriculture, Environmental Protec-
tion Agency, National Aeronautics and Space
Administration, and the Occupational Safety
and Health Administration.

Parenthetically, it should be mentioned
that representatives of the National Academy
of Sciences and National Science Foundation
are already attending meetings of the Re-
combinant Advisory Committee on a regular
basis, and the National Aeronautics and
Space Administration and the Energy Re-

38441

search and Development Administration have
sent a representative to several of the meet-
ings.

VII. Non-Federal and international rela-
tionships. ORDA will attempt to develop in-
terrelationships with private foundations,
professional societies, scientific journais and
industry, and to coordinate NIH policies with
international bodies concerned with recom-
binant DNA technology. The Executive Sec-
retary of the Recombinant Advisory Com-
mittee already has a working relationship
with the European Molecular Blology Orga-
nization.

VIII. Institutional Biohazards Committees.
ORDA will receive directly from each Iinsti-
tution involved in recombinant DNA activi-
ties the roster of its institutional biohazards
committee. The minimum information
should include the names, addresses, occu-
pations and qualifications of the chairman
and members of the committee. Institutions
will be notified by the appropirate instru-
ment (NIH Guide, NIH Manual, etc.) of the
necessity for filing this information with
ORDA.

As stipulated in the Guidelines, ORDA will
assist in the formation of area biohazards
committees composed of members of a given
institution and/or other organizations be-
yond its own staff. An area committee will
be necessary when expertise from outside a
given institution 1s necessary for the bio-
hazards committee to fulfill Its functions.

ORDA will review the composition of in-
stitutional biohazards committees for com-
pliance with the recommendations stated in
the Guidelines, and will maintain updated
lists of such complying committees. Serious
questions about the suitability of a commit-
tee will be brought to the attention of the
Executive Committee.

IX. Information on accidents, containment
and innovations. As stipulated by the Gulde-
lines, investigators are required to report to
ORDA and to their institutional biohazards
committees any serious or extended illness
of a worker, or any accidents of the type de-
scribed in the Guidelines. The Guidelines
also require that investigators report to
ORDA and their bichazards committee any
problems pertaining to operation and imple-
mentation of biological and physical con-
tainment safety practices and procedures, or
equipment or facility failure.

ORDA will receive from investigators in-
formation on purported EK2 and EK3 sys-
tems, and information bearing on the Guide-
lines, such as technical information relating
to hazards and new safety procedures or in-
novations.

ORDA will receive and file these reports
and, as appropriate bring them to the at-
tention of the Deputy Director for Science,
NIH, the Office of Research Safety, NCI, and
the Recombinant Advisory Committee or
subcommittees thereof. ORDA may, after re-
view, recommend appropirate action to the
Deputy Director for Science, NIH.

X. Recombinant DNA molecule program
advisory committee. ORDA will have the
management responsibilities for the Recom-
binant Advisory Committee, will serve as its
staff, and will provide the Executive Secre-
tary. Staff functions will include the gather-
ing, analysis and dissemination of informa-
tion, the presentation of issues, etc.

XI. Transition and implementation. Pro-
posals for initial gathering of information on
NIH activities in this area and implementa-
tion of the Guidelines are discussed in this
Appendix C.

The relationships, responsibilities and pro-
cedures proposed in this memorandum and
its appendices are wide-ranging and complex.
They are, however, an attempt to describe
how NIH might administer the Guidelines
governing this controversial technology re-
sponsibly and effectively. I look forward to

FEDERAL REGISTER, VOL. 43, NO. 176—THURSDAY, SEPTEMBER 9, 1976
38442

hearing your comments and those of your
staff on these proposals.

WILLIAM J. GARTLAND, Jr., Ph. D.
APPENDIX A TO APPENDIX C

PROCESSING, REVIEW, AND MANAGEMENT OF
EXTRAMURAL PROJECTS INVOLVING RECOM-
BINANT DNA TECHNOLOGY

I. General. The purpose of this appendix is
to discuss procedures for the processing, re-
view and management of NIH supported
projects which involve the use of recombi-
nant DNA technology. The term “application”,
as used here, refers to all contract proposals
and grant applications for research projects,
program projects, centers, training, fellow-
ships, research career development, etc., as
defined in NIH Manual Issuance 4101: “Ac-
tivity Codes, Organizational Codes and Defi-
nitions used in Extramural Programs.” The
procedures would apply to applications re-
viewed by DRG study sections and Institute
and Division initial review groups.

Il. Capture of information. One of the
primary functions of the Office of Recombi-
nant DNA Activities (ORDA) is to maintain
a central register of NIH supported projects
which involve recombinant DNA technology
so that the NIH will know where these proj-
ects are located and will have the capability
of rapidly communicating with project di-
rectors should the need arise.

In order to maintain an updated central
register of projects it will be necessary for
the NIH to modify application forms to in-
dicate on the face sheet whether or not re-
combinant DNA technology is involved in
the project. This could be modeled after the
statement currently in use regarding re-
search involving human subjects. The infor-
mation would be captured, as is the case for
human experimentation, and permit sorting
by different parameters such as awarding
component, geographic location of projects,
ete.
III. Receipt of applications. Through the
use of appropriate instrument (NIH
Guide, NIH Manual, etc.), the NIH will in-
form applicants of the necessity for assess-
ing the physical and biological containment
required for the proposed experiments as
stipulated in the NIH guidelines. This assess-
ment must be incorporated into the applica-
tion.

All applications requesting support for
projects. involving recombinant DNA tech-
nology will be required to have on file two
documents: A Memorandum of Understand-
ing and Agreement (MUA) and a Certifica-
tion Statement (Certification) from the in-
stitutional biohazards committee. The NIH
must and will require that a properly exe-
cuted MUA and Certification accompany all
applications proposing to use recombinant
DNA technology. This will eliminate the need
for tracking these documents after the appli-
cation is accepted for review. The originals of
these documents will be placed in the official
files of the NIH awarding components with
copies on file in ORDA.

IV. Review of applications. The executive
secretaries of initial review groups are re-
sponsible for identifying all applications in-
volving recombinant DNA technology, and
for placing on the first page of every sum-
mary statement the following special note:

RECOMBINANT DNA MOLECULES—POTENTIAL
BIOHAZARD

The executive secretaries are responsible
for ensuring that the initial review groups
make an independent assessment of the bio-
logical and physical containment levels re-
quired for the proposed experiments, and for
stating in the text of the summary state-
ment the initial review group’s determina-
tion as to whether the containment levels
proposed by the investigator meet the levels

NOTICES

stipulated in. the NIH guidelines. If the pro-
posed containment levels are inadequate, the
initial review group should discuss, in as
much detail as possible, the inadequacies of
the proposed containment and under what
circumstances, if at all, the application
should be eligible for funding. Initial review
groups should be encouraged to disapprove
applications if the proposed containment
levels are so inadequate as to be irresponsi-
ble. It will be the responsibility of the
awarding unit to inform applicants directly
as to the fact that this was the reason for
disapproval, .

Executive secretaries and members of rele-
vant initial review groups will receive a copy
of the Guidelines to permit them to make
these judgments. Problems relating to as-
sessment of biological and physical contain-
ment levels proposed by investigators versus
those required by the Guidelines will be re-
ferred to ORDA.

As in the case of human experimentation,
national advisory councils and boards and
final review bodies are expected to carefully
scrutinize proposals, involving recombinant
DNA technology, and make appropriate
recommendations.

V. Award of new and competing renewal
projects. Prior to the award of any project
involving recombinant DNA technology, the
NIH awarding component will forward to
ORDA one copy of each of the following
documents: The application, summary state-
ment, MUA, Certification, any comments of
the final review body, and a request for
clearance to award. In those cases in which
the initial or final review group, or staff of
an awarding BID finds that a project re-
quires a higher level of containment than
that originally proposed by the applicant or
the institution, a properly executed revised
MUA and Certification Statement will be
required prior to a request for approval to
award, ORDA will review the documents and
indicate concurrence or non-concurrence
with the request for clearance to award. In
the latter case, ORDA will prepare a memo-
randum outlining the reasons for disapprov-
al but emphasizing that the action is inde-
pendent of scientific merit or other reasons.
Such a memorandum should be forwarded to
the applicant through the awarding unit. In
those cases in which ORDA is not able to
reach a decision, or in which the awarding
component or applicant disputes the deci-
sion, the decision will be submitted to the
Executive Committee, and, if necessary, to
the Recombinant Advisory Committee, for
review. The final decision will rest with the
Deputy Director for Science, NIH.

BIDs will forward to ORDA a copy of all
award statements Involving these applica-
tions.

ORDA will retain in its files the documents
cited above. In the event that the volume of
filed documents becomes excessive, ORDA
will retain a copy of the face sheet of a
funded application rather than the entire
application. In those cases in which the
proposed project involving recombinant DNA
technology is but one component of a mul-
tiproject application, ORDA will retain in
its files the face sheet of the application and
the section(s) describing the project(s) in-
volving recombinant DNA technology.

It must be emphasized that the primary
responsibility for ensuring that applications
have been properly reviewed, and that re-
quired documents are properly executed lies
with NIH staff involved with the initial re-
view groups and program areas. The proce-
dures proposed above are intended to serve
as a final review prior to award. ORDA will
be available to NIH staff for advice and con-
sultation, but it can not be expected to make
decisions for review units and awarding
components.

VI. Award of non-competing renewals and
incrementally-funded contracts. Bach non~
competing renewal of a grant and subsequent
budget. period of an incrementally-funded
contract utilizing recombinant DNA tech-
nology must be accompanied by an updated
Certification Statement from the institu-
tional biohazards committee. Prior to any
award of this type the program official in
the awarding component has the responsi-
bility for reviewing the application for con-
formity with the Guidelines, for determin-
ing whether the proposed protocols do or
do not require a higher level of containment
thfin was required in the application as re-
viewed by the initial review group, and for
ensuring that the required documents are
properly executed. The program official will
then forward to ORDA one copy of the ap-
plication and the certification statement,
along with a request for clearance to award.
The latter will include a statement to the
effect that the program official has reviewed
the application for conformity with the
Guidelines, and that the proposed contain-
ment levels are adequate. Thereafter, the
procedures described in V will be followed,
and BIDs will forward to ORDA a copy of
all award statements involving these proj-
ects.

If the investigator proposes to significantly
alter an approved protocol at the time of the
non-competing renewal or subsequent budg-
et period of an incrementally-funded con-
tract, then the procedures described in VI
must be followed. It is the responsibility of
the program official to ensure that all the in-
formation and properly executed documents
required in VII are present in the application
prior to forwarding the request to ORDA.

VII. Changes in awarded projects. Since in
many cases the NIH supports projects for
project periods longer than one year, a num-
ber of situations will arise in funded projects.
One situation arises when an investigator
makes a decision to utilize recombinant
DNA technology after the project has been
reviewed and awarded. Another situation
arises when an investigator decides to close
DNA segments other than those originally
reviewed, and for which higher levels of con-
tainment may be required. In these cases,
and in all cases in which an investigator
wishes to significantly alter an approved pro-
tocol, the investigator must first apply to
the NIH awarding component for permission
before proceeding. This requirement should
be stated in the appropriate NIH instrument
(NIH Manual, NIH Guide, etc.) The investi-
gator will be required to submit to the award-
ing component a proposed protocol, an
assessment of the levels of physical and bio-
logical containment required by the Guide-
lines, an MUA, and a Certification Statement
from the institutional biohazards commit-
tee. The program official in the awarding
component has the responsibility for review-
ing the request in light of the Guidelines, for
ensuring that the required documents are
properly executed, and for forwarding to
ORDA a copy of all the documents along
with a recommendation. The latter should in-
clude the program official's independent
assessment of the levels of physical and
biological containment required by the NIH
Guidelines, and a recommendation as to how
to proceed. ORDA will review the request,
and, when appropriate, refer the request to
the initial review group, the Recombinant
Advisory Committee, or ad hoc consultants.

VIII. Requests for lowering of containment
levels. Under “‘characterized clones of DNA
recombinants derived from shotgun experi-
ments,” the Guidelines state:

* * * before containment conditions lower
than the ones used to clone the DNA can be
adopted, the investigator must obtain ap-
proval from the granting agency. Such ap-

FEDERAL REGISTER, VOL. 41, NO. 176——THURSDAY, SEPTEMBER 9, 1976
proval would be contingent upon data con-
cerning: (a) The absence of potentially
harmful genes (e.g., sequences contained in
indigenous tumor viruses or which code for
toxic substances), (b) the relation between
the recovered and desired segment (e.g., hy-
bridization and restriction endonuclease
fragmentation analysis where applicable),
and (c) maintenance of the biological prop-
erties of the vector.

This stipulation for NIH approval may be
one of the most difficult sections of the
Guidelines to implement. This is because of
the technical nature of the data to be eval-
uated, and because of the volume of requests
which can be anticipated. Therefore, the fol-
lowing proposed procedures are especially
viewed as a feasibility trial.

An investigator who wishes to use lower
jJevels of containment for characterized
clones derived from shotgun experiments
must state, in writing, the justification for
the request to the program official of the NIH
awarding component. Such justification will
provide data on (a), (b) and (c) as
stated above. The program official will
retain the original request in the award-
ing component’s file, and forward a capy
to ORDA which will submit the request
to the Recombinant Advisory Committee or
to a subcommittee therecf for evaluation, or,
if a precedent has been established, will
make a decision independently. The decision
will be forwarded to the program official
who may appeal. The final decision rests with
the Deputy Director for Science, NIH.

IX. Large-scale experiments. The Guide-
lines state that:

* * * at this time large-scale experiments
(e.g., more than 10 liters of culture) with re-
combinant DNAs known to make harmful
products are not to be carried out * * *,
However, specific experiments in this cate-
gory may be exempted from this rule if spe-
cial biological containment precautions and
equipment designed for large-scale opera-
tions are used, and provided that these ex-
periments are expressly approved by the Re-
combinant DNA Molecule Program Advisory
Committee.

An investigator who wishes to conduct such
experiments must submit a request, along
with a properly executed MUA and Certifica-
tion Statement from the institutional bio-
hazards committee, to the program official
of the NIH awarding component. The pro-
gram official will retain the original request
in the awarding component’s file, and for-
ward copies to ORDA, ORDA will bring the
request to the attention of the Recombinant
Advisory Committee or subcommittees
thereof, by mail, telephone, or presentation
at the next meeting or, if a precedent has
been established, will make a decision in-
@ependently.

APPENDIX B TO APPENDIX C
NIH INTRAMURAL RESEARCH

Because NIH intramural research projects
are reviewed in a very different fashion than
extramural projects, different procedures are
applicable than those proposed in Appen-
dix A,

At present, the Chief of the Laboratory
in which an investigator plans to utilize
recombinant DNA technology requests ap-
proval through the Scientific Director of the
relevant BID to the Deputy Director for
Science, NIH with copies to the Associate
Director for Environmental Health and
Safety, DRS. The request for approval is in
the form of a draft Memorandum of Under-
standing and Agreement (MUA) which de-
scribes the type of experiment, nature of
host-vector system, assessment of potential
risk, proposed safety measures, proposed
training of personnel, etc. The Deputy Direc-

NQTCES

tor for Science then requests the NIH Bio-
hazards Committee to review the research
plan and procedures proposed in the draft
MUA. The recommendations of the NIH Bio-
hazards Committee are forwarded to the
Deputy Director for Science, NIH. Recom-
mendations of the NIH Biohazards Commit-
tee must be included in a final MUA, and
the Associate Director for Environmental
Health and Safety, DRS must certify that
the safety measures included in the final
MUA are available. The research cannot pro-
ceed until the final MUA is fully approved.
The original copy of the MUA is sent to the
Associate Director for Environmental Health
and Safety, DRS with copies to the requesting
investigator, the Laboratory Chief, the Scien-
tific Director and the-Executive Secretary of
the NIH Biohazards Committee.

It is proposed here that a copy of the
final MUA be forwarded to ORDA for review.
If ORDA does not concure with the recom-
mendations of the NIH Biohazards Commit-
tee, it may request the Deputy Director for
Science, NIH to bring the matter to the
attention of the Executive Committee or the
Recombinant Advisory Committee for resolu-
tion.

ORDA will assist the NIH Biohazards Com-
mittee with problems relating to assessment
of biological and physical containment levels
proposed by investigators versus those re-
quired by the Guidelines, with requests for
the use of lower containment levels for
characterized clones derived from shotgun
experiments, and with requests for permission
to do large-scale experiments with recom-
binants known to make harmful products.
ORDA will also assist the NIH Biohazards
Committee in periodic review and revisions
of MUAs. If ORDA does not concur with the
decisions of the NIH Biohazards Committee,
it may request the Deputy Director for
Science, NIH to bring the matter to the
attention of the Executive Committee or the
Recombinant Advisory Committee.

APPENDIX C TO APPENDIX C
TRANSITION AND IMPLEMENTATION

The procedures proposed in Appendices A
and B should be implemented as soon as
possible. However, clearly there will be an
interim period after the Guidelines are issued
and before all the procedures are function-
ing. It is the purpose of this Appendix to
Propose how the Office of Recombinant DNA
Activities (ORDA) might initiate coordina-
tion and gathering of information during this
period.

I. Intramural research. ORDA will brief
the Scientific Directors of the BIDs who will
be expected to assure ORDA and the Deputy
Director for Science, NIH of present and
future compliance by intramural research
scientists with the Guidelines.

ORDA will request the Deputy Director
for Science, NIH to provide a copy of the
final MUA on all intramural projects, utiliz-
ing recomingant DNA technology, which are
already in progress. After review of the MUAs,
ORDA will report any concerns to the Deputy
Director for Science, NIH.

Il. Extramural programs. ORDA will brief
the Executive Committee for Extramural Af-
fairs on NIH policies and procedures.

BIDs will be required to report to ORDA
all presents or planned workshops, training
courses, conferences, etc., relating to recom-
binant DNA technology. BIDs must also re-
port ail present or planned RPFs and RF As
likely to result in projects utilizing recom-
binant DNA technology. After review of this
information, ORDA will report any concerns
to the Deputy Director for Science, NIH and/
or the Executive Committee.

With regard to active grants and contracts,
BIDs will be required to submit to ORDA a
copy of the application, summary state-

ment and award statement for each cur-
rently funded project involving recombinant
DNA technology. NIH awarding components
will be responsible for ensuring that this
reporting is as complete as possible.

BIDs will send a letter to investigators
identified in the paragraph above to deter-
mine whether active research projects are in
compliance with the Guidelines. Responses
to this query will be retained in BID of-
ficial files, and a copy will be forwarded to
ORDA for review. If ORDA is satisfied that a
project is in compliance with the Guide-
lines, no further action is required. If the
investigator reports that the project is not
in full compliance with the guidelines, those
aspects of the project which are not in com-
Pliance will have to be terminated. However,
investigators will have the opporéunity to
petition the Recombinant Advisory Commit-
tee to permit continued use cf characterized
clones already in existence and constructed
under Asilomar guidelines. Presumably, the
use of these clones will be permitted to con-.
tinue until the Recombinant Advisory Com-
mittee or a subcommittee thereof, has ren-
dered its opinion.

The above procedures assume that all in-
vestigators are already at least in compli-
ance with Asilomar guidelines. If projects
are identified which appear not to be in com-
pliance with Asilomar guidelines, they will be
brought to the immediate attention of the
Deputy Director for Science, NTH and the Re-
combinant Advisory Committee.

APPENDIX D
RECOMBINANT DNA RESEARCH

Guidelines
as published in the
FEDERAL REGISTER, Part IT,
July 7, 1976

On Wednesday, June 23, 1976, the Director,
National Institutes of Health, with the con-
currence of the Secretary of Health, Educa-
tion, and Welfare, and the Assistant Secre-
tary for Health, issued guidelines that will
govern the conduct of NIH-supported re-
search on recombinant DNA molecules. The
NIH is also undertaking an environmental
impact assessment of these guidelines for
recombinant DNA research in accordance
with the National Environmental Policy Act
of 1969,

The NIH Guidelines establish carefully
controlled conditions for the conduct of ex-
periments involving the production of such
molecules and their insertion into organisms
such as bacteria. These Guidelines replace
the recommendations contained in the 1975
“Summary Statement of the Asilomar Con-
ference on Recombinant DNA Molecules.”
The latter would have permitted research
under less strict conditions than the NIH
Guidelines.

The chronology leading to the present
Guidelines is described in detail in the NIH
Director’s decision document that follows.
In summary, scientists engaged in this re-
search called, in 1974, for a moratorium on
certain Kinds of experiments until an inter-
national meeting could be convened to con-
sider the potential hazards of recombinant
DNA melecules. They also called upon the
NIH to establish a committee to provide ad-
vice on recombinant DNA technology.

The international meeting was held at the
Asilomar Conference Center, Pacific Grove,
California, in February 1975. The consensus
of this meeting was that certain experiments
should not be done at the present time, but
that most of the work on construction of re-
combinant DNA molecules should proceed
with appropriate physical and biological bar-
riers. The Asilomar Conference report also

FEDERAL REGISTER, VOL. 41, NO. 176——THURSDAY, SEPTEMBER 9, 1976
38444

made interim assignments of the potential
risks associated with different types of experi-
ments. The NIH then assumed responsibility
for translating the broadly based Asilomar
recommendations into detailed guidelines for
research.

The decision by the NIH Director on these

Guidelines was reached after extensive scien- -

tific and public airing of the issues during
the sixteen months which have elapsed since
the Asilomar Conference. The issues were
discussed at public meetings of the Re-
combinant DNA Molecule Program Advisory
Committee (Recombinant Advisory Commit-
tee) and the Advisory Committee to the NIH
Director. The Recombinant Advisory Com-
mittee extensively debated three different
versions of the Guidelines during this period.

The Ad¥isory Committee to the NIH Direc-
tor, augmented with consultants representing
law, ethics, consumer affairs and ‘the environ-
ment, was asked to advise as to whether
the proposed Guidelines balanced re-
sponsibility to protect the public with the
potential benefits through the pursuit of
new knowledge. The many different points
of view expressed at this meeting were taken
into consideration in the decision.

The NIH recognizes a special obligation to
disseminate information on these guidelines
as widely as possible. Accordingly, the Guide-
jines will be sent to all of the approximately
95,000 NIH grantees and contractors. Major
professional societies which represent scien-
tists working in this area will also be asked
to endorse the Guidelines. The Guidelines
will be sent to medical and scientific journals
and editors of these journals will be asked
to request that investigators include a de-
scription of the physical and biological con-
tainment procedures used in any recombin-
ant research they report on. International
health and scientific organizations will also
receive copies of the guidelines for their
review.

Filing of an environmental impact state-
ment will provide opportunity for the scien-
tific community, Federal, State and local
agencies and the general public to address the
potential benefits and hazards of this re-
search area. In order for there to be fur-
ther opportunity for public comment and
consideration, these guidelines are being
offered for general comment in the FEDERAL
RecisTer. It must be clearly understood by
the reader that the material that follows is
not proposed rulemaking in the technical
sense, but is a document on which early
public comment and participation is invited.

Please address any comments on these
draft policies and procedures to the Director,
National Institutes of Health, 9000 Rockville
Pike, Bethesda, Maryland 20014. All comments
should be received by November 1, 1976.

Additional copies of this notice are avail-
able from the Acting Director, Office of Re-
combinant DNA Activities, National Institute
of General Medical Sciences, National Insti-
tutes of Health, 9000 Rockville Pike,
Bethesda, Maryland 20014.

Donatp S, FREDRICKSON, M.D.,

Director,
National Institutes of Healtn.

JUNE 25, 1976.

DECISION OF THE DrrEctToR, NATIONAL
INSTITUTES OF HEALTH TO RELEASE
GUIDELINES FOR RESEARCH ON RECOM-
BINANT DNA MOLECULES

JUNE 23, 1976.
CONTENTS

Introduction
I. General Policy Considerations
A. Science Policy
B. Implementation Within the NIH

NOTICES

C. Implementation Beyond the Purview

of NIH

D. Environmental Policy

Methods of Containment (See Guide-
lines II)

Prohibited Experiments
lines IIT, A)

Permissible Experiments: E. Coli K-12
Host-Vector Systems (See Guidelines
Tif, B, 1)

. Classification of Experiments Using the
E, Coli K-12 Containment Systems
(See Guidelines III, B, 2)

Classification of Experiments Using Con-
tainment Systems Other than E. Coli
K-12 (See Guidelines III, B, 4)

Roles and Responsibilities (See Guide-
lines IV)

INTRODUCTION

on

Ift. (See Guide-

Iv.

VI.

Vil.

Today, with the concurrence of the Secre-
tary of Health, Education, and Welfare and
the Assistant Secretary for Health, I am re-
leasing guidelines that will govern the con-
duct of NIH-supported research on recom-
binant DNA molecules (molecules resulting
from the recombination in cell-free systems
of segments of deoxyribonucleic acid, the ma-
terial that determines the hereditary charac-
teristics of all known cells). These guidelines
establish carefully controlled conditions for
the conduct of experiments involving the in-
sertion of such recombinant genes into orga-
nisms, such as bacteria. The chronology lead-
ing to the present guidelines and the deci-
sion to release them are outlined in this in-
troduction.

In addition to developing these guidelines,
NIH has undertaken an environmental im-
pact assessment of these guidelines for re-
combinant DNA research in accordance with
the National Environmental Policy Act of
1969 (NEPA). The guidelines are being re-
leased prior to completion of this assessment,
They will replace the current Asilomar guide -
lines, discussed below, which in many in-
stances allow research to proceed under less
strict conditions. Because the NIH guidelines
will afford a greater degree of scrutiny and
protection, they are being released today, and
will be effective while the environmental im-
pact assessment is under way.

Recombinant DNA research brings to the
fore certain problems in assessing the poten-
tial impact of basic science on society as @
whole, including the manner of providing
public participation in those assessments.
The field of research involved is a rapidly
moving one, at the leading edge of biological
science. The experiments are extremely tech-
nical and complex. Molecular biologists active
in this research have means of keeping in-
formed, but even they may fail to keep
abreast of the newest developments. It is not
surprising that scientists in other fields and
the general public have difficulty in under-
standing advances in recombinant DNA re-
search. Yet public awareness and understand-
ing of this line of investigation is vital.

It was the scientists engaged in recombi-
nant DNA research who called for a morato-
rium on certain kinds of experiments in
order to assess the risks and devise appropri-
ate guidelines. The capability to perform
DNA recombinations, and the potential haz-
ards, had become apparent at the Gordon
Research Conference on Nucleic Acids in July
1973. Those in attendance voted to send an
open letter to Dr. Philip Handler, President
of the National Academy of Sciences, and to
Dr. John R. Hogness, President of the Insti-
tute of Medicine, NAS. The letter, appearing
in “Science 181,” 1114, (1973), suggested
“that the Academies [sic] establish a study
committee to consider this problem and to
recommend specific actions. or guidelines,

should that seem appropriate.”

In response, NAS formed a committee, and
its members published another letter in
“Science 185,”" 303, (1974). Entitled “Poten-
tial Biohazards of Recombinant DNA Mole-
cules,” the letter proposed:

First, and most important, that until the
potential hazards of such recombinant DNA
molecules have been better evaluated or
until adequate methods are developed for
preventing their spread, scientists through-
out the world join with the members of this
committee in voluntarily deferring * * *
[certain] experiments * * *.

Second, plans to link fragments of animal
DNAs to bacterial plasmid DNA or bacterio-
phage DNA should be carefully weighed * * *.

Third, the Director of the National Insti-
tutes of Health is requested to give immedi-
ate consideration to establishing an advisory
committee charged with (i) overseeing an
experimental program to evaluate the po-
tential biological and ecological hazards of
the above types of recombinant DNA mole-
cules; (ii) developing procedures which will
minimize the spread of such molecules
within human and other populations; and
(ii) devising guidelines to be followed by in-
vestigators working with potentially hazard-
ous recombinant DNA molecules.

Fourth, an international meeting of in-
volved scientists from all over the world
should be convened early in the coming year
to review scientific progress in this area and
to further discuss appropriate ways to deal
with the potential biohazards of recombi-
nant DNA molecules.

On October 7, 1974, the NIH Recombinant
DNA Molecule Program Advisory Committee
(hereafter “Recombinant Advisory Commit-
tee”) was established to advise the Secretary,
HEW, the Assistant Secretary for Health, and
the Director, NIH, “concerning a program
for developing procedures which will mini-
mize the spread of such molecules within
human and other populations, and for de-
vising guidelines to be followed by investi-
gators working with potentially hazardous
recombinants.”

The international meeting proposed in the
“Science” article (185, 303, 1974) was held in
February 1975 at the Asilomar Conference
Center, Pacific Grove, California. It was
sponsored by the National Academy of Sci-
ences and supported by the National Insti-
tutes of Health and the National Science
Foundation. One hundred and fifty people
attended, Including 52 foreign scientists from
15 countries, 16 representatives of the press,
and 4 attorneys.

The conference reviewed progress in re-
search on recombinant DNA molecules and
discussed ways to deal with the potential
biohazards of the work. Participants felt that
experiments on construction of recombinant
DNA molecules should proceed, provided that
appropriate biological and physical contain-
ment is utilized. The conference made rec-
ommendations for matching levels of con-
tainment with levels of possible hazard for
various types of experiments. Certain ex-
periments were judged to pose such serious
potential dangers that the conference recom-
mended against their being conducted at
the present time.

A report on the conference was submitted
to the Assembly of Life Sciences, National
Research Council, NAS, and approved by its
Executive Committee on May 20, 1975. A
summary statement of the report was pub-
lished in “Science 188,” 991 (1975), “Nature
925,” 442, (1975), and the “Proceedings of
the National Academy of Sciences 72,” 1981,
(1975). The report noted that “in many
countries steps are already being taken by
national bodies to formulate codes of prac-
tice for the conduct of experiments with

Known or potential biohazard. Until these are

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
established, we urge individual scientists to
use the proposals in this document as a
guide.”

The NIH Recombinant Advisory Committee
held its first meeting in San Francisco im-
mediately after the Asilomar conference. It
proposed that NIH use the recommendations
of the Asilomar conference as guidelines for
research until the committee had an oppor-
tunity to elaborate more specific guidelines,
and that NIH establish a newsletter for in-
formal distribution of information. NIH ac-
cepted these recommendations.

At the second meeting, held on May 12-13,
1975, in Bethesda, Maryland, the committee
received a report on biohazard-containment
facilities in the United States and reviewed a
proposed NIH contract program for the con-
struction and testing of microorganisms that
would have very limited ability to survive in
natural environments and would thereby
limit the potential hazards. A subcommittee
chaired by Dr. David Hogness was appointed
to draft guidelines for research involving re-
combinant DNA molecules, to be discussed at
the next meeting.

The NIH committee, beginning with the
@raft guidelines prepared by the Hogness sub-
committee, prepared proposed guidelines for
research with recombinant DNA molecules at
its third meeting, held on July 18-19, 1975,
in Woods Hole, Massachusetts.

Following this meeting, many letters were
received which were critical of the guidelines.
The majority of critics felt that they were
too lax, others that they were too strict. All
letters were reviewed by the committee, and
& new subcommittee, chaired by Dr. Eliza-
beth Kutter, was appointed to revise the
guidelines.

A fourth committee meeting was held on
December 4-5, 1975, in La Jolla, California.
For this meeting a ‘‘variorum edition” had
been prepared, comparing line-for-line the
Hogness, Woods Hole, and Kutter guidelines.
The committee reviewed these, voting item-
by-item for their preference among the three
variations and, in many cases, adding new
material. The result was the “Proposed
Guidelines for Research fnvolving Recom-
binant DNA Molecules,” which were referred
to the Director, NTH, for a final decision in
December 1975.

As Director of the National Institutes of
Health, I called a special meeting of the Ad-
visory Committee to the Director to review
these proposed guidelines. The meeting was
held at NIH, Bethesda, on February 9-10,
1976. The Advisory Committee is charged to
advise the Director, NIH, on matters relating
to the broad setting—scientific, technological,
and socioeconomic—in which the continuing
development of the biomedical sciences. edu-
cation for the health professions, and bio-
medical communications must take place,
and to advise on their implications for NIH
policy, program development, resource allo-
cation, and administration. The members of
the committee are knowledgeable in the fields
of basic and clinical biomedical sciences, the
social sciences, physical sciences, research,
education, and communications. In addition
to current members of the committee, I in-
vited a number of former committee mem-
bers as well as other scientific and public rep-
resentatives to participate in the special
February session.

The purpose of the meeting was to seek
the committee’s advice on the guidelines
proposed by the Recombinant Advisory Com-

. mittee. The Advisory Committee to the Di-
rector was asked to determine whether, in
their Judgment, the guidelines balanced
scientific responsibility to the public with
scientific freedom to pursue new knowledge.

FEDERAL REGISTER, VOL. 41, NO. 17

NOTICES

Public responsibility weighs heavily in this
genetic research area. The scientific commu-
nity must have the public’s confidence that
the goals of this profoundly important re-
search accord respect to important ethical,
legal, and social values of our society. A key
element in achieving and maintaining this
public trust is for the scientific community to
ensure an openness and candor in its pro-
ceedings. The meetings of the Director's Advi-
sory Commitiee, the Asilomar group, and the
Recombinant Advisory Committee have re-
flected the intent of science to be an open
community in considering the conduct of
recombinant DNA experiments. At the Direc-
tor's Advisory Committce meeting, there was
ample opportunity for comment and an air-
ing of the issues, not only by the committee
members but by public witnesses as well. All
major points of view were broadly repre-
sented.

I have been reviewing the guidelines in
light of the comments and suggestions made
by participants at that meeting, as well as
the written comments received afterward. As
part of that review I asked the Recombinant
Advisory Committee to consider at their
mecting of April 1-2, 1976, a number of
selected issues raised by the commentators.
I have taken those issues and the response
of the Recombinant Advisory Committee into
account in arriving at my decision on the
guidelines. An analysis of the issues and the
basis for my decision follow:

I. GENERAL POLICY CONSIDERATIONS

A word of explanation might be interjected
at this point as to the nature of the studies
in question. Within the past decade, enzymes
capable of breaking DNA strands at specific
sites and of coupling the broken fragments
in new combinations were discovered, thus
making possible the insertion of foreign genes
into viruses or certain cell particles (plas-
mids). These, in turn, can be used as vec-
tors to introduce the foreign genes into bac-
teria or into cells of plants or animals in test
tubes. Thus transplanted, the genes may im-
part their hereditary properties to new hosts.
These cells can be isolated and cloned—that
is, bred into a geneticaly homogeneous cul-
ture, In general, there are two potential uses
for the clones so produced: as a tool for
studying the transferred genes, and as a new
useful agent, say for the production of a
scarce hormone.

Recombinant DNA research offers great
promise, particularly for improving the un-
derstanding and possibly the treatment of
various diseases. There is also a potential
risk—that microorganisms with transplanted
genes may prove hazardous to man or other
forms of life. Thus special provisions are
necessary for their containment.

All commentators acknowledged the ex-
emplary responsibility of the scientific com-
munity in dealing publicly with the poten-
tial risks in DNA recombinant research and
in calling for a self-imposed moratorium on
certain experiments in order to assess po-
tential hazards and devise appropriate guide~
lines. Most commentators agreed that the
process leading to the formulation of the
proposed guidelines was a most responsible
and responsive che. Suggestions by the com-
mentators on broad policy considerations are
presented below. They relate to the science
policy aspects of the guidelines, the imple-
mentation of the guidelines for NIH erantees
and contractors, and the scope and impact
of the guidelines nationally and interna-
tionally.

A. SCIENCE POLICY CONSIDERATIONS

Commentators were divided on how best to
steer a& course between stifling research
through excessive regulation and alloy ing

cs
“4
a
e
ma
an
uu
x

ofthis

it to continue with sufficient controls. Sev-
eral emphasized that the public must have
assurance that the controls afford adequate
protection against potential hazards. In the
views of these commentators, the burden is
on the scientific community te show that the
danger is minimal and that the benefits are
substantial and far outweigh the risks.

Opinion differed on whether the proposed
guidelines were an appropriate response to
the potential benefits and hazards. Several
found the guidelines to so exagzgegrate safety
procedures that inquiry would be unneces-
sarily retarded, while others found the guide-
lines weighted toward promoting research.
The issue was how to strike a reasonable bal-
ance—in fact, a proper policy ‘‘bias”"—be-
tween concerns to “go slow” and those to
progress rapidly.

There was strong disagreement about the
nature and level of the possible hazards of
recombinant DNA research. Several com-
mentators believed that the hazards posed
were unique. In their view, the occurrence of
an accident or the escape of a vector could
initiate an irreversible process, with a po-
tential for creating problems many times
greater than those arising from the multi-
tude of genetic recombinations that occur
spontaneously in nature. These commenta-
tors stress the moral obligation on the part
of the scientific community to do no harm.

Other commentators, however, found the
guidelines to be adequate to the hazards
posed. In their view, the guidelines struck
an appropriate balance so that research could
proceed cautiously. Still other commenta-
tors found the guidelines too onerous and
restrictive in light of the potential benefits
of this research for medicine, agriculture, and
industry. Some felt that the guidelines are
perhaps more stringent than necessary given
the available evidence on the likelihood of
hazards, but supported them as a compromise
that would best serve the scientific com-
munity and the public at large. Many com-
mentators urged that the guidelines be
adopted as soon as possible to afford more
specific direction to this research area.

I understand and appreciate the concerns
of those who urge that this research proceed
because of the benefits and of those who urge
caution because of potential hazards. The
guidelines issued today allow the research
to go forward in a manner responsive and
appropriate to hazards that may be realized
in the future.

The object of these guidelines is to ensure
that experimental DNA recombination will
have no ill effects on those engaged in the
work, on the general public, or on the en-
vironment. The essence of their construction
is subdivision of potential experiments by
class, decision as to which experiments should
be permitted at present, and assignment to
these of certain procedures for containment
of recombinant organisms.

Containment is defined as physical and
biological. Physical containment involves the
isolation of the research by procedures which
have evolved over many years of experience
in laboratories studyi infectious micro-
organisms. Pl containment—the first physi-
cal containment level—is that used in most
routine bacteriology leboratories. P2 and P3
afford increasing isolation of the research
from the environment. P4 represents the
most ex:reme measures used for containing
virulent pathogens, and permits no escape
of contaminated air, wastes, or untreated
materials. “Biological’’ containment is the
use of vectors or hosts that are crippled by
mutation so that the recombinant DNA is
incapable of surviving under natural con-
ditions.

The experiments now permitted under the
guidelines involve no known additional haz-

  
  

ie
1S

 

  
38416

ard to the workers or the environment beyond
the relatively low risk known to be associated
with the source materials. The additional
hazards are speculative and therefore not
quantifiable: In a real sense they are consid-
erably less certain than are the benefits now
clearly derivable from the projected research.

For example, the ability to produce,
through “molecular cloning,” relatively large
amounts of pure DNA from the chromosomes
of any living organism will have a profound
effect in many areas of biology. No other pro-
gedure, not even chemicai synthesis, can pro~
vide pure material corresponding to particu-
lar genes. DNA “probes,” prepared from the
clones will yield precise evidence on the
presence or absence, the organization, and
the expression of genes in health and disease.

Potential medical advances were outlined
by scientists active in this research area who
were present at the meeting of the Director’s
Advisory Committee. Of enormous impor-
tance, for example, is the opportunity to ex-
plore the malfunctioning of cells in compli-
cated diseases. Our ability to understand a
variety of hereditary defects may be signif-
icantly enhanced, with amelioration of their
expression a real possibility. There is the
potential to elucidate mechanisms in certain
cancers, particularly those that might be
caused by viruses.

Instead of mere propagation of foreign DNA,
the expression of the genes of one organism
by the cell machinery of another may alter
the new host and open opportunities for
manipulating the biological properties of
cells. In certain prokaryotes (organisms with
& poorly developed nucleus, like bacteria),
this exchange of genetic information occurs
in nature. Such exchange explains, for in-
stance, an important mechanism for the
changing and spreading of resistance to anti-
biotics in bacteria. Beneficial effects of this
mechanism might be the production of med-
ically important compounds for the treat-
ment ‘and control of disease. Examples fre-
quently cited are the production of insulin,
growth hormone, specific antibodies, and
clotting factors absent in victims of hemo-
philia.

Aside from the potential medical benefits, a
whole host of other applications in science
and technology have been envisioned. Ex-
amples are the large-scale production of en-
zymes for industrial use and the development
of bacteria that could ingest and destroy ofl
spills in the sea. Potential benefits in agri-
culture include the enhancement of nitrogen
fixation in certain. plants, permitting in-
creased food production.

While the projected research offers the pos-
sibility of many benefits, it must proceed
only with assurance that potential hazards
can be controlled or prevented. Some com-
mentators are concerned that nature may
maintain a barrier to the exchange of DNA
between prokaryotes and eukaryotes (higher
organisms, with a well-formed nucleus)-—a
barrier that can now be crossed by experi-
mentalists. They further argue that expres-
sion of the foreign DNA may alter the host
in unpredictable and undesirable ways. Con-
ceivable harm could result if the altered
host has a competitive advantage that would
foster its survival in some niche within the
ecosystem. Other commentators believe that
the endless experiments in recombination
of DNA which nature has conducted since
the beginning of life on the earth, and which
have accounted in part for the evolution of
species, have most likely involved exchange of
DNA between widely disparate species. They
argue that prokaryotes such as bacteria in
the intestines of man do exchange DNA with
this eukaryotic host and that the failure of
the altered prokaryotes to be detected at-
tests to a sharply limited capacity of such
recombinants to survive. Thus nature, this

NOTICES

argument runs, has already tested the proba-
bilities of harmful recombination and any
survivors of such are already in the ecosys-
tem. The fact is that we do not know which
of the above-stated propositions is correct.

The international scientific community, as
exemplified by the Asilomar conference and
the deliberations attendant upon prepara-
tion of the present guidelines, has indica-
ted a desire to proceed with research in a
conservative Manner. And most of the con-
siderable public commentary on the subject,
while urging caution, has also favored pro-
ceeding. Three European groups have inde-
pendently arrived at the opinion that re-
combinant DNA research should proceed
with caution, These are the Working Party
on Experimental Manipulation of the Ge-
netic Composition of Micro-Organisms, whose
“Ashby Report” was presented to Parliament
in the United Kingdom by the Secretary of
State for Education and Science in January
1975; the Advisory Committee on Medical
Research of the World Health Organization,
which issued a press release in July 1975;
and the European Molecular Biology Organi-
zation Standing Committee on Recombinant
DNA, meeting in February 1976.

There is no means for a flat proscription
of such research throughout the world com-
munity of science. There is also no need to
attempt it. It is likely that the evaluation en-
gendered in the preparation and application
of these guidelines will lead to beneficial re-
view of some of the containment practices
in other work that is not technically defined
as recombinant DNA research.

Recombinant DNA research with which
these guidelines are concerned involves mi-
croorganisms such as bacteria or viruses or
cells of higher organisms growing in tissue
culture. It is extremely important for the
public to be aware that his résearch is not
directed to altering of genes in humans al-
though some of the techniques developed in
this research may have relevance if this is
attempted in the future.

NIH recognizes its responsibility to con-
duct and support research designed to deter-
mine the extent to which certain potentially
harmful effects from recombinant DNA mole-
cules may occur. Among these are experi-
ments, to be conducted under maximum
containment, that explore the capability of
foreign genes to alter the character of host
or vector, rendering it harmful, as through
the production of toxic products.

Given the general desire that no rare and
unexpected event arising from this research
shall cause irreversible damage, it is obvious
that merely to establish conservative rules of
conduct for one group of scientists is not
enough. The precautions must be uniformly
and unanimously observed. Second, there
must be full and timely exchange of experi-
ences so that guidelines can be altered on the
basis of new knowledge. The guidelines must
also be implemented in a manner that pro-
tects all concerned—the scientific workers
most likely to encounter unexpected hazards
and all forms of life within our biosphere.
The responsibility of the scientists involved
is an inescapable and extreme as is their op-
portunity to beneficially enrich our under-
standing.

B. IMPLEMENTATION CONSIDERATIONS WITHIN
THE NIK

All the commentators had suggestions con-
cerning the structure and function of deci-
sion making as it relates to the principal in-
vestigator, the local bichazards committee,
the peer review group, and the NIH Recom-
binant Advisory Committee. These com-~-
ments and my response on the section of the
guidelines relating to roles and responsibili-
ties of investigators, their institutions, and
the National Institutes of Health are pre-

sented below.

Of considerable concern to all commenta-
tors was the process by which NIH would
proceed to implement the guidelines. The
Scientific community generally urged that
there be no Federal regulations, while some
of the public commentators recommended
the regulatory process.

Many who opposed changing the proposed
guidelines into Federal regulations expressed
concern for flexibility and administrative
efficiency, which could best be achieved, in
their view, through voluntary compliance.
Other commentators, however, believed it im-
perative to proceed toward regulation. In
their view, the guidelines could be imple-
mented for purposes of NIH funding and
would govern the conduct of experiments
until regulations were in effect. Another com-
mentator who thought regulation would be
harmful rather than helpful suggested that
if there were to be regulations, they should
be along lines similar to those that govern
the sale, distribution, use, and disposal of
radioisotopes.

The question of how best to proceed now
that the guidelines have been released de-
serves careful attention. I share the concern
of those who feel that the guidelines must
remain flexible. It is especially important
that there be opportunity to change them
quickly, based on new information relating
to scientific evidence, potential risks, or
safety aspects of the research program.

The suggestions for regulation need fur-
ther attention at this time. The process for
regulation not only involves the Director of
NIH, but also the Assistant Secretary for
Health and the Secretary of Health, Educa-
tion, and Welfare. These guidelines are being
promulgated now in order to afford additional
protection to all concerned. Consideration of
their conversion to regulations can proceed
with continuing review of their content and
present and future implications. Meanwhile,
the NIH shall continue to provide the oppor-
tunity for public comment and participation
at least equivalent to that provided if steps
towards regulations were to proceed immedi-
ately. The guidelines will be published in the
FepreraL REGISTER forthwith to allow for fur-
ther public comment.

 

Cc. IMPLEMENTATION CONSIDERATIONS BEYOND
THE PURVIEW OF NIH

Special concern has been expressed by
many commentators regarding the applica-
tion of the guidelines to research outside
NIH by investigators other than its grantees
or contractors. It has been urged that the
guidelines be made applicable to recombinant
DNA research conducted or supported by
other agencies in HEW and by NSF, ERDA,
DoD, and other governmental departments.
Most commentators believe that these or
“similar guidelines should also govern research
in the private sector, including industry.
voluntary organizations, and foundations.
Many feel that experiments conducted in col-
leges, universities, and even in high schools
require some form of monitoring. And
finally, all agree that in view of the potential
hazards of recombinant DNA research to the
biosphere, some form of international under-
standing on guidelines for the research is
essential.

The committee, in the proposed guidelines.
has suggested as one means of control that a
description of the physical and biological
containment procedures practiced in a re-
search project be included in the publication
of research results. In the scientific com-
munity this can be a powerful force for con-
formity, and we will undertake to present the
recommendation to all appropriate journals.
We are also prepared to take steps to dis-
seminate the guidelines widely, and to ar-
range for a continual flow of information
outward concerning the activities of the Re-

FEDERAL REGISTER, VOL. 41, NO. 176——-THURSDAY, SEPTEMBER 9, 1976
combinant Advisory Committee and the Ad-
visory Committee to the Director, NIH, in
the evolution of the guidelines and their
implementation.

In response to these suggestions, I have
already held a meeting with relevant HEW
agencies and with representatives from other
departments of the Federal Government. The
purpose of the meeting was to exchange in-
formation on recombinant DNA research and
to discuss the NIH guidelines. It served as
an important beginning to address a com-
mon concern of these public institutions. A
number of the representatives indicated that
various departments might very well adopt
the guideline for research conducted both
in-house and supported outside, Following
up, I have begun preliminary discussions
with the Assistant Secretary for Health and
the Secretary of HEW, to determine possible
methods to ensure adoption of the guidelines
by all Federal agencies. Encouraged by these
efforts, we held a meeting on June 2 with
representatives of industry to provide them
with full information about the guidelines
and to help determine the present and future
interests of industrial laboratories in this
type of research. The meeting provided one
of the first opportunities for industry rep-
resentatives to convene for a discussion of
this research area, and an industry commit-
tee under the auspices of the Pharmaceutical
Manufacturers Association will be formed
to review the guidelines for potential ap-
plication to the drug industry. Further meet-
ings will be scheduled with other groups that
have an active interest in recombinant DNA
research,

It is my hope that the guidelines will be
voluntarily adopted and honored by all who
support or conduct such research through-
out the United States, and that at least very
similar guidelines will obtain throughout the
rest of the world. NIH places the highest
priority on efforts to inform and to work
with international organizations, such as the
World Health Organization and the Inter-
national Council of Scientific Unions, with a
review to achieving a consensus on safety
standards in this most important research
area.

There has been considerable international
cooperation and activity in the past, and I
expect it to continue in the future. The
aforementioned Ashby Report, presented to
Parliament in January 1975, describes the
advances in knowledge and possible bene-
fits to society of the experiments involving
recombinant DNA molecules, and attempts
to assess the hazards in these techniques.
The Asilomar meeting also had a number of.
international representatives, as mentioned
previously. The European Molecular Biology
Organization (EMBO) has been involved in
considering guidelines for recombinant DNA
research. They have closely followed the ac-
tivities of NIH, and will thus be encouraged,
I believe, to monitor their research with aug-
mented cooperation and coordination. For
example, EMBO recently announced plans
for a voluntary registry of recombinant DNA
research in Europe. Following this EMBO
initiative, NIH shall similarly maintain a
voluntary registry of investigators and insti-
tutions engaged in such research in the
United States. Plans for establishing this reg-
istry are under way.

D. ENVIRONMENTAL POLICY

A number of commentators urged NIH to
consider preparing an environmental im-
pact statement on recombinant DNA re-
search activity. They evoked the possibility
that organisms containing recombinant DNA
molecules might escape and affect the en-
vironment in potentially. harmful ways.

Iam in full agreement that the potentially
harmful effects of this research on the en-
vironment shauld be assessed. As discussed

CONSIDERATIONS

NOTICES

throughout this paper, the guidclines are
premised on physical and biological contain-
ment to prevent the release or propagation
of DNA recombinants outside the laboratory.
Deliberate release of organisms into the en-
vironment is prohibited. In my view, the
stipulated physical and biological contain-
ment ensures that this research will proceed
with a high degree of safety and precaution.
But I recognize the legitimate concern of
those urging that an environmental impact
assessment be done. In view of this concern
and ensuing public debate, I have reviewed
the appropriateness of such an assessment
and have directed that one be undertaken.

The purpose of this assessment will be to
review the environmental effects, if any, of
research that may be conducted under the
guidelines. The assessment will provide fur-
ther opportunity for all concerned to address
the potential benefits and hazards of this
most important research activity. I expect a
draft of the environmental impact statement
should be completed by September 1 for
comment by the scientific community, Fed-
eral and State agencies, and the general
public.

It should be noted that the development of
the guidelines was in large part tantamount
to conducting an environmental impact as-
sessment. For example, the objectives of re-
combinant DNA research, and alternate ap-
proaches to reach those objectives, have been
considered. The potential hazards and risks
have been analyzed. Alternative approaches
have been thoroughly considered, to maxi-
mize safety and minimize potential risk. And
an elaborate review structure has been cre-
ated to achieve these safety objectives. From
a@ public policy viewpoint, however, the envi-
ronmental impact assessment will be yet
another review that will provide further op-
portunity for the public to participate and
comment on the conduct of this research.

Il. METHODS OF CONTAINMENT

Comments on the containment provisions
of the proposed guidelines were directed to
the definitions of both physical and biological
containment and to the safety and effective-
mess of the prescribed levels. Several com-
mentators found the concept of physical con-
tainment imprecise and too subject to the
possibility for human error. Others ques-
tioned the concept of biological containment
in terms of its safety and purported effective-
ness in averting potential hazards. The com-
mentators were divided on which method of
containment would provide the most effective
and safe system to avoid hazards. Several sug-
gested that each of the physical containment
levels be more fully explained.

W. Emmett Barkley, Ph.D., Director of the
Office of Research Safety, National Cancer In-
stitute, was asked to review the section on
physical containment in light of these com-
ments. Dr. Barkley convened a special com-
mittee of safety and health experts, who met
to consider not only this section of the
guidelines but also the section of the roles
and responsibilities of researchers and their
Institutions. The committee thoroughly re-
viewed the section on physical containment
and recommended a number of changes. The
Recombinant Advisory Committee, meeting
on April 1-2, 1976, reviewed the recommenda-
tions of the Barkley group. These are incor-
porated, with editorial revisions, in the final
version of the guidelines.

The present section on physical contain-
ment is directly responsive to those com-
mentators who asked for greater detail and
explanation. Although different in detail, the
four levels of containment approximate those
given by the Center for Disease Control for
human etiologic agents and by the National
Cancer Institute for oncogenic viruses. For
each of the proposed levels, optional items
have been excluded, and only those items

38147

deemed absolutely necessary for safety are
presented. Necessary facilities, practices, and
equipment are specified. To give further
guidance to investigators and their institu-
tions, a supplement to the guidelines explains
more fully safety practices appropriate to re-
combinant DNA research. And a new section
has been added to ensure that shipment of
recombinant DNA materials conforms, where
appropriate, to the standards, prescribed by
the U.S. Public Health Service, the Depart-
ment of Transportation, and the Civil Aero-
nautics Board.

The section on physical containment is
carefully designed to offer a constructive ap-
proach to meeting potential hazards for re-
combinant experiments at all levels of pre-
sumed risk. Certain commentators had sug-
gested that the first level of physical con-
tainment (Pl) be merged with the second
level (P2). This suggestion, however, would
tend to apply overly stringent standards for
some experiments and might result in a
lowering of standards necessary at the second
level. I believe the level of control must be
consistent with a reasonabie estimate of the
hazard; and the section on physical contain-
ment does provide this consistency.-Accord-
ingly, the first and second levels of physical
containment remain as separate sections in
the guidelines.

Because of the nature and operation of
facilities required for experiments to be done
at the fourth level of containment (P4), a
provision has been included that the NIH
shall review such facilities prior to funding
them for recombinant DNA studies. The
situation merits the special attention of ex-
perts who have maximum familiarity with
the structure, operation, and potential prob-
Jems of P4 installations. Several com-
mentators advocated that NIH arrange for
sharing of P4 facilities, both in the NIH
intramural program and in institutions sup-
ported through NIH awards. In response to
these suggestions, we are currently review-
ing our facilities, including those at
the Frederick Cancer Research Center (Fort
Detrick), to determine how such a program
can best be devised. It is most important that
P4 facilities be made available to investiga-
tors. It should be noted that incidents of in-
fection by even the most highly infectious
and dangerous organisms are extremely infre-
quent at P4 facilities, and therefore the
potential for hazard in certain complex ex-~
periments in recombinant DNA research is
considerably reduced.

TTI. PROHIBITED EXPERIMENTS

1. Practically all commentators supported
the present prohibition of certain experi-
ments. There were suggestions for a clearer
definition of the prohibition of certain ex-
periments where increased antibiotic resist-
ance May result. And it was urged by some
that the prohibition be broadened to include
experiments that result in resistance to any
antiblotic, irrespective of its use in medicine
or agriculture. Consideration of such a sug-
gestion must take into account that antt-
biotic resistance occurs naturally among bac-
teria, and that resistance is a valuable marker
in the study of microbial genetics in general
and recombinants in particular.

In view of these concerns, however, the
Recombinant Advisory Committee was asked
to reconsider carefully the prohibition and
related sections concerning antibiotic resist-
ance. The committee noted that the prohibi-
tion relating to drug resistance was intended
to ban those experiments that could com-
Promise drug use in controlling disease
agents in veterinary as well as human medi-
cine and this ts now clearly stated.

In the draft guidelines there were two
statements concerning resistance to drugs
which related to experiments with EF. col.

The statements appeared to allow experi-

FEDERAL REGISTER, VOL. 41,-NO. 176—-THURSDAY, SEPTEMBER 9, 1976
38448

ments that would extend the range of resist-
ance of this bacterlum to therapeutically
useful drugs and disinfectants, and thus
seemed to be in conflict with the general
prohibition on such research. There are
numerous reports in the scientific literature
indicating that E. coli can acquire resistance
to all antibiotics known to act against it.
Since E. coli acquires resistance naturally,
the prohibition directed against increasing
resistance does not apply. The ambiguous
statements have been deleted from the pres-
ent guidelines. On the other hand, new
language has been inserted in the section
dealing with other prokaryote species to set
containment levels for permitted experi-
ments.

2. The Recombinant Advisory Committee
was also asked to clarify whether the pro-
hibition of use of DNA derived from patho-
genic organisms (those classified as 3, 4, and
§ by the Center for Disease Control, USPHS)
also included the DNA from any host in-
fected with these organisms. The committee
explained that this prohibition did extend
to experiments with’ cells known to be 50
infected. To avoid misunderstanding, the
prohibition as now worded includes such
cells. In addition, the prohibitions have been
extended to include moderate-risk oncogenic
viruses, as defined by the National Cancer
Institute, and celis known to be infected
with them.

3. Two other issues relating to the section
on prohibited experiments were raised by
Roy Curtiss III, Ph.D., Professor, Depart-
ment of Microbiology, University of Alabama
School of Medicine, Birmingham, who is a
member of the Recombinant Advisory Com-
mittee. Dr. Curtiss noted that for the class
of experiments prohibited on the basis of
production of highly toxic substances, only
substances from microorganisms were cited
as examples. He suggested that other exam-
pies be included, such as venoms from insects
and snakes. The committee approved the
suggestion and I concur.

In the proposed guidelines, release of or-
ganisms containing recombinant DNA mole-
cules into the environment was prohibited
unless a series of controlled tests had been
done to leave no reasonable doubt of safety.
Dr. Curtiss felt that the guidelines should
provide greater specificity for testing and
should include some form of review prior
to release of the organism. I have decided
that the guidelines should, for the present,
prohibit any deliberate release of organisms
containing recombinant DNA into the en-
vironment. With the present limited state
of knowledge, it seems highly unlikely that
there will be in the near future, any re-
combinant organism that is universally ac-
cepted as beig beneficial to introduce into
the environment. When the scientific evi-
dence becomes available that the potential
benefits of recombinant organisms, particu-
larly for agriculture, are about to be realized,
then the guidelines can be altered to meet
the need for release. It is most important
that the potential environmental impact of
the release be considered.

IV. PERMISSIBLE EXPERIMENTS: E. cour K-12
Host-VectTor SyYsTeEMs

The continued use of E. coli as a host has
drawn considerable comment, including
some suggestions that its use be prohibited
presently or within a specified time limit.
It should be stressed that the use of E. coli
as detailed in the guidelines is limited to E.

1 Specifically, experiments that would ex-
tend resistance to therapeutically useful
drugs must use P3 physical containment plus
a host-vector comparable to EK1, or P2 con-
tainment plus a host-vector comparable to
EK2.

NOTICES

coli K-12, a strain that has been carried in
the laboratory for decades, and does not in-
volve the use of any strain of E. coli that is
freshly isolated from a natural source. E. coli
K-12 does not usually colonize the normal
bowel, even when given in large doses, and
exhibits little if any muliplication while
passing through the alimentary canal. For
years it has been the subject of more intense
investigation than any other single organism,
and Knowledge of its genetic markup and
recombinant behavior exceeds greatly that
pertaining to any other organism, I believe
that because of this experience, E. coli K~12
will provide a host-vector system that Is
safer than other candidate microorganisms.

NIA recognizes the importance of support-
ing the development of alternative host-vec-
tor systems (such as B. subtilis, which has
no ecological niche in man) and will en-
courage such development. It should be
noted, however, that for each new host-vec-
tor system, the same questions of risk from
altered properties attendant upon the pres-
ence of recombinant genes will apply as ap-
ply to E. coli. NIH does not believe it wise
to set a time limit on replacement of E. coli
systems by other organisms.

There were specific suggestions concerning
the three levels of biological containment
prescribed for use of E. coli K-12 vectors.
Some commentators requested a more de-~
tailed explanation of the adequacy of protec-
tion for laboratory personnel with the first
level of containment (EK1).? Sections of the
guidelines dealing with physical containment
and roles and responsibilities now specify the
need for safety practices and accident plans.

For the second level of containment (EK2),
it is required that a cloned DNA fragment be
contained in a host-vector system that has
no greater than a 10-8 probability of sur-
vival in a nonpermissive or natural environ-
ment. It was suggested that the selection of
this level of biological containment and the
appropriate tests for vertification be more
fully explained in the guidelines. The com-
mittee, in responding to a request for fur-
ther examination of this point, reviewed at
considerable length the testing for an EK2
system and recommended certain modifica-
tions. We have accepted the committee’s new
language that better explains testing of sur-
vival of a genetic marker carried on the vec-
tor, preferably on an inserted DNA fragment.

Possible tests to determine the level of bio-
logical containment afforded by these al-
tered host-vector systems are outlined in this
section. Because this is such a new area of
scientific research and development, however,
it is inappropriate to standardize such test-
ing at the present time. Standards will grad-
ually be set as more experience with EK2
host-vector systems is acquired. The commit-
tee, for example, during its April 1976 meet-
ings gave its first approval to an EK2 host-
vector system. What is necessary is that new

 

?The EK1 system presently consists of 4
battery of different vectors and of E. coli
K-12 mutants, all of which afford a consider-
able degree of biological containment. The
diversity of vectors and of host mutants in
this battery has permitted a wide range of
important scientific questions to be attacked.
For example, the availability of different vec-
tors with cleavage sites for different restric-
tion endonucleases have increased the kind
of DNA segments that can be cloned. By con-
trast, the first EK2 host-vector systems are
only now being considered by the Recom-
binant Advisory Committee. While NIH is
supporting the development of more EK2
host-Vector systems, it is not expected that a
battery equivalent to that available for the
EK1 system will be certified by the Recom-
binant Advisory Committee in the near fu-

ture.

and more effective tests be devised by investi-
gators, and this effort is very likely to occur
under the present guidelines. For example,
one task recognized by the committee is to
clarify how survival of the organism and the
cloned DNA should be defined in terms of
temperature, medium, and other variables.

It is also very important to note here that
the stringent requirements set by the com-
mitteesfor EK2 biological containment jeop-
ardize considerably the capacity of such crip-
pled organisms to survive and replicate even
under permissive laboratory conditions. More
experience will-be required to determine
whether EK2 containment will permit some
lines of important research to be followed.

Several commentators suggested that
methods and procedures to confirm an EK
system at the third level of containment
(EK3) be more fully explained. The Recom-
binant Advisory Committee was asked to con-
sider this suggestion. After considerable dis-
cussion the committee declined to define the
procedures more fully at this time, because
development of an EK3 system is still far
enough in the future not to warrant specific
testing procedures. Further, it is not clear
what tests are best suited. The language,
therefore, remains general. The committee,
however, is aware of the concerns for a more
completely defined system of testing, and has
eonsidered the possibility of organizing a
symposium for purposes of designating tests.
In my view, more fully developed protocols
for testing EK3 systems are warranted, and
it is necessary that guidelines here be more
fully developed before the committee pro-
, ceeds to certify such a system. In this regard
the NIH fs prepared through the National
Institute of Allergy and Infectious Diseases
to support contracts to accomplish this task.
We will seek the advice and assistance of the
committee to define the scope of necessary
work.

These guidelines also include a statement
that for the time being no EK2 or EK3 host-
vector system will be considered bona fide
until the Recombinant Advisory Committee
has certified it. I share the concern of the
commentators that new host-vector systems
require the highest quality of scientific re-
view and scrutiny. At this early stage of
development, it is most important that the
committee provide that scrutiny. Further, I
beHleve that until more experience has been
gained, the committee should encourage and
the NIH support research that will Independ-
ently confirm and augment the data on
which certification of EK2 host-vector sys-
tems are based.

V. CLASSIFICATION OF EXPERIMENTS USING THE
E. Cott K-12 ConTaAINMENT SYSTEMS

The guidelines assign different levels of
containment for experiments in which DNA
from different sources is to be introduced
into an £. coli K-12 host-vector system. The
variation is based on both facts and as-
sumptions. There are some prokaryotes (bac-
teria) which constantly exchange DNA with
E. coli. Here 1t is assumed that experimen-
tal conditions beyond those obtained in care-
ful, routine microbiology laboratories are
superfluous, because any exchange experi-
ments have undoubtedly been performed al-
ready in nature.

In every instance of artificial recombina-
tion, consideration must be given to the pos-
sibility that foreign DNA may be translated
into protein (expressed), and also to the
possibility that normally repressed genes of
the host may be expressed and thus change,
undesirably, the characteristics of the cell.
It is assumed that the more similar the
DNAs of donor and host, the greater the
probability of expression of foreign DNA, or of
possible derepression of host genes. In those
cases where the donor exchanges DNA with
E. coli in nature, it is unlikely that recom-

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
bination experiments will create new genetic
combinations. When prokaryote donors not
Known to exchange DNA with £. colt in
nature are used, however, there is a greater
potential for new genetic combinations to
be formed and be expressed. Therefore, it is
required that experiments involving prokary-
otic DNA from a donor that is not Known
to exchange DNA with £. coli in nature be
carried out at a higher level of containment,
Recombination using prokaryotic DNA from
an organism known to be highly pathogenic
is prohibited.

There are only limited data available con-
cerning the expression of DNA from higher
forms of life (eukaryotes) in Z. coli (or any
other prokaryote). Therefore, the contain-
ment prescriptions for experiments inserting
eukaryotic DNA into prokaryotes are based
on risks having quite uncertain probabilities.

On the assumption that a prokaryote host
might translate eukaryotic DNA, it is fur-
ther presumed that the product of that for-
elgn gene would be most harmful to man
if it were an enzyme, hormone, or other
protein that was similar (homologous) to
proteins already produced by or active in
Man. An example is a bacterium that could
produce insulin. Such a “rogue” bacterlum
could be of benefit if contained, a nuisance or
possibly dangerous if capable of surviving
in nature. This is one reason that the higher
the phylogenetic order of the eukaryote, the
higher the recommended containment, at
least until the efficiency of expression of DNA
from higher eukaryotes in prokaryotes can
be determined.

‘There is a second, more concrete reason for
scaling containment upward as the eukaryote
host becomes similar to man. This is the
concern that viruses capable of propagating
in human tissue, and possibly causing dis-
eases, can contaminate DNA, replicate in
prokaryote hosts and infect the experimen-
talist. Such risks are greatest when total
DNA from donor tissue is used in “shot-
gun” recombinant experiments; it diminishes
tw much lower levels when pure cloned DNA
is used.

The commentators were clearly divided on
the classification of containment criteria for
different Kinds of recombinant DNAs. Many
commentators considered the guidelines too
stringent and rigid. Others viewed the guide-
lines in certain instances as too permissive.
And still others endorsed the guidelines as
sensible and reasonable, affording the public
an enormous degree of protection from the
speculative risks. Several suggestions were
made for the specific classes of experiments,
and they follow:

1. Comment on the use of DNA from ani-
mals and plants In recombinant experiments
varied widely. Some commentators suggested
banning the use of DNA from primates, other
mammals, and birds. Others suggested that
higher levels of the containment be used
for all such experiments. Still others believed
that the guidelines were too strict for ex-
periments of this class. I have carefully re-
viewed the issues raised by the commentators
and the response of the committee to certain
queries concerning use of animal and plant
DNA in these experiments.

In my view, the classification for the use
of DNA from primates, other mammals, and
birds is appropriate to the potential hazards
that might be posed. The physical and bio-
logical containment levels are very strict. For
example, biological containment levels are at
EK2 or EK3, and will effectively preclude ex-
perimentation until useful EK2 and EK3 sys-
tems are available. EK2 systems are still in
the initial stages of development, and the
first system was only certified at the most re-
cent meeting of the Recombinant Advisory
Committee. An EK3 host-vector system has
yet to be tested, and its certification is far
enough in the future to place a moratorium

NOTICES

on those experiments requiring biological
containment at an EK3 level. The physical
containment levels of P3 or P4 themselves
afford a very high degree of protection. I am
satisfied that the guidelines demonstrate the
caution and prudence that must govern the
conduct of experiments in this category.

The guidelines allow reduced containment
levels for primate DNA when it is derived
from embryonic tissue or germ-line cells. This
is based on evidence that embryonic material
is less Iikely to contain viruses than Is tissue
from the adult. Obviously, the embryonic
tissue must be free of adult tissue, and the
present guidelines so indicate.

I have also carefully considered the special
concerns arising from the use of DNA from
cold-blooded vertebrates and other cold-
blooded animals, because several commenta-
tors questioned the basis of lower physical
and biological containment levels for DNA
from these species. The Recombinant Ad-
visory Committee has debated this exten-
sively, and they were asked to do so once
again in April. The committee has now rec-
ommended high containment levela (P3+
EK2) when the DNA is from a cold-blooded
vertebrate known to produce a potent toxin.
That recommendation is included in the
present guidelines. Where no toxin is in-
volved the committee supported lower con-
tainment levels. The guidelines specify P2-+
EK2 levels for such work. There was consider-
able discussion concerning the advisability of
recommending lower containment (P2-+ EK1)
when the DNA is isolated from embryonic
tissue or germ-line cells from cold-blooded
vertebrates. Those supporting lower con-
tainment levels argued that the justification
for P2+EK2 was the possibility that cold-
blooded vertebrates may carry viruses and
that the distinction between adult and germ
cell tissue is real. Others argued that, con-
trary to the situation with primate DNA,
viruses are not a central problem with cold-
blooded vertebrates and therefore no dis-
tinction should be made on the basis of tis-
sue origin. Finally, the committee recom-
mended, on a divided vote (8 to 4), to adopt
P2+4+EK1 when the cold-blooded vertebrate
DNA is isolated from embryonic tissue or
germ-line cells. Upon reviewing these con-
siderations, I have decided to retain the con-
tainment levels for embryonic or germ-line
DNA from cold-blooded vertebrates as rec-
ommended by the committee.

In April the committee also reviewed, at
our request, the classification of exper!-
ments where DNA ts derived from other cold-
blooded animals or lower eukaryotes. Sev-
eral commentators, for example, had been

2A committee member, David 8. Hogness,
Ph. D., Professor, Department of Biochemts-
try, Stanford University, California, submit-
ted a statement in support of lower contaln-
ment levels based on current scientific evi-
dence. That evidence is based on certain
differences between cold- and warm-blooded
vertebrates. One of the criteria used for the
evaluation of the relative risk that might be
encountered with different levels of shotgun
experiment is the degree of sequence homol-
ogy between the DNA of the given species and
that of humans. This criterion is used to
estimate the likelihood that segments of
DNA from the given species might be inte-
grated into the human genome by recom-
bination; the greater the homology, the
greater the likelihood of integration. Studies
of sequence homologies indicate that there
is a considerable degree of homology be-
tween human DNA and DNA from other pri-
mates, much less homology between pri-
mates and other mammals, and even lower
but detectable homology between birds and
primates. By contrast, no significant homolo-
gies between cold-blooded vertebrates and
primates have been detected.

38449

concerned about the fact that insects are
Known to carry agents pathogenic to man.
In the committee review, it was noted that
viruses carried by insects and Known to
transnut disease to man are RNA rather than
DNA viruses and do not reproduce via DNA
copied from RNA. In order, however, to make
the intent clearer, the guidelines have been
rewritten for experiments of this class. New
language is inserted to ensure that strict
containment levels are employed when the
DNA comes from known pathogens or species
known to carry them. Further, to reduce the
potential hazards, we have also included in
the guidelines the requirement that any in-
sect must be grown under laboratory condi-
tions for at least 10 generations prior to ite
use as a DNA source.

2. As alluded to above, certain commenta-
tors expressed concern that when E. coli be-
comes the host of recombinant DNA from
prokaryotes with which DNA is not usually
exchanged, there is hazard of altered host
characteristics resulting from translation of
the DNA into functioning proteins. The
committee was asked to review the guide-
lines and take into account this potential
hazard. They agreed that the containment
levels should be increased for this category
of experiment, from P2+EK1 to either P2+
EE2 or P3-+EK1. That recommendation is
included in the present guidelines.

Comments were made concerning that class
of experiments in which the recombinant
DNA, regardless of source, has been cloned.
A clone is a population of cells derived from
s single cell and therefore all the cells are
presumed to be genetically identical. As out-
lined in the proposed guidelines, clones could
be used at lower containment levels if they
had been rigorously characterized and shown
to be free of harmful genes. Several com-
mentators inquired how the characterization
was to be performed and the freedom from
harmful genes demonstrated. Although the
committee acknowledges that these terms are
unavoidably vague, they do cite appropriate
scientific methods to make relevamt deter-
minations. Again, this is a rapidly chang-
ing area and more clarity and precision can
be expected with experience. Reduced con-
tainment requirements for this class of ex-
periment are warranted because of the purl-
fied nature of clones. Further, the granting
agency must approve the clone before con-
talnment conditions can be reduced, thus
providing an additional element of review.

4. Another comment was related to the use
of DNA from organelles (intracellular ele-
ments that contain special groups of genes
for particular cell functions). Concern was
expressed about the potential contamination
of purified organelle DNA with DNA from
viruses because of the similarity of thelr
structures. The committee agrees, and the
guidelines now specify a requirement, that
the organelles be tsolated prior to extracting
DNA, as a further means of reducing the
hazard of viral contamination.

5. Some commentators were troubled about
the lowering of containment for that class of
experiments involving recombinations with
cell DNA segments purified by chemical or
physical methods. They asked that proce-
dures for determining the state of purifica-
tion be more fully detailed and that the Re-
combinant Advisory Committee certify the
purity. There are, however, appropriate tech-
niques, such as gel electrophoresis, with
which a purity of 99 percent by mass can be
achieved and ascertained. There is no way
for the committee to certify these results
beyond repeating the experiments themselves.
These techniques are well documented and
described In the literature. I do not believe
it 8s necessary or feasible for the committee
to review each procedure for purification of
DNA.

6. Cominents were made concerning the
use of DNA derived from animal viruses. It

FEDERAL REGISTER, VOL. 41, NO. 176-—-THURSDAY, SEPTEMBER 9, 1976
; 38450

was urged that containment levels for this
class of experiment be increased. On the
basis of my review, I find the containment
conditions appropriate to the potential haz-
ard posed. As defined in the guidelines, exper-
iments are to be done at very. strict levels of
containment and these can be lowered only
when the cloned DNA recombinants have
been shown to be free of possibly harmful
genes by suitable biochemical and biological

tests. This also pertains to DNA that is-

copied from RNA viruses. In no instance are
the guidelines more lenient, and in most in-
stances they are more stringent than condi-
tions obtaining in many laboratories where
such viruses are studied in non-DNA-recom-
binant experiments.

VI. CLASSIFICATION OF EXPERIMENTS USING
CONTAINMENT SYSTEMS OTHER THAN E. COLr
K-12

1. No issue with regard to these guidelines
raised more comment than the use of animal
viruses as vectors. Of special concern to many
commentators was the use of the simian
(monkey) virus 40 (hereafter “SV40”). Some
suggested a complete ban on the use of this
virus; others urged its retention as a vector.
SvV40 is not known to produce any disease in
man, although it can be grown in human cells
and on very rare occasions has been isolated
from humans. Many humans have received
SV40 virus inadvertently in vaccines prepared
from virus grown in monkey kidney-cell cul-
tures. An intensive search has been made and
1s continuing for evidence that SV40 might
cause cancer or be otherwise pathogenic for
man. At present, 1t is my view that the ex-
tensive knowledge we have of SV40 virus pro-
vides us with sufficient sophistication to en-
sure its safe handling under the conditions
developed for its use in the guidelines.

I believe work with SV40 should continue
under the most careful conditions, but I do
recognize and appreciate the concerns ex-
pressed over its possible harmful effects in
humans. In light of these concerns, I asked
the Recombinant Advisory Committee to
review this section of the guidelines. The
committee reconsidered the containment
conditions for this class of experiments and
judged them appropriate to meet the poten-
tial hazards.‘

This class of experiments will proceed un-
der the most careful and stringent condi-
tions. Work with SV40 virus will be done at
the maximum level of physical containment
(P4). The extraordinary precautions required
in a P4 facility lessen the likelihood of a
potential hazard from this work. Only defec-
tive SV40 virus will be used as vector; that is,
the SV40 virus particles that carry the foreign
DNA cannot multiply by themselves. When a
number of strict conditions are met, this
work will be permitted to go on at the third
level of containment (P3), which in itself re-
quires care and precision. It should be noted
that SV40 virus and its DNA can be efficiently
disinfected by Clorox and autoclaving. These
are customary procedures for disinfecting
glassware and other items used in SV40 ani-
majl-ce]l work.

Some commentators suggested that the
containment criteria for experiments using
polyoma virus as the vector be strengthened.
There is no evidence that polyoma infects
humans or replicates to any significant extent
in human cells. It holds promise as a vector,
as is more fully documented in an appendix
to these guidelines.

«One member dissented from this position.
During the discussion, additional language
was recommended (and adopted) to ensure
that the defective SV40-virus/helper-virus
system, with its inserted non-SV40 DNA seg-
ment, does not replicate in human cells with
significantly more efficiency than does SV40.

NOTICES

2. Several commentators found the guide-
lines inadequate regarding experiments with
plant host-yector systems. Because NIH
shared these concerns, a group of extensive
experience with plants was appointed to re~
view this section. The group met concur-
rently with the Recombinant Advisory Com-
mittee in April 1976 and made several modi-
fications. The suggested revisions were ac-
ceptable to the full committee, and we have
included them in the guidelines.

The modifications are responsive to the
stated concerns of the commenators. A de-
scription of greenhouse facilities is given, and
physical containment conditions have been
modified to take into account operations with
whole plants. On the whole the respec-
tive portions of the guidelines relating to
plants are more fully explained and the in-
tent is clarified.

I have also accepted the recommendation
of the subcommittee to lower the biological
containment level from EK2 to EK1 for ex-
periments in which the DNA from plants is
used in conjunction with the EZ. coli K-12
host-vector system, thereby setting contain-
ment in this instance at the same level re-
quired for experiments with lower-eukaryote
DNA.

VII. RoLes AND RESPONSIBILITIES

1. Most commentators had suggestions for
the section on the roles and responsibilities of
investigators, their local institutions, and
NIH. Commentators generally urged open-
ness, candor, and public participation in the
process, emphasizing shared responsibility
and accountability from the local to the na-
tional level. We reviewed that section of the
guidelines in light of these comments and
have asked the Recombinant Advisory Com-
mittee to review certain issues.

It is clear that much of the success of the
guidelines will lie in the wisdom with which
they are implemented. Because of the im-
portance of this section, especially in terms
of safety programs and plans, we have care-
fully weighed the comments and suggestions
made in this regard. NIH has a special re-
sponsibility to take a leading role in ensuring
that safety programs are part of all recombi-
nant DNA research. Dr. Barkley and a spe-
cially convened committee were asked to pro-
vide greater detail for safety, accident, and
training plans for this section of the guide-
nes, Based on their recommendations, the
section has been extensively rewritten to
clarify the respective responsibilities of the
principal investigator, the institution (in-
cluding the institutional blohazards com-
mittee), the NIH initial review group (study
section), the NIH Recombinant DNA Mole-
cule Program Advisory Committee, and NIH
staff.

This section has a definitive administra-
tive framework for assuring that safety is an
essential and integrated component of re-
search involving recombinant DNA molecules.
The guidelines require investigators to in-
stitute, monitor, and evaluate containment
and safety practices and procedures. Before
research is done, the investigator must have
safety and accident plans in place and train-
ing exercises for the staff well under way.

Some commentators suggested that the in-
vestigator be required to obtain informed
consent of laboratory personnel prior to their
participation. Rather than rely explicitly on
an informed consent document, the guide-
lines now make the investigator responsible
for advising his program and support staff as
to the nature and assessment of the real and
potential biohazards. He must explain and
provide for any advised or requested precau-
tionary medical policies, vaccinations, or
serum collections. Further, an appendix to
the guidelines includes detailed explanations
for dealing with accidents, as well as instruc-

 

tions for the training of staff in safety and
accident procedures.

In response to suggestions for epidemiolog-
ical monitoring, the guidelines now require
the principal investigator to report certain
categories of accidents, in writing, to appro-
priate officials. NIH ts investigating proce-
dures for long-term surveillance of workers
engaged in recombinant DNA research.

2. A number of comments on the role and
responsibilities of the institutional biohaz-
ards committee were received. Comments
were directed to the structure of the com-
mittee, the scope of its responsibility, and
the methods for operation. Comments on
structure included suggestions that the com-
mittee have a broadly based representation,
especially in terms of health and safety ex-
pertise. Some others suggested NIH require
certain classes of representation. In response
_to these suggestions, the guidelines now rec-
ommend membership from a diversity of dis-
ciplines relevant to recombinant DNA mole-
cule technology, biological safety, and engi-
neering.

For broader representation beyond the im-
mediate scientific expertise, the guidelines
now recommend that local committees should
possess, or have available, the competence
necessary to determine the acceptability of
their findings in terms of applicable laws,
regulations, standards of practice, communi-
ty attitudes, and health and environmental
considerations. The names of and relevant
background information on the committee
members wiil be reported to NIH. |

In response to suggestions that decisions
of the committee be made publicly available,
the guidelines now recommend that minutes
of the meetings should be kept and made
available for public inspection.

Commentators generally approved of the
responsibility given to the institutional bio-
hazards committee to serve as a source of
advice and reference to the investigator on
scientific and safety questions. It was fur-
ther suggested that the committee’s respon-
sibility be broadened in the development,
monitoring, and evaluation of safety stand-
ards and procedures. In response to these
suggestions, the guidelines now indicate that
the institutional biohazards committee has
the responsibility to certify, and recertify an-
nually, to NIH that the facilities, procedures,
practices, training, and expertise of involved
personnel have been reviewed and approved.
The Recombinant Advisory Committee sug-
gested that examination might be unneces-

. Sary for Pl facilities, but we believe that all

facilities should be reviewed to emphasize the
importance of safety programs.

Some commentators suggested that the
guidelines should stipulate that the local
committees be required to determine the con-
tainment conditions to be imposed for a
given project (which the draft guidelines
specifically noted was not their responsibil-
ity). The Recombinant Afivisory Committee
took exception to this suggestion. They urged
NIH not to include these conditions as local
requirements, arguing among other things
that review by the NIH study sections would
provide the necessary scrutiny at the national
level and assure uniformity of standards in
application of the guidelines. I do not believe
that NIH should require the local institution
to have Its biohazards committee assess what
containment conditions are required for a
given project. On the other hand, the guide-
lines should not prohibit the local institution
from having its biohazards committee per-
form this function. Accordingly, I have de-
leted the prohibition that appeared in the
proposed guidelines.

Another suggestion was that the local com-
mittee ensure that research js carried out in
accordance with standards and procedures
under the Occupational Safety and Health

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
Act (OSHA). This is an area of importance to
the local institutions under Federal and
State law, but need not be included as a re-
quirement in the guidelines. NIH will main-
tain Maison with the Occupational Safety and
Health Administration (Department of La-
bor) to ensure maximum Federal cooperation
in this venture.

I would also encourage all institutions, as
suggested by several commentators, to review
their insurance compensation programs to
determine whether their laboratory person-
nel, in the research area, are covered for
injuries.

3. The commentators approved of having
the NIH study sections responsible for mak-
ing an independent evaluation of the classi-
fication of the proposed research under the
guidelines, along with the customary judg-
ment of the scientific merit of each grant
application, This additional element of review
will ensure careful attention to potential
hazards in the research activity. The study
sections will also scrutinize the proposed
safeguards. Biological safety expertise shal!
be available to the study section for con-
sultation and guidance in this regard.

4. Several commentators made suggestions
concerning the structure, function, and scope
of responsibility of the NIH Recombinant
DNA Molecule Program Advisory Committee.

Comments on possible structural mechan-
isms for decision making included sugges-
tions that there be a scientific and technical
committee and a general advisory public
policy committee. It was also suggested that
the scientific committee include scientists
who are not actively engaged in recombinant
research, and that the public policy com-
mittee have a broad scientific and public
representation,

I have carefully reviewed these comments
and suggestions. In response, the following
structure has been devised. 'The Recombinant
Advisory Committee shall serve as the scien-
tific and technical committee. Its member-
ship shall continue to include scientists who
represent disciplines actively engaged in re-
combinant DNA research. In my view, it is
most important that this committee have the
necessary expertise to assure that the guide-
lines are of the highest scientific quality. The
committee has provided this expertise in the
past, and it must continue to do so. The com-
mittee shall also include members from other
scientific disciplines.

It should be noted that the present com-
mittee recommended on its own initiative
that a nonscientist be appointed. Emmette
S. Redford, Ph. D., LL. D., Ashbel Smith Pro-
fessor of Government and Public Affairs at
the Lyndon B. Johnson School of Public Af-
fairs, University of Texas at Austin, serves in
that capacity. An ethicist has also been
nominated for appointment.

The Advisory Committee to the Director,
NIH, shall serve to provide the broader public
policy perspectives. This committee, at its
meeting on February 9-10, 1976, reviewed the
proposed guidelines with the participation of
public witnesses, and shall continue to pro-
vide such review for future activities of the
Recombinant Advisory Committee.

In response to suggestions, the responsi-
bilities of the Recombinant Advisory Com-
mittee have been expanded. In addition to
reviewing the guidelines for possible modi-
fication as scientific evidence warrants, the
committee will certify EK2 and EK3 systems.
In response to requests by the investigator,
local committee, or study section, the com-
mittee will also provide evaluation and re-
view in order to advise on levels of required
containment, on lowering of requirements
when coloned recombinants are to be used,
and on questions concerning potential bilo-
hazard and adequacy of containment provi-
sions.

NOTICES

Commentators also asked that the com-
mittee review ongoing research initiated
prior to the implementation of the guide-
lines. Now that the guidelines are being re-
leased, NIH-funded investigators in this field
will be asked to give assurance, within a
given period, that they will comply. Any in-
vestigators who constructed clones under the
Asilomar guidelines will be asked to petition
NIH for special consideration of their case,
if the new guidelines require higher contain~
ment than did the Asilomar guidelines. Here
the advice of the Recombinant Advisory
Committee will be sought.

There were also suggestions that the com-
mittee certify chemical purification of recom-
binant DNA, but as I indicated earlier, these
procedures are too well known to require
NIH monitoring.

6. In Ught of comments received, NIH will
provide review, through appropriate NIH
offices, of data from institutional biohazards
committees (including accident reports) and
will ensure dissemination of these findings as
appropriate. Dr. William Gartland will head
the newly created NIH Office of Recombi-
nant DNA Activities for these purposes. In
addition, NIH will provide for rapid dis-
semination of Information through its Nu-
cleic Acid Recombinant Scientific Memo-
randa (NARSM), distributed by the National
Institute for Allergy and Infectious Diseases.
NIH will also provide an appropriate mech-
anism for approving and certifying clones
before containment conditions can be
lowered.

With these extended modifications, the
section of the guidelines dealing with roles
and responsibilities now sets forth a more
fully developed review structure involving
the principal investigator, local biohazards
committees, and the Recombinant Advisory
Committee, as well as peer review commit-
tees. Guidelines now provide extensive op-
portunity for advice, from the local to the
national level. Several levels of review and
scrutiny are provided, ensuring the highest
standards for scientific merit and conditions
for safety.

The Recombinant Advisory Committee in
conjunction with the Director’s Advisory
Committee shall continue to serve as an
ongoing forum for examining progress in
the technology and safety of recombinant
DNA research. Their responsibility, and that
of the NIH Director, is to ensure that the
guidelines, through modification when called
for, reflect the soundest scientific and safety
evidence as it accrues in this area. Their

task, in a sense, is Just beginning.

DONALD S. FREDRICKSON, M.D.,
Director,
National Institutes of Heaith.

NaTIONAL INSTITUTES OF HEALTH

GUIDELINES FOR RESEARCH INVOLVING
RECOMBINANT DNA MOLECULES

JUNE 1976.
CONTENTS

I.
Introduction.
II.
Containment,
A. Standard practices and training.
B. Physical containment levels.
Pi Level (Minimal).
P2 Level (Low).
P3 Level (Moderate).
P4 Level (High).
Cc. Shipment.
D. Biological containment levels.

ri.

Experimental Guidelines.
A. Experiments that are not to be per-
formed. .

38451

B. Containment guidelines for permissible
experiments.
1, Biological containment criteria using E.
Colt K~12 host-vectors,
EK1 host-vectors.
EK2 host-vectors.
EK3 host-vectors.
3. Classification of experiments using the
£. Coli K-12 containment systems.
<a> Shotgun experiments. ,
(1) Eukaryotic DNA recombinants.
(ii) Prokaryotic DNA recombinants.
(iii) Characterized clones of DNA recom-
binants derived from shotgun experiments.
<b> Purified cellular DNAs other than
“plasmids, bacteriophages, and other viruses.
<e> Plasmids, bacteriophages, and other
viruses.
(1) Animal viruses.
(ii) Plant viruses.
(iii) Eukaryotic organelle DNAs.

(iv) Prokaryotic plasmid and phage
DNAs.
3. Experiments with other prokaryotic host-
vectors.
4. Experiments with eaukaryotic host-
vectors.

<a> Animal host-vector systems.
<b> Plant host-vector systems.
<e> Fungal or similar lower eukaryotic
systems. :
Iv.

Roles and Responsibilities.

A. Principal investigator.

B. Institution.

Cc. NIH Initial Review Group (Study Sec-
tions).

D. NIH Recombinant DNA Molecule Fro-
gram Advisory Committee.

E. NIH Sta/f.
Vv.
Footnotes.
VIL
References.
Vil.

Members of the Recombinant DNA Molec:vie
Program Advisory Committee.

APPENDICES

A. Statement on the use of Bacillus subtilis
in recombinant molecule technology,

B. Polyoma and SV40 Virus.

Cc. Summary of Workshop on the Design &
Testing of Safer Prokaryotic Vehicles
& Bacterial Hosts for Research on Re-
Combinant DNA Molecules.

D. Supplementary Information on Physical
Containment (Including Detaved
Contents).

I. INTRODUCTION

The purpose of these guidelines is to rec-
commend safeguards for research on recom-
binant DNA molecules to the National In-
stitutes of Health and to other institutions
that support such research. In this context
we define recombinant DNAs as molecules
that consist of different segments of DNA
which have been joined together in cell-free
systems, and which have the capacity to in-
fect and replicate in some host cell, either
autonomously or as an integrated part of
the host’s genome.

This is the first attempt to provide a de-
tailed set of guidelines for use by study sec-
tions as well as practicing scientists for evai-
uating research on recombinant DNA mole-
cules. We cannot hope to anticipate al) pos-
sible lines of imaginative research that are
Possible with this powerful new methad--
ology. Nevertheless, a considerable volume of
written and verbal contributions from sci-
entists in a variety of disciplines has been
received. In many instances the views pre-~
sented to us were contradictory. At present,
the hazards may be guessed at, speculated
about, or voted upon, but they cannet be

FEDERAL REGISTER, VOL. 41, NO. 176——THURSDAY, SEPTEMBER 9, 1976
: 88a52

: Known absolutely in the absence of firm ex-
perimental data—and, unfortunately, the
needed data were, more often than not, un-
available. Our problem then has been to con-
struct guidelines that aliow the. promise of
the methodology to be realized while advocat-
ing the considerable caution that 1s de-
manded by what we and others view as po-
tential hazards. .

In designing these guidelines we have
adopted the following principles, which are
consistent with the general conclusions that
were formulated at the International Con-
ference Center, Pacific Grove, California, in
February 1975 (3): (i) There are certain ex-
periments for which the assessed potential
hazard is so serious that they are not to be
attempted to the present time. (ii) The re-
mainder can be undertaken at the present
time provided that the experiment is justifi-
able on the basis that new knowledge or
benefits to humankind will accrue that can-
not readily be obtained by use of conven-
tional methodology and that appropriate
safeguards are incorporated into the design
and execution of the experiment. In addi-
tion to an insistence on the practice of good
microbiological techniques, these safeguards
consist of providing both physical and biclo-
gical barriers to the dissemination of the po-
tentially hazardous agents. (ili) ‘The level of
containment provided by these barriers is to
match the estimated potential hazard for
each of the different classes of recombinants,
For projects in a given class, this level is to
be highest at initiation and modified subse-
quently only if there is a substantiated
change in the assessed risk or in the applied
methodology. (iv) The guidelines will be sub-
jected to periodic review (at least annually)
and modified to reflect improvements in our
Knowledge of the potential biohazards and of
the available safeguards.

In constructing these guidelines it has
been necessary to define boundary conditions
for the different levels of physical and bio-
logical containment and for the classes of
experiments to which they apply. We recog-
nize that these definitions do not take into
account existing and anticipated special pro-
cedures and information that will allow par-
ticular experiments to be carried out under
different conditions than indicated here
without sacrifice of safety. Indeed, we urge
that individual investigators devise simple
and more effective containment procedures
and that study sections give consideration
to such procedures which may allow change
in the containment levels recommended here.

It is recommended that all publications
dealing with recombinant DNA work include
e description of the physical and biological
containment procedures practiced, to aid and
forewarn others who might consider repeat-
ing the work.

II. CoNTAINMENT

Effective biological safety programs have
been operative in a variety of laboratories for
many years. Considerable Information there-
fore already exists for the design of physical
containment facilities and the selection of
laboratory procedures applicable to orga-
nisms carrying recombinant DNAs (4-17).
The existing programs rely upon mechanisms
that, for convenience, can be divided into
two categories: (1) A set of standard prac-
tices that are generally used in microbiolog-
ical laboratories, and (ii) special procedures,
equipment, and laboratory installations that
provide physical barriers which are applied
in varying degrees according to the estimated
biohazard.

Experiments on recombinant DNAs by
their very nature lend themselves to a third
containment mechanism—namely, the ap-
plication of highly specific biological barriers.
In fact, natural barriers do exist which either
limit the infectivity of a vector or vehicle

“NOTICES

(plasmid, bacteriophage or virus) to specific
hosts, or its dissemination and survival in
the environment. The vectors that provide
the means for replication of the recombinant
DNAs and/or the host cells in which they
replicate can be genetically designed to de-
crease by many orders of magnitude the
probability of dissemination of recombinant
DNAs outside the laboratory.

As these three means of containment are
complementary, different levels of contain-
ment appropriate for experiments with dif-
ferent recombinants can be established by
applying different combinations of the physi-
cal and biological barriers to a constant use
of the standard practices. We consider these
categories of containment separately here in
order that such combinations can be con-
veniently expressed in the guidelines for re-
search on the different kinds of recombinant
DNAs (Section III).

A. Standard practices and training. The
first principle of containment is a strict ad-
herence to good microbiological practices (4—
13). Consequently, all personnel directly or
indirectly involved in experiments on re-
combinant DNAs must receive adequate in-
struction. This should include at least train-
ing in aseptic techniques and instruction in
the biology of the organisms used in the ex-
periments so that the potential biohazards
can be understood and appreciated.

Any research group working with agents
with a known or potential biohazard should
have an emergency plan which describes the
procedures to be followed if an accident con-
taminates personnel or environment. The
principal investigator must ensure that
everyone in the laboratory is familiar with
both the potential hazards of the work and
the emergency pian. If a research group is
working with a known pathogen for which
an effective vaccine is available, all workers
should be immunized. Serological monitor-
ing, where appropriate, should be provided.

B. Physical containment levels. A variety
of combinations (levels) of special practices,
equipment, and laboratory installations that
provide additional physical barriers can be
formed. For example, 31 combinations are
listed in “Laboratory Safety at the Center for
Disease Control” (4); four levels are associ-
ated with the “Classification of Etiologic
Agents on the Basis of Hazard” (5), four
levels were recommended in the “Summary
Statement of the Asilomar Conference on
Recombinant DNA Molecules” (3); and the
National Cancer Institute uses three levels
for research on oncogenic viruses (6). We
emphasize that these are an aid to, and not a
substitute for, good technique. Personnel
must be competent in the effective use of all
equipment needed for the required contain-
ment level as described below. We define only
four levels of physical containment here,
both because the accuracy with which one
can presently assess the biohazards that may
result from recombinant DNAs does not war-
Tant a more detailed classification, and be-
cause additional flexibility can be obtained
by combination of the physical with the bio-
logical barriers. Though different in detail,
these four levels (P1<P2<P3<P4) approxl-
mate those given for human etiologic agents
by the Center for Disease Control (i.e., classes
1 through 4; ref. 5), in the Asilomar sum-
mary statement (1e., minimal, low moderate,
and high; ref. 3), and by the National Can-
cer Institute for oncogenic viruses (Le., low,
moderate, and high; ref. 6), as is indicated
by the P-number or adjective in the fol-
lowing headings. It should be emphasized
that the descriptions and assignments of
physical containment detafled below are
based on existing approaches to containment
of hazardous organisms.

We anticipate, and indeed already Know
of, procedures (14) which enhance physical

containment capability in novel ways. For
example, miniaturization of screening,
handling, and analytical procedures provides
substantial containment of a given host-
vector system. Thus, such procedures should
reduce the need for the standard types of
physical containment, and such innovations
will be considered by the Recombinant DNA
Molecule Program Advisory Committee.

The special practices, equipment and facil-
ity installations indicated for each level of
physical containment are required for the
bafety of laboratory workers, other persons,
and for the protection of the environment.
Optional items have been excluded; only
those items deemed absolutely necessary for
safety are presented. Thus, the listed require-
ments present basic safety criteria for each
level of physical containment. Other micro-
biological practices and laboratory tech-
niques which promote safety are to be en-
couraged. Additional information giving
further guidance on physical containment is
provided in a supplement to the guidelines
(Appendix D).

Pi Level (Minimal). A laboratory suitable
for experiments involving recombinant DNA
molecules requiring physical containment at
the P1 level is a laboratory that possesses
no special engineering design features. It is a
laboratory commonly used for microorga-
nisms of no or minimal biohazard under
ordinary conditions of handling. Work tn this
laboratory is generally conducted on open
bench tops. Special containment equipment
is neither required nor generally available in
this laboratory. The laboratory is not sepa-
rated from the general traffic patterns of the
building. Public access is permitted.

The control of biohazards at the Pl level
is provided by standard microbiological prac-
tices of which the following are examples: (1)
Laboratory doors should be kept closed while
experiments are in progress. (ii) Work sur-
faces should be decontaminated daily and
following spills of recombinant DNA mate-
rials, (111) Liquid wastes containing recom-
binant DNA materials should be decon-
taminated before disposal. (1v) Solid wastes
contaminated with recombinant DNA mate-
rials should be decontaminated or packaged
in a durable leak-proof container before pe-
moval from the laboratory. (v) Although
pipetting by mouth is permitted, it is prefer-
able that mechanical pipetting devices be
used. When pipetting by mouth, cotton-
plugged pipettes shall be employed. (vi) Eat-
ing, drinking, smoking, and storage of food
in the working area should be discouraged.
(vil) Facilities to wash hands should, be
available. (viil) An Insect and rodent con-
trol program should be provided. (ix) The
use of laboratory gowns, coats, or uniforms
is discretionary with the laboratory super-
visor.

P2 hevel (Low), A laboratory suitable for
experiments involving recombinant DNA
molecules requiring physical containment at
the P2 level is similar in construction and
design to the P1 laboratory, The P2 laboratory
must have access to an autoclave within the
building; 1t may have a Biological Safety
Cabinet.1 Work which does not produce a
considerable aerosol is conducted on the open
bench. Although this laboratory is not sepa-
rated from the general traffic patterns of the
building, access to the laboratory is limited
when experiments requiring P2 level physical
containment are being conducted. Experi-
ments of lesser biohazard potential can be
carried out concurrently in carefully demar-
cated areas of the same laboratory.

The P2 laboratory 1s commonly used for
experiments involving microorganisms of low
bichazard such as those which have been
classified by the Center for Disease Control
as Class 2 agents (5).

See footnotes on p. 38459.

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
~

The following practices shall apply to all
experiments requiring P2 level physical con-
tainment: (i) Laboratory doors shall be kept
closed while experiments are in progress.
(it) Only persons who have been advised
of the potential biohazard shall enter the
laboratory. (iit) Children under 12 years of
age shall not enter the laboratory. (iv) Work
surfaces shall be decontaminated daily and
immediately following spills of recombinant
DNA materials. (v) Liquid wastes of recom-
binant DNA materials shall be decontami-
nated before disposal. (vi) Solid wastes con-
taminated with recombinant DNA inaterials
shall be decontaminated or packaged in a
durable leak-proof container before removal
from the laboratory. Packaged materials shall
be disposed of by incineration or sterilized
before disposal by other methods. Contami-
nated materials that are to be processed
and reused (ie., glassware) shall be decon-
taminated before removal from the labora-
tory. (vii) Pipetting by mouth is prohibited;
mechanical pipetting devices shall be used.
(vili) Eating, drinking, smoking. and stor-
age of food are not permitted in the working
area, (ix) Facilities to wash hands shall be
available within the laboratory. Persons han-
dling recombinant DNA materials should be
encouraged to wash their hands frequently
and when they leave the laboratory. (x) An
insect and rodent control program shall be
provided. (xi) The use of laboratory gowns,
coats, or uniforms is required. Such cloth-
ing shall not be worn to the lunch room
or outside the building. (xii) Animals net
related to the experiment shall not be per-
mitted in the laboratory. (xiii) Biological
Safety Cabinets‘ and/or other physical con-
tainment equipment shall be used to mini-
mize the hazard of aerosolization of recom-
binant DNA materials from operations or
devices that produce a considerable aerosol
{e.g., blender, lyophilizer, sonicator, shaking
machine, etc.). (xiv) Use of the hypodermic
needie and syringe shall be avoided when
alternate methods are available.

P3 Level (Moderate). A laboratory suitable
tor experiments involving recombinant DNA
molecules requiring physical containment at
the P3 level has special engineering design
features and physical containment equip-
ment. The laboratory is separated from areas
which are open to the general public. Sep-
aration is generally achieved by controlled
access corridors, air locks, locker rooms or
other double-doored facilities which are not
available for use by the general public, Ac-
cess to the laboratory is controlled. Biological
Safety Cabinets! are available within the
controlled laboratory area. An autoclave shall
be available within the building and prefer-
ably within the controlled laboratory area.
The surfaces of walls, floors, bench tops,
and ceilings are easily cleanable to facilitate
housekeeping and space decontamination.

Directional air flow is provided within the
controlled laboratory area. The ventilation
system is balanced to provide for an inflow
of supply air from the access corridor into
the laboratory. The general exhaust air from
the laboratory Is discharged outdoors and so
dispersed to the atmosphere as to prevent
reentry into the building. No recirculation
of the exhaust air shall be permitted without
appropriate treatment.

No work in open vessels involving hosts
or vectors containing recombinant DNA
molecules requiring P3 physical containment
js conducted on the open bench. All such
procedures are confined to Biological Safety
Cabinets?

The following practices shall apply to all
experiments requiring P3 level physical con-
tainment: (1) The universal biohazard sign
is required on all laboratory access doors.
Only persons whose entry into the laboratory

“See footnotes on p. 38459.

NOTICES

is required on the basis of program or Sup-
port needs shall be authorized to enter. Such
persons shall be advised of the potential bio-
hazards before entry and they shall comply
with posted entry and exit procedures. Chil-
dren under 12 years of age shall not enter the
laboratory. (ii) Laboratory doors shall be kept
closed while experiments are in progress. (iit)
Biological Safety Cabinets! and other phys-
ical containment equipment shall be used
for all procedures that produce aerosols of re-
combinant DNA materials (e.g., pipetting,
plating, flaming, transfer operations, grind-
ing, blending, drying, sonicating, shaking,
ete.). (iv) The work surfaces of Biological
Safety Cabinets? and other equipment shall
be decontaminated following the completion
of the experimental activity contained with-
in them. (v) Liquid wastes containing re-
combinant DNA matertiais shall be decontam-
inated before disposal. Solid wastes con-
taminated with recombinant DNA materials
shall be decontaminated or packaged in a
durable leak-proof container before removal
from the laboratory. Packaged material shall
‘be sterilized before disposal. Contaminated
materials that are to be processed and re-
used (Le., glassware) shall be sterilized in
the controlled laboratory area or placed in a
durable leak-proof container before removal
from the controlled laboratory area. This
container shall be sterilized before the ma-
terials are processed, (vii) Pipetting by mouth
is prohibited; mechanical pipetting devices
shall be used, (viii) Eating, drinking, smok-
ing, and storage of food are not permitted
in the laboratory. (ix) Facilities to wash
hands shall be available within the labora-
tory. Persons shall wash hands after experi-
ments involving recombinant DNA materials
and before leaving the laboratory. (x) An in-
sect and rodent control program shall be pro-
vided. (xi) Laboratory clothing that protects
street clothing (1e., long sleeve solid-front
or wrap-around gowns, no-button or slipover
jackets, ete.) shall be worn in the laboratory.
FRONT-BUTTON LABORATORY COATS
ARE UNSUITABLE. Gloves shall be worn
when handling recombinant DNA materials.
Provision for laboratory shoes is recom-
mended. Laboratory clothing shall not be
worn outside the laboratory and shall be de-
contaminated before it is sent to the laundry.
(xii) Raincoats, overcoats, topcoats, coats,
hats, caps, and such street outerwear shall
not be kept in the laboratory. (xfil) Animals
and plants not related to the experiment
shall not be permitted in the laboratory. (xiv)
Vacuum lines shall be protected by filters and
liquid traps. (xv) Use of the hypodermic
needle and syringe shall be avoided when
alternate methods are available. (xvi) If ex-
periments of lesser biohazard potential are to
pe conducted in the same laboratory con-
currently with experiments requiring P3 level
physical containment they shall be con-
ducted only in accordance with all P3 level
requirements, (xvii) Experiments requiring
P3 level physical containment can be con-
ducted in laboratories where the directional
air flow and general exhaust air conditions
described above cannot be achieved, provided
that this work is conducted In accordance
with all other requirements listed and is
contained in a Biological Safety Cabinet1
with attached glove ports and gloves. All ma-
terials before removal from the Biological
Safety Cabinet‘ shall be sterilized or trans-
ferred to a non-breakable, sealed container,
which is then removed from the cabinet
through a chemical decontamination tank,
autoclave, ultraviolet air lock, or after the
entire cabinet has been decontaminated.

P4 Level High. Experiments involving re-
combinant DNA molecules requiring physical
containment at the P4 level shall be con-
fined to work areas in a facility of the type
designed to contain microorganisms that are

38453

extremely hazardous to man or may cause
serious epidemic disease, The facility is either
a separate building or it is a controlled area,
within a building, which is completely iso-
lated from all other areas of the building.
Access to the facility is under strict control.
A specific facility operations manual is avail-
able, Class III Biological Safety Cabinets!
are available within work areas of the facility.

A P4 facility has engineering features
which are designed to prevent the escape of
microorganisms to the environment (14, 15,
16, 17}, These features include: (1) Mono-
lithic walls, floors, and ceilings in which all
penetrations such as for air ducts, electrical
conduits, and utility pipes are sealed to as-
sure the physical isolation of the work area
and to facilitate housekeeping and space
decontamination; (ii) air locks through
which supplies and materials can be brought
safely into the facility; (iti) contiguous
clothing change and shower rooms through
which personnel enter into and exit from
the facility; (iv) double-door autoclaves to
sterilize and safely remove wastes and other
materials from the facility; (v) a biowaste
treatment system to sterilize liquid effluents
if facility drains are installed; (vi) a separate
ventilation system which maintains nega-
tive air pressures and directional air flow
within the facility; and (vii) a treatment
system to decontaminate exhaust air before
it is dispersed to the atmosphere. A central
yacuum utility system is not encouraged; if
one is installed, each branch line leading to a
laboratory shall be protected by a high effi-
ciency particulate air filter.

The following practices shall apply io all
experiments requiring P4 level physical con-
tainment: (i) The universal biohazard sign
is required on all facility access doors and
all interior doors to individual laboratory
rooms where experiments are conducted.
Only persons whose entry into the facility or
individual laboratory rooms is required on
the basis of program or support needs shail
be authorized to enter. Such persons shall
be advised of the potential biohazards and
instructed as to the appropriate safeguards
to ensure their safety before entry. Such per-
sang shall comply with the instructions and
all other posted entry and exit procedures.
Under no condition shall children under 15
years of age be allowed entry. (if) Personnel
shall enter into and exit from the facility
only through the clothing change and show-
er rooms. Personnel shall shower at each
exit from the facility. The air locks shall not
he used for personnel entry or exit except
for emergencies. (1i1) Street clothing shall be
removed in the outer facility side of the
clothing change area and kept there. Com-
plete laboratory clothing including under-
garments, pants and shirts or Jumpsuits,
shoes, head cover, and gloves shall be pro-
vided and used by all persons who enter into
the facility shall be placed in an entry air
pe stored in lockers provided for this pur-
pose or discarded into collection hampers
pefore personnel enter into the shower area.
(iv) Supplies and materials to be taken into
the facility shall be placed in an entry air
lock. After the outer door (opening to the
corridor outside of facility) has been secured,
personnel occupying the facility shall re-
trieve the supplies and materials by opening
the interior air Jock door. This door shall be
secured after supplies and materials are
prought into the facility. (v) Doors to labora-
tory rooms within the facility shall be kept
closed while experiments are in progress. (vi)
Experimental procedures requiring P4 level
physical containment shall be confined to
Class III Biological Safety Cabinets.’ All ma-
terials, before removal from these cabinets,
shall be sterilized or transferred to @ non-
breakable sealed container, which is then re=
moved from the system through a chemical
decontaminated tank, autoclave, or after the

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
38st

entire system has been decontaminated.
(vli) No materials shall be removed from the
facility unless they have been sterilized or
decontaminated in a manner to prevent the
release of agents requiring P4 physical con-
tainment. All wastes and other materials and
equipment not damaged by high tempera-
ture or steam shall be sterilized in the dou-~
ble-door autoclave. Biological materials to be
removed from the facility shall be transferred
to a non-breakable sealed container which
is then removed from the facility through a
chemical decontamination tank cr a cham-
ber designed for gas sterilization. Othey ma-
terials which may be damaged by temipera-
ture or steam shall be sterilized by gaseous
or vapor methods in an air lock or chamber
designed for this purpose. (viii) Eating,
drinking, smoking, and storage of food are
not permitted in the facility. Foot-operated
water fountains located in the facility cor-
ridors are permitted. Separate potable water
piping shall be provided for these water
fountains. (ix) Facilities to wash hands shall
be available within the facility. Persons shall
wash hands after experiments. (x) An insect
and rodent control program shall be provid-
ed. (xi) Animals and plants not related to
the experiment shall not be permitted in the
facility. (xil) If a central vacuum system is
provided, each vacuum outiet shall be pro-
tected by a filter and liquid trap in addition
to.the branch line HEPA filter mentioned
above. (xitl) Use of the hypodermic needle
and syringe shall be avoided when alternate
methods are available. (xiv) If experiments
of lesser biohazard potential are to be con-
ducted in the facility concurrently with ex-
periments requiring P4 level containment,
they shall be confined in Class I or Class II
Biological Safety Cabinets! or isolated by
other physical containment equipment.
Work surfaces of Biological Safety Cabinets +
and other equipment shall be decontami-

nated following the completion of the ex--

perimental activity contained within them.
Mechanical pipetting devices shall be used.
All other practices listed above with the ex-
ception of (vi) shall apply.

C. Shipment. To protect product, per-
sonnel, and the environment, all recom-
binant DNA material will be shipped in con-
tainers that meet the requirements issued by
the U.S. Public Health Service (Section 72.25
of Part 72, Title 42, Code of Federal Regula-
tions), Department of Transportation (Sec-
tion 173.387 (b) of Part 173, Title 49, Code of
Federal Regulations) and the Civil Aero-
nautics Board (C.A.B. No. 82, Official Air
Transport Restricted Articles Tariff No. 6—-D)
for shipment of etiologic agents. Labeling re-
quirements specified in these Federal regu-
lations and tariffs will apply to all viable re-
combinant DNA materials in which any por-
tion of the material is derived from an etio-
logic agent listed in paragraph (c) of 42 CFR
72.25, Additional information on packing and
shipping is given in a supplement to the
guidelines (Appendix D, part X).

D. Biological containment levels. Biologi-
cal barriers are specific to each host-vector
system. Hence the criteria for this mecha-
nism of containment cannot be generalized
to the same extent as for physical contain-
ment. This is particularly true at the present
time when our experience with existing host-
vector systems and our predictive knowledge
about projected systems are sparse. The clas-
sification of experiments with recombinant
DNAs that is necessary for the construc-
tion of the experimental guidelines (Sec-
tion III) can be accomplished with least con-
fusion if we use the host-vector system as the
primary element and the source of the in-
serted DNA as the’secondary element in the
classification. It is therefore convenient to.
specify the nature of the biological contain-~
ment under host-vector headings such as

those given below for Escherichia coli K-12.

NOTICES

TID. ExrerRtmENTAL GUIDELINES

A general rule that, though obvious, de-
serves statement is that the level of contain-
ment required for any experiment on DNA
recombinants shall never be less than that
required for the most hazardous component
used to construct and clone the recombinant
DNA (Le. vector, host, and inserted DNA). In
most cases the level of containment will be
greater, particularly when the recombinant
DNA is formed from species that ordinarily
do not exchange genetic information. Han-
dling the purified DNA will generally require
less stringent precautions than will propa-
gating the DNA. However, the DNA itself
should be handled at least as carefully as one
would handle the most dangerous of the
DNAs used to make it.

The above rule by itself effectively pre-
cludes certain experiments—namely, those in
which one of the components fs in Class 5
of the “Classification of Etiologic Agents on
the Basis of Hazard” (5), as these are ex-
cluded from the United States by law and
USDA administrative-policy. There are ad-
ditional experiments which may engender
such serious biohazards that they are not
to be performed at this time. These are
considered prior to presentation of the con-~
tainment guidelines for permissible ex-
periments.

A. Experiments that are not to be per-
formed, We recognize that it can be argued
that certain of the recombinants placed in
this category could be adequately contained
at this time. Nonetheless, our estimates of
the possible dangers that may ensue if that
containment fails are of such magnitude
that we consider it the wisest policy to at
least defer experiments on these recombi-
nant DNAs until there is more information to
accurately assess that danger and to allow
the construction of more effective biological
barriers. In this respect, these guidelines are
more stringent than those initially recom-
mended (1).

The following experiments are not to be
initiated at the present time: (1) Cloning of
recombinant DNAs derived from the patho-
genic organisms in Classes 8, 4, and 5 of
“Classification of Etiologic Agents on the
Basis of Hazard” (5), or oncogenic viruses
classified by NCI as moderate risk (6), or cells
known to be infected with such agents, re-
gardless of the host-vector system used. (il)
Deliberate formation of recombinant DNAs
containing genes for the biosynthesis of po-
tent toxins (e.g., botulinum or diphtheria
toxins; venoms from insects, snakes, etc.).
(ili) Deliberate creation from plant patho-
gens of recombinant DNAs that are likely to
increase virulence and host range. (iv) Delib-
erate release Into the environment of any
organism containing a recombinant DNA
molecule. (v) Transfer of a drug resistance
tralt to microorganisms that are not known
to acquire it naturally if such acquisition
could compromise the use of a drug to con-
trol disease agents In human or veterinary
medicine or agriculture.

In addition, at this time large-scale ex-
periments (e¢.g., more than 10 liters of cul-
ture) with recombinant DNAs known to
make harmful products are not to be carried
out. We differentiate between smali- and
large-scale experiemnts with such DNAs be-
cause the probability of escape from contain-
ment barriers normally increases with in-
creasing scale. However, specific experiments
in this category that are of direct societal
benefit may be excepted from this rule if
special biological containment precautions
and equipment designed for large-scale op-
erations are used, and provided that these
experiments are expressly approved by the
Recombinant DNA Molecule Program Ad-
visory Committee of NIH.

B. Containment guidelines for permissible
experiments. It ts antictpated that most re-

combinant DNA experiments initiated before
these guidelines are next reviewed (Le., with-
in the year) will employ £. coli K-12 host-
vector ssytems. These are also the systems for
which we have the most experience and
knowledge regarding the effectiveness of the
containment provided by existing hosts and
vectors necessary for the construction of
more effective biological barriers.

For these reasons, FE. coli K-12 appears to
be the system of choice at this time, although
we have carefully considered arguments that
many of the potential dangers are com-
pounded by using an organism as intimately
connected with a man as is E. coli. Thus,
while proceeding cautiously with E. coli,
serious efforts should be made toward devel-
oping alternate host-vector systems; this suh-
ject is discussed in considerable detail in Ap-
pendix A,

We therefore consider DNA recombinants
in E. coli K-12 before proceeding to other
host-vector systems.

1. Biological containment criteria using E.
eolt K-12 host-vectors EK1 host-vectors.
These are host-vector systems that can be
estimated to already provide a moderate level
of containment, and include most of the
presently available systems. The host is al-
ways £. coli K-12, and the vectors include
nonconjugative plasmids [e.g., pSC101, ColE1
or derivatives thereof (19-26)] and variants
of bacteriophage 4 (27-29).

The £. coli K-12 nonconjugative plasmid
system is taken as an example to tlustrate
the approximate level of containment refer-
red to here. The availatde data from experi-
ments involving the feeding of bacteria to
humans and calves (30-32) indicate that EF.
colt K-12 did not usually colonize the normal
bowel, and exhibited little, if any, multiplica-
tion while passing through the alimentary
tract even after feeding high doses (i.e., 10°
to 10 bacteria per human or calf). However,
general extrapolation of these results may not
be warranted because the implantation of
bacteria into the intestinal tract depends on
a@ number of parameters, such as the nature
of the intestinal flora persent in a given in-
dividual and the physiological state of the
inoculum, Moreover, since viable E. coli K-12
can be found in the feces after humans are
fed 10' bacteria in broth (30) or 3x10 bac-
terla protected by suspension in milk (31),
transductional and conjugational transfer of
the plasmid vectors from EF. coli K-12 to resi-
dent bacteria in the fecal matter before and
after excretion must also be considered.

The nonconjugative plasmid vectors can-
not promote their own transfer, but require
the presence of a conjugative plasmid for
mobilization and transfer to other bacteria.
When present in the same cell with dere-
pressed conjugative plasmids such as F or
Rldrdi9, the nonconJugative ColE1, ColE1-
trp and pSC101 plasmids are transferred to
suitable recipient strains under ideal labora-
tory conditions at frequencies of about 0.5,
10-4 to 10-5, and 10-* per donor cell, respec-
tively. These frequencies are reduced by
another factor of 10? to 104 if the conjugative
Rlasmid employed is repressed with respect to
expression of donor fertility.

The experimental transfer system which
most closely resembles nonconjugative plas-
mid transfer in nature is a triparental mat-
ing. In such matings, the bacterial cell pos-
sessing the nonconjugative plasmid must
first acquire a conjugative plasmid from
another cell before it can transfer the non-
conjugative plasmid to a secondary recipient.
With ColE1, the frequencies of transfer are
10— and 10-+ to 105 when using conjugative
plasmid donors possessing derepressed and
repressed plasmids, respectively. Mobilization
of ColEl-trp and pSC101 under similar lab-
oratory conditions is so low as to be usually
undetectable (33). Since most conjugative
plasmids in nature are repressed for expres-

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1975
sion of donor fertility, the frequency at which
monconjugative plasmids are mobilized and
transferred by this sequence of events in
vivo is difficult to estimate. However, in calves
fed on an antibiotic-supplemented diet, it
has been estimated that such triparental
nonconjugative R plasinid transfer occurs at
frequencies of no more than 10-% to 10—#
per 24 hours per calf (32). In terms of con-
sidering other means for plasmid transmis-
sion in nature, it should be noted that trans-
duction does operate in vivo for Staphylococ-
cus aureus (34) and probably for EF. coli as
well. However, no data are available to indi-
cate the frequencies of plasmid transfer in
vivo by either transduction or transforma-
tion.

Thesé observations indicate the low prob-
abilities for possible dissemination of such
plasmid vectors by accidental ingestion,
which would probably involve only a few
hundred or thousand bacteria provided that
at least the standard practices (Section II-A
above) are followed, particularly the avoid-
ance of mouth pipetting. The possibility of
colonization and hence of transfer are in-
creased, however, if the normal flora in the
bowel is disrupted by, for example, anti-
biotic therapy (35). For this reason, persons
recelving such therapy must not work with
DNA recombinants formed with any E. coli
K-12 host-vector system during the therapy
period and for seven days thereafter; simi-
larly, persons who have achlorhydria or who
have had surgical removal of part of the
stomach or bowel should avoid such work,
as should those who require large doses of
antacids.

The observations on the fate of E. coli
K-12 in the human alimentary tract are also
relevant to the containment of recombinant
DNA formed with bacteriophage \ variants.
Bacteriophage can escape from the labora-
tory either as mature infectious phage par-
ticles or in bacterial host cells in which the
phage genome is carried as a plasmid or
prophage. The fate of FE. coli K-12 host cells
carrying the phage genome as a plasmid or
prophage is similar to that for plasmid-con-
taining host cells as discussed above. The
survival of the \ phage genome when released
as infectious particles depends on their sta-
bility in nature, their infectivity and on the
probability of subsequent encounters with
naturally occurring \-sensitive E. coli strains.
Although the probability of survival of )
and its infection of resident intestinal E. coli
in animals and humans has not been meas-
ured, it 1s estimated to be small given the
high sensitivity of , to the low pH of the
stomach, the insusceptibility to , infection
of smooth £. coli cells (the type that nor-
mally resides in the gut), the infrequency of
naturally occurring ,-Sensitive E. coli (36)
and the failure to detect infective , particles
jn human feces after ingestion of up to 10"
X Particles (37).

Moreover, \ particles are very sensitive to
desiccation.

Establishment of ) as a stable lysogen is a
frequent event (10° to 10-1) for the att* int+
cI*+ phage so that this mode of escape would
be the preponderant laboratory hazard; how-
ever, most EK1 ) vectors currently in use
lack the att and int functions (27-29) thus
reducing the probability of lysogenization to
abcut 10 to 10-* (38-40). The frequency for
the conversion of , to a plasmid state for
persistence and replication is also only about
10°* (41). Moreover, the routine treatment of
phage lysates with chloroform (42) should
eliminate all surviving bacteria including
lysogens and ) plasmid carriers. Lysogeniza-
tion could also occur when an infectious
contalning cloned DNA infects a )-sensitive

cell in nature, and recombines with a resident

FEDERAL REGISTER, VOL. 41, NO. 176-——THURSDAY, SEPTEMBER 9,

NOTICES

lambdoid prophage. Although )-sensitive EF.
coli straing seem to be rare, a significant
fraction do carry lambdoid prophages (43—
44) and thus this route of escape should be
considered.

While not exact, the estimates for con-
tainment afforded by using these host-vec-
tors are at least as accurate as those for
physical containment, and are sufficient to
indicate that currently employed plasmid
and ) vector systems provide a moderate level
of biological containment. Other noncon-
jugative plasmids and bacteriophages that,
in association with £. coli K-12 can be esti-
mated to provide the same approximate level
of moderate containment are included in the
EK1 class.

EK2 host-vectors. These are host-vector
systems that have been genetically con-
structed and shown to provide a high level of
biological containment as demonstrated by
data from suitable tests performed in the
laboratory. The genetic modifications of the
E. colt K-12 host and/or the plasmid or
phage vector should not permit survival of a
genetic marker carried on the vector, prefer-
ably a marker within an inserted DNA frag-
ment, in other than specially designed and
carefully regulated laboratory environments
at a frequency greater than 10-%. This meas~
ure of biological containment has been se-
lected because it is a measurable entity. In-
deed, by testing the contributions of pre-
existing and newly introduced genetic prop-
erties of vectors and hosts, individually or in
various combinations, it should be possible
to estimate with considerable precision, that
the specially designed host-vector system can
provide a margin of biological containment
in excess of that required. For the time being,
no host-vector system will be considered to
be a bona fide EK2 host-vector system until
it is so certified by the NIH Recombinant
DNA Molecule Program Advisory Committee.

For EK2 host-vector systems in which the
vector is a plasmid, no more than one in 10
host cells should be able to perpetuate the
vector and/or a cloned DNA fragment under
non-permissive conditions designed to repre-
sent the natural environment either by sur-
vival of the original host or as a consequence
of transmission of the vector and/or a cloned
DNA fragment by transformation, transduc-
tion or conjugation to a host with properties
common to those in the natural environment.

In terms of potential EK2 plasmid-host
systems, the following types of genetic modi-
fications should reduce survival of cloned
DNA. The examples given are for illustrative
purposes and should not be contrued to en-
compass all possibilities. The presence of the
non-conjugative plasmids ColEl-trp and
pS101 in an £. coli. K-12 strain possessing
& mutation eliminating host-controlled re-
striction and modification (hsdS) results tn
about 10°-fold reduction in mobilization to
restriction-proficient recipients. The com-
bination of the dapD8, AbioH-asd, Agal-chi*
and rfb mutations in Z. coli K-12 results in
no detectable survivors in feces of rats follow-
ing feeding by stomach tube of 10° celis in
milk and similarly leads to complete lysis of
cells suspended in broth medium lacking
diaminopimelic acid. E. coli K-12 strains with
AthyA and deoC(dra) mutations undergo
thymineless death in growth medium lack-
ing thymine and give a 10°-fold reduced sur-
vival during passage through the rat intes-
tine compared to wild-type thy* FE. coli K-12.
(However, the AthyA mutation alone or in
combination with a deoB(drm) mutation
only reduces in vivo survival by a factor of
102.) Other host mutations, as yet untested,
that might further reduce survival of the
plasmid-host system or reduce plasmid trans-
mission are: the combination polA(TS)
recA(TS) AthyA which might interfere with
ColE! replication and lead to DNA degrada-

381355

tion at body temperatures; Con mutations
that reduce the ability of conjugative
plasmids to enter the plasmid-host complex
and thus should reduce mobilization of the
cloned DNA to other strains; and mutations
that confer resistance to known transducing
phages Mutations can also be introduced into
the plasmid to cause it to be dependent on
& specific host, to make its replication
thermosensitive and/or to endow it with a
killer capability such that all cells (other
than its host) into which it might be trans-
ferred will not survive.

In the construction of EK2 plasmid-host
systems it is Important to use the most
stable mutations available, preferably dele-
tions. Obviously, the presence of all muta-
tions contributing to higher degrees of bio-
logical containment must be verified pe-
riodically by appropriate tests. In testing the
level of biological containment afforded by a
proposed EK2 plasmid-host system, it is im-
portant to design relevant tests to evaluate
the survival of the vector and/or a cloned
DNA fragment under conditions that are pos-
sible in nature and that are also most advan-
tageous for its perpetuation. For example, one
might conduct a triparental mating with a
primary donor possessing a redepressed F-
type or I-type conjugative plasmid, the safer
host with AbioH-asd, dapD8, Agal-chl', rfb,
AthyA, deoc, trp and hsdS mutations and a
plasmid vector carrying an easily detectable
marker such as for ampicillin resistance or an
inserted gene such as trp*, and a secondary
recipient that is Su- hsdS trp (i.e., permissive
for the recombinant plasmid). Such matings
would be conducted in a medium lacking dia-
mino pimelic acid and thymine and surviva)
of the Ap’ or trp marker in any of the three
strains followed as a function of time. Sur-
vival of the vector and/or a cloned marker »\
transduction could also be evaluated by in-
troducing a known generalized transducing
phage into the system. Similar experiments
should also be done using a secondary recipi-
ent that is restrictive for the plasmid vector
as well as with primary donors possessing re-
pressed conjugative plasmids with incom-
patibility group properties like those com-
monly found in enteric microorganisms.
Since a common route of escape of plasmid-
host systems in the laboratory might be by
accidental ingestion, it is suggested that the
same types of experiments be conducted in
sultable animal-model systems. In addition
to these tests on survival of the vector and;
or a cloned DNA fragment, it would be useful
to determine the survival of the host strain
under nongrowth conditions such as in water
and as a function of drying time after a
culture has been spilled on a lab bench.

For EK2 host-vector systems in which the
vector is a phage, no more than one in 10
phage particles should be able to perpetuate
itself and/or a cloned DNA fragment under
non-permissive conditions designed to repre-
sent the natural environment either (a) as
&@ prophage or plasmid in the laboratory host
used for phage propagation or (b) by surviv-
ing in natural environments and transferring
itself and/or a cloned DNA fragment to a
host (or its resident lambdoid prophage)
with properties common to those in the na-
tural environment.

In terms of potential EK2 \-host systems,
the following types of genetic modification
should reduce survival of cloned DNA, The
examples given are for illustrative purposes
and should not be construed to encompass all
possibilities. The probability of establishing
AX lysogeny in the normal laboratory host
should be reduced by removal of the phage
att site, fhe Int function, the repressor
gene(s) and adding virulence-enhancing
mutations. The frequency of plasmid forma-
tion, although normally already less than
10-*, could be further reduced by defecte 15

1976
38456

the Pr-Q region, including mutations such
as vir-s, cro(TS), c17, rit, O(TS}, P(TS), and
nin. Moreover, chloroform treatment used
routinely following cell lysis would reduce
the number of surviving cells, including pos-
sible lysogens or plasmid carriers, by more
than 10%. The host may also be modified by
deletion of the host Aatt site and inclusion
of one or more of the mutations described
above for plasmid-host systems to further
reduce the chance of formation and survival
of any lysogen or plasmid carrier cell.

The survival of escaping phage and the
chance of encountering a sensitive host in
nature are very low, as discussed for EK1 sys-
tems. The infectivity of the phage particles
could be further reduced by introducing mu-
tations (eg., suppressed ambers) which
would make the phage particles extremely
unstable except under special laboratory con-
ditions (e.g., high concentrations of salts or

putrescine). Another means would be to

make the phage itself a two-component sys-
tem, by eliminating the tail genes and re~
producing the phage as heads packed with
DNA; when necessary and under specially
controlled conditions, these heads could be
made infective by adding tail preparations.
An additional safety factor in this regimen
is the extreme instability of the heads, unless
they are stored in 10mM putrescine, a con-
dition easy to obtain in the laboratory but
not in nature. The propagation of the escap-
ing phage in nature could further be blocked
by adding various conditional mutations
which would permit growth only under spe-
cial laboratory conditions or in a special
permissive laboratory host with suppressor or
gro-type (mop, dnaB, rpoB) mutations. An
additional safety feature would be the use
of an r- m- (ksdS) laboratory host, which
produces phage with unmodified DNA which
should be restricted in r+ m+ bacteria that
are probably prevalent in nature. The likelt-
hood of recombination between the » vector
and lambdoid prophages which are present
in some E, coli strains might be reduced by
elimination of the Red function and the pres-
ence of the recombination-reducing Gam
function together with mutations contribut-
ing to the high lethality of the A phage.
However, these second-order precautions
might not be relevant if the stability and
infectivity of the escaping A particles are
reduced by special mutations or by propa-
gating the highly unstable heads.

Despite multiple mutations in the phage
vectors and laboratory hosts, the yield of
phage particles under suitable laboratory
conditions should be high (10° —10" parti-
cles/ml). This permits phage propagation
in relatively small volumes and constitutes
an additional safety feature.

The phenotypes and genetic stabilities of
the mutations and chromosome alterations
included in these \-host systems indicate that
containment well in excess of the required
10-* or lower survival frequency for the A
vector with or without a cloned DNA frag-
ment should be attained. Obviously the pres-
. ence of all mutations contributing to this
high degree of biological containment must
be verified periodically by appropriate tests.
Laboratory tests should be performed with
the bacterial host to measure all possible
routes of escape such as the frequency of
lysogen formation, the frequency of plasmid
formation and the survival of the lysogen or
carrier bacterium, Similarly, the potential for
perpetuation of a cloned DNA fragment car-
ried by infectious phage particles can be
tested by challenging typical wild-type £Z. coli
strains or 8 A-sensitive nonpermissive labora-
tory K-12 strain, especially one lysogenic for
a lambdoid phage.

In view of the fact that accurate assess-

ment of the probabilities for escape of in-

FEDERAL REGISTER, VOL. 41,

NOTICES

fectious \} grown on r- m~ Su: hosts is de-
pendent upon the frequencies of r-, Su+, and
A-sensitive strains in nature, investigators
need to screen £. coli strains for these prop-
erties. These data will also be useful in pre-
dicting frequencies of successful escape of
plasmid cloning vectors harbored in r- m- Su:
strains.

When any investigator has obtained data
on the level of containment provided by a
proposed EK2 system, these should be re-
ported as rapidly as possible to permit gen-
eral awareness and evaluation of the safety
features of the new system. Investigators are
also encouraged to make such new safer clon-
ing systems generally available to other scien-
tists. NIH will take appropriate steps to aid
in the distribution of these safer vectors and

-hosts.

EK3 host-vectors. These are EK2 systems
for which the specified containment shown
by laboratory tests has been independently
confirmed by appropriate tests in animals, in-
cluding humans or primates, and in other
relevant environments in order to provide
additional data to validate the levels of con-
tainment afforded by the EK2 host-vector
systems, Evaluation of the effects of indi-
vidual or combinations of mutations con-
tributing to the biological containment
should be performed as a means to confirm
the degree of safety provided and to further
advance the technology of developing even
safer vectors and hosts. For the time being,
no host-vector system will be considered to
be & bona fide EK3 host-vector system, until
it is so certified by the NIH Recombinant
DNA Molecule Program Advisory Committee.

2. Classification of experiments using the
E. coli K-2 containment systems. In the fol-
lowing classification of containment criteria
for different kinds of recombinant DNAs, the
stated levels of physical and biological con-
tainment are minimums. Higher levels of
biological containment (EK3>EK2>EK1) are
to be used if they are available and are
equally appropriate for the purposes of the
experiment.

<a>Shotgun experiments. These experi-
ments involve the production of recombinant
DNAs between the vector and the total DNA
or (preferably) any partially purified fraction
thereof from the specified cellular source.

(1) Eukaryotic DNA recombinants—Pri-
mates. P3 physical containment+an EK3
host-vector, or P4 physical containment+an
EK2 host-vector, except for DNA from un-
contaminated embryonic tissue or primary
tissue cultures therefrom, and germ-line cells
for which P3 physical containment-++an EK2
host-vector can be used. The basis for the
lower estimated hazard in the case of DNA
from the latter tissues (if freed of adult
tissue) is their relative freedom from. hori-
zontally acquired adventitious viruses.

Other mammals. P3 physical containment
-+-an EK2 host-vector.

Birds. P3 physical containment!an EK2
host-vector.

Cold-blooded vertebrates. P2 physical con-
tainmenttan EK2 host-vector except for
embryonic or germ-line DNA which require
P2 physical containment+an EK1 host-
vector, If the eukaryote is known to produce
a potent toxin, the containment shall be
increased to P3+EkK2.

Other cold-blooded animals and lower
eukaryotes. This large class of eukaryotes 1s
divided into the following two groups:

{1) Species that are known to produce a
potent toxin or are known pathogens (l.e.,
an agent Usted in Class 2 of ref. § or a plant
pathogen) or are known to carry such patho-
genic agents must use P3 physical contain-
ment+an EK2 host-vector. Any species that
has a demonstrated capacity for carrying par-
ticular pathogenic agents Js included in this

group unless it has been shown that those
organisms used as the source of DNA do not
contain these agents; in this case they may
be placed in the second group,

{2) The remainder of the species in this
class can use P2--EK1. However, any insect
in this group should have been grown under
laboratory conditions for at least 10 genera-
tions prior to its use as a source of DNA.

Plants. P2 physical containment-+an EK1
host-vector. If the plant carries a known
pathogenic agent or makes a product known
to be dangerous to any species, the contain-
ment must be raised to P3 physical contain-
ment-+-an EK2 host-vector.

(il) Prokaryotes DNA
Prokaryotes that exchange genetic
matio with E. coli?.

The level of physical containment is di-
rectly determined by the rule of the most
dangerous component (see introduction to
Section III). Thus Pl conditions can be used
for DNAs from those bacteria in Class 1 of
ref. 5. (‘Agents of no or minimal haz-
ard * * *’) which naturally exchange genes
with KE. coli; and P2 conditions should be
used for such bacteria if they fall in Class
2 of ref. 5 (“Agents of ordinary potential
hazard * * *"), or plant pathogens or sym-
bionts. EK1 host-vectors can be used for
all experiments requiring only Pi physical
containment; in fact, experiments in this
category can be performed with E. coli K-12
vectors exhibiting a lesser contaiment (e.z.,
conjugative plasmids) than EK1 vectors. Ex-
periments with DNA from species requiring
P2 physical containment which are of low
pathogenicity (for example, enteropathogenic
Escherichia coli, Salmonella typhimurium,
and Klebsiella pneumoniae) can use EKi
host-vectors, but those of moderate path-
ogenicity (for example, Salmonella typhi,
Shigella dysenteriae type I, and Vibrio
cholerae) must use EK2 host-vectors? A
specific example of an experiment with a
plant pathogen requiring P2 physical con-
tainment--an EK2 host-vector would be
cloning the tumor gene of Agrobacterium
tumefaciens.

Prokaryotes that do not exchange genetic
information with E. coli. The minimum con-
tainment conditions for this class consist
of P2 physical containment +an EK2 host-
vector or P3 physical containment--an EK1
host-vector, and apply when the risk that the
recombinant DNAs will increase the path-
ogenicity or ecological potential of the host
is fudged to be minimal. Experiments with
DNAs from pathogenic species (Class 2 ref.
5 plus plant pathogens) must use P3-- EK2.

(ili) Characterized clones of DNA recombi-
nants derived from shotgun experiments.
When a cloned DNA recombinant has been
rigorously characterized ‘* and there ts suffi-
cient evidence that it is free of harmful
genes,+ then experiments involving this re-
combinant DNA can be carried out under
P1+EK1 conditions if the inserted DNA is
from a species that exchanges genes with
&, coli, and under P2+EK1 conditions if not.

<b> Purified cellular DNAs other than
plasmids, bacteriophages, and other viruses.
The formation of DNA recombinants from
cellular DNAs that have been enriched® by
physical and chemical techniques (Le., not
by cloning) and which are free of harmful
genes can be carried out under lower con-
tainment conditions than used for the cor-
responding shotgun experiment. In general,
the containment can be decreased one step
in physical containment (P4>P3+P2>P1)
while maintaining the biological contain-
ment specified for the shotgun experiment,
or one step in biological containment (EK3~>
EK2~>EK1) while maintaining the specified
physical containment—provided that the

recombinants—
infor-

See footnotes on p. 38459.

NO, 176—-THURSDAY, SEPTEMBER 9, 1976
new condition is not less than that specified
above for characterized clones from shotgun
experiments (Section <a> —4Hli). .
<c> Plasmids, bacteriophages, and other
viruses. Recombinants formed between EK-
type vectors and other plasmid or virus DNAs
have in common the potential for acting as
double vectors because of the replication
functions in these DNAs. The containment
conditions given below apply only to propa-
gation of the DNA recombinants in £. coli K-
12 hosts. They do not apply to other hosts
where they may be able to replicate ag a re-
sult of functions provided by the DNA in-
serted into the EK vectors. These are con-
6idered under other host-vector systems.

(1) Animal viruses. P4+EK2 or P3+EK3
shall be used to isolate DNA recombinants
that include all or part of the genome of an
animal virus. This recommendation applies
not only to experiments of the “shotgun”
type but also to those involving partially
characterized subgenomic segments of. viral
DNAs (for example, the genome of defective
viruses, DNA fragments isolated after treat-
ment of viral genomes with restriction
enzymes, etc). When cloned recombinants
have been shown by suitable biochemical and
biological tests to be free of harmful regions,
they can be handled in -P3+EK2 conditions.
In the case of DNA viruses, harmless regions
Include the late region of the genome; in
the case of DNA copies of RNA viruses, they
might include the genes coding for capsid
proteins or envelope proteins.

(il) Plant viruses. P3+EK1 or P2+EK2
conditions shall be used to form DNA re-
combinants that include all or part of the
genome of a plant virus.

(iil) Eukaryotic organelle DNAs. The con-
tainment conditions given below apply only
when the organelle DNA has been purified *
from isolated organelles. Mitochondrial DNA
from primates: P3+-EK1 or P2-+-EK2. Mito-
chondrial or chloroplast DNA from other
eukaryotes: P24-EK1. Otherwise, the condi-
tions given under shotgun experiments
apply.

(iv) Prokaryotic plasmid and phage DNAs.
Plasmids and phage from hosts that ex-
change genetic—information with E. coli,
Experiments with DNA recombinants formed
from plasmids or phage genomes that have
not been characterized with regard to
presence of harmful genes or are known to
contribute significantly to the pathogenicity
of their normal hosts must use the contain-
ment conditions specified for shotgun experi-
ments with DNAs from the respective host.
If the DNA recombinants are formed from
plasmids or phage that are known not to con-
tain harmful genes, or from purified* and
characterized plasmid or phage DNA segments
known not to contain harmful genes, the
experiments can be performed with P1 physi-
cal containment-tan EK1 host-vector.

Plasmids and phage from hosts that do
not exchange genetic information with
E. colt. The rules for shotgun experiments
with DNA from the host apply to their plas-
mids or phages. The. minimum containment
conditions for this category (P2--EK2, or
P3+4+EK1) can be used for plasmid and
phage, or for purified* and characterized
segments of plasmid and phage DNAs, when
the risk that the recombinant DNAs will
increase the pathogenicity or ecological po-
tential of the host is judged to be minimal.

Note: Where applicable, cDNAs (i.e.,
complementary DNAs) synthesized in vitro
from cellular or viral RNAs are included
within each of the above classifications.
For example, cDNAs formed from cellular
RNAs that are not purified and character-
ized are included under <a>, shotgun ex-
periments; cDNAs formed from purified and

 

See footnotes on p, 38459,

NOTICES

characterized RNAs are included under
<b>; cDNAs formed from viral RNAs are
included under <e>; etc.

3. Experiments with other prokaryotic
host-vectors, Other prokaryotic host-vector
systems are at the speculative, planning,
or developmental stage, and consequently
do not warrant detailed treatment here at
this time. However, the containment cri-
teria for different types of DNA recombi-
nants formed with EF. coli K-12 host-vectors
can, with the aid of some general principles
given here, serve as a guide for containment
conditions with other host-vectors when
appropriate adjustment is made for their
different habitats and characteristics. The
newly developed host-vector systems should
offer some distinct advantage over the E.
coli K-12 host-vectors—tfor instance, ther-
mophilic organism or other host-vectors
whose major habitats do not include humans
and/or economically important animals and
plants. In general, the strain of any pro-
Karyotic species used as the host is to con-
form to the definition of Class 1 etiologic
agents given in ref. 5 (ie., “Agents for no
or minimal hazard * * *”), and the plasmid
or phage vector should not make the host
more hazardous. Appendix A gives a de-
tailed discussion of the B. subtilis system,
the most promising alternative to date.

At the initial stage, the host-vector must
exhibit at least a moderate level of biological
containment comparable to EK1 systems,
and should be capable of modification to ob-
tain high levels of containment comparable
to EK2 and EK8. The type of confirmation
test(s) required to move a host-vector from
an EK2-type classification to an EK3-type
will clearly depend upon the preponderant
habitat of the host-vector. r example, if
the unmodified host-vector propagates
mostly in, on, or around higher plants, but
not appreciably 1n warm-blooded animals,
modification should be designed to reduce
the probability that the host-vector can es-
cape to and propagate in, on, or around such
plants, or transmit recombinant DNA to
other bacterial hosts that are able to occupy
these ecological niches, and it is these lower
probabilities which must be confirmed. The
following principles are to be followed in
using the containment criteria given for ex-
periments with F. colt K-12 host-vectors as a
guide for other prokaryotic systems, Experi-
ments with DNA from prokaryotes (and their
plasmids or viruses) are classified accord-
ing to whether the prokaryote in question
exchanges genetic information with the host-
vector or not, and the containment condi-
tions given for these two classes with EF. coli
K-12 host-vectors applied. Experiments with
recombinants between plasmid or phage vec-
tors and DNA that extends the range of re-
sistance of the recipient species to thera-
peutically useful drugs must use P3 physical
containment + a host-vector comparable to
EK1 or P2 physical containment + a host-
vector comparable to EK2. Transfer of re-
combinant DNA to plant pathogens can be
made safer by using nonreverting, doubly
auxotrophic, non-pathogenic variants. Ex-
periments using a plant pathogen that af-
fects an element of the local flora will re-
quire more stringent containment than if
carried out in areas where the host plant is
not common.

Experiments with DNAs from eukaryotes
(and their plasmids or viruses) can also fol-
low the criteria for the corresponding experi-
ments with F. coli K-12 vectors if the major
habitats of the given host-vector overlap
those of FE. colt. If the host-vector has a
major habitat that does not overlap those of
E. coli (e.g., root nodules in plants), then the
containment conditions for some eukaryotic
recombinant DNAs ned to be increased (for

instance, higher plants and their viruses in

38457

the preceding example) while others can be
reduced.

4. Experiments with eukaryotic host-vec-
tors-—<a> Antmal host-vector systems. Be-
cause host cell lines generally have little if
any capacity for propagation outside the
laboratory, the primary focus for contain-
ment is the vector, although cells should also
be derived from cultures expected to be of
minimal hazard. Given good microbiological
practices, the most likely mode of escape of
recombinant DNAs from a physically con-
tained laboratory is carriage by humans;
thus vectors should be chosen that have lit-
tle or no ability to replicate in human cells.
To be used as a vector in a eukaryotic host, a
DNA molecule needs to display all of the fol-
lowing properties:

(1) It shall not consist of the whole ge-
nome of any agent that is infectious for hu-
mans or that replicates to a significant ex-
tent in human cells in tissue culture.

(2) Its functional anatomy should be
known—that is, there should be a clear idea
of the location within the molecule of:

(a) The sites at which DNA synthesis
originates and terminates,

(b) The sites that are cleaved by restric-
tion endonucleases, - :

{c) The template regions for the major
gene products.

(3) It should be well studied genetically.
It is desirable that mutants be available in
adequate number and variety, and that
quantitative studies of recombination have
been performed.

(4) The recombinant must be defective,
that is, its propagation as a virus is depend-
ent upon the presence of a complementing
helper genome. This helper should either (a)
be integrated into the genome of a stable
line of host cells (a situation that would
effectively limit the growth of the vector to
that particular cell line) or (b) consist of a
defective genome or an appropriate condi-
tional -lethal mutant virus (in which case
the experiments would be done under non-
permissive’ conditions), making vector and
helper dependent upon each other for prop-
agation. However, if none of these is avail-
able, the use of a non-defective genome as
helper would be acceptable,

Currently only two viral DNAs can be con-
sidered as meeting these requirements: These
are the genomes of polyoma virus and SV40.

Of these, polyoma virus is highly to be
preferred. SV40 is known to propagate in
human cells, both in vivo and in vitro, and
to infect laboratory personnel, as evidenced
by the frequency of their conversion to pro-
ducing SV40 antibodies, Also, SV40 and re-
lated viruses have been found in association
with certain human neurological and ma-
lignant diseases. SV40 shares many prop-
erties, and gives complementation, with the
common human papova viruses. By contrast,
there is no evidence that polyoma infects
humans, nor does it replicate to any signifi-
cant extent in human cells in vitro. How-
ever, this system still needs to be studied
more extensively. Appendix B gives further
details and documentation,

Taking account of all these factors:

i. Polyoma virus. a Recombinant DNA
Molecules consisting of defective polyoma
virus genomes plus DNA sequences of any
nonpathogenic organism, including Class 1
viruses (5), can be propagated in or used ta
transform cultured cells. P3 conditions are
required. Appropriate helper virus can be
used if needed. Whenever there is a choice, it
is urged that mouse cells, derived preferably
from embryos, be used as the source of eu-
Karyotic DNA. Polyma virus is a mouse virus
and recombinant DNA molecules containing
both viral and cellular sequences are already
Known to be present in virus stocks grown at
a high multiplicity. Thus, recombinants
forméd in vitro between polyoma virus DNA

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
38458

and mouse DNA are presumably not nove)
irom an evolutionary point of view.

b. Such experiments are to be done under
P4 conditions if the recombinant DNA con-
tains segments of the genomes of Class 3
animal viruses (5). Once it has been shown
by suitable biochemical and biological tests
that the cloned recombinant contains only
harmless regions of the viral genome (see
Section IIIB-2-c-i} and that the host range
of the polyoma virus vector has not been al-
tiered, experiments can be continued under
P3 conditions.

3, SV40 Virus. a. Defective SV40 genomes,
with appropriate helper, can be used as a@
vector for recombinant DNA molecules con-
taining sequences of any non-pathogenic
organism or Class I virsus (5), (i.e. a shot-
gun type experiment). P4 conditions are re-
quired. Established HMnes of cultured cells
should be used. \

b. Such experiments are to be carried
out in P3 (or P4) conditions if the non-
5V40 DNA segment is (a) a purified® seg-
ment of prokaryotic DNA lacking toxigenic
genes, or (b) a segment of eukaryotic DNA
whose function has been established, which
does not code for a toxic product, and which
has been previously cloned in a prokaryotic
host-vector system. It shall be confirmed
that the defective virus—helper virus sys-
tem does not replicate significantly more ef-
ficiently in human cells in tissue culture
than does SV40, following infection at a mul-
tiplicity of infection of one or more helper
SV40 viruses per cell.

c. A recombinant DNA molecule consist-
ing of defective SV40 DNA lacking sub-
stantial segments of the late region, plus
DNA from non-pathogenic organisms or Class
I viruses (5), can be propagated as an auton-
omous cellular element in established lines
of cells under P3 conditions provided that
there is no exogenous or endogenous helper,
and that it is demonstrated that no infec-
tious virus particles are heing produced. Un-
til this has been demonstrated, the appro-
priate containment conditions specified in
2, a. and 2. b. shall be used.

d. Recombinant DNA molecules consisting
of defective SV40 DNA and sequences from
non-pathogenic prokaryotic or eukaryotic
organisms or Class I viruses (5) can be used
to transform established lines of non-permis-
sive cells under P3 conditions. It. must be
demonstrated that no infectious virus par-
ticles are being produced; rescue of SV40
from such transformed cells by co-cultiva-
tion or transfection techniques must be car~
ried out in P4 conditions.

8. Efforts are to be made to ensure that all
cell lines are free of virus particles and myco-
plasma.

Since SV40 and polyoma are Hmited tn
their scope to act as vectors, chiefly because
the amount of foreign DNA that the normal
virions can carry probably cannot exceed
2%10¢ daltons, the development of systems
in which recombinants can be cloned and
propagated purely in the form of DNA, rather
than in the coats of infectious agents 1s
necessary. Plasmid forms of viral genomes or
organelle DNA need to be explored as possible
eloning vehicles in eukaryotic cells.

<b> Plant host-vector systems. For cells
in tissue cultures, seedlings, or plant parts
(eg., tubers, stems, fruits, and detached
leaves) or whole mature plants of small
species (¢.g., Arabidopsis) the P1—P4 contain-
ment conditions that we have specified previ-
ously are relevant concepts. However, work
with most plants poses additional problems.

 

See footnotes on p. 38459.

NOTICES

The greenhouse facilities accompanying P2
laboratory physical containment conditions
ean be provided by: (1) Insect-proof green-
houses, (ii) appropriate sterilization of con-
taminated plants, pots, soil, and runoff water,
and (lil) adoption of the other standard
practices for microbiological work. P3 physi-
cal containment can be sufficiently approxi-
mated by confining the operations with
whole plants to growth chambers like those
used for work with radioactive isotopes: Pro-
vided, That (1) such chambers are modified
to produce a negative pressure environment
with the exhaust air appropriately filtered,
Gi) that other operations with infectious
materials are carried out under the specified
P3 conditions, and (iii) to guard against in-
advertent insect transmission of recombinant
DNA, growth chambers are to be routinely
fumigated and only used in insect proof
rooms. The P2 and P3 conditions specified
earlier are therefore extended to include
these cases for work on higher plants.

The host cells for experiments on re-
combinant DNAs may be cells in culture, in
seedling or plant parts. Whole plants or plant
parts that cannot be adequately contained
shall not be used as hosts for shotgun ex-
periments at this time, and attempts to in-
fect whole plants with recombinant DNA
shall not be initiated until the effects on
host cells in culture, seedlings or plant parts
have been thoroughly studied.

Organelle or plasmid DNAs and DNAs of
viruses of restricted host range may be used
as vectors. In general, similar criteria for
selecting host-vectors to those given in the
preceding section on animal systems are to
apply to plant systems.

DNA recombinants formed between the
initial moderately contained vectors and DNA
from cells of species in which the vector DNA
can replicate, require P2 physical contain-
ment. However, if the source of the NA is
ltself pathogenic or known to carry patho-
genic agents, or to produce products dan-
gerous to plants, or if the vector is an un-
modified virus of unrestricted host range, the
experiments shall be carried out under P3
conditions.

Experiments on recombinant DNAs formed
between the above vectors and DNAs from
other species can also be carried out under
P2 if that DNA has been purified* and de-
termined not to contain harmful genes.
Otherwise, the experiments shall be carried
out under P3 conditions if the source of the
inserted DNA is not itself s pathogen, or
known to carry such pathogenic agents, or
to produce harmful products—and under P4
conditions if these conditions are not met.

The development and use of host-vector
systems that exhibit a high level of biologi-
cal containment permit a decrease of one
step in the physician containment specified
above (P4>P3>P2>P1). :

<e> Fungal or similar lower euharyotic
host-vector systems, The containment cri-
teria for experiments on recombinant DNAs
using these host-vectors most closely re-
semble those for prokaryotes, rather than
those for the preceding eukaryotes, in that
the host cells usually exhibit a capacity for
dissemination outside the laboratory that is
similar to that for bacteria. We therefore
consider that the containment guidelines
given for experiments with EF. coli K-12 and
other prokaryotic host-vectors (Sections
ITIB-1 and ~-2, respectively) provide adequate

direction for experiments with these lower
eukaryotic host-vectors. This is particularly
true at this time since the development of
these host-vectors is presently in the specu-
jative stage.

IV. Roies anv RESPONSIBILITIES

Safety in research involving recombinant
DNA molecules depends upon how the re~«
search team applies these guidelines. Motiva-
tion and critical Judgment are necessary, In
addition to specific safety knowledge, to en-
sure protection of personnel, the public, and
the environment.

The guidelines given here ave to help the
principal investigator determine the nature
of the safeguards that should be imple-
mented. These guidelines will be incomplete
iu same respects because all conceivable ex-
periments with recombinant DNAs cannect
now be anticipated. Therefore, they cannot
substitute for the investigator’s own Knowi-
edgeable and discriminating evaluation.
Whenever this evaluation calls for an In-
crease in containment over that indicated
in the guidelines, the investigator has a
responsibility to institute such an increase.
In contrast, the containment conditions
called for in the guidelines should not be
decreased without review and approval at
the institutional and NIH levels,

The following roles and responsibilities de-
fine an administrative framework in which
safety is an essential and integrated fune-
tion of research involving recombinant DNA
molecules.

A. Principal investigator. The principal in-
vestigator has the primary responsibility for:
(i) Determining the real and potential bio-
hazards of the proposed research, (HM) de-
termining the appropriate level of biologica
and physical containment, (iil) selecting the
microbiological practices and laboratory

_ techniques for handling recombinant DNA

materials, (iv) preparing procedures for deai-
ing with accidental spills and overt personne}
contamination, (v) determining the appli-
cability of various precautionary medical
practises, serological monitoring, and im-
munization, when available, (vi) securing
approval of the proposed research prior to
initiation of work, (vii) submitting Informa-
tion on purported EK2 and EK3 systems to
the NIH Recombinant DNA Molecule Pro-
gram Advisory Committee and making the
strains available to others, (viii) reporting
to the institutional biohazards committee
and the NIH Office of Recombinant DNA Ac-
tivities new information bearing on the
guidelines, such as technical information re-
lating to hazards and new safety procedures
or innovations, (ix) applying for approval}
‘trom the NIH Recombinant DNA Molecule
Program Advisory Committee for large scale
experiments with recombinant DNAs known
to make harmful products (1.e., more than 10
liters of culture), and (x) applying to NIH
for approval to lower containment levels
when a cloned DNA recombinant derived
from a shotgun experiment has been rigor-
ously characterized and there is sufficient
evidence that it is free of harmful genes,

Before work is begun, the principal i1-
vestigator is responsible for: (i) Making
available to program and support staff copies
of those portions of the approved grant ap-
plication that describe the bichazards and
the precautions to be taken, (ii) advising the
program and support staff of the nature and
assessment of the real and potential bio-
hazards, (iif) instructing and training thie
staff in the practices and techniques required
to ensure safety, and in the procedures for
dealing with accidentally created biohazards,
and (iv) informing the staff of the reasons
and provisions for any advised or requested
precautionary medical practises, vaccina-
tions, or serum collection.

During the conduct of the recearch, the
principal investigator is responsible for: (1)

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
Supervising the safety performance of the
staff to ensure that the required safety prac-
tices and techniques are employed, (ii) In-
vestigating and reporting in writing to the
NIH Office of Recombinant DNA Activities
and the institutional biohazards committee
any serious or extended illness of a worker
or any accident that results in (a) inocula-
tion of recombinant DNA materials through

utaneous penetration, (b) ingestion of
recombinant DNA materials, (c) probable
inhalation of recombinant DNA materials
following gross aerosolization, or (d) any
incident causing serious exposure to per-
sonnei or danger of environmental contami-
nation, (ili) invesigating and reporting in
writing to the NIH Office of Recombinant
DNA Activities and the institutional bio-
hazards committee any problems pertaining
to operation and implementation of bio-
logical and physical containment safety prac-
tices and procedures, or equipment or facil-
ity failure, (iv) correcting work errors and
conditions that may result in the release of
recombinant DNA materials, and (v) ensur-
ing the integrity of the physical contain-
ment (e.g., biological safety cabinets) and
the biological containment (e.g., genotypic
and phenotypic characteristics, purity, etc.).

B. Institution. Since in almost all cases,
NIH grants are made to institutions rather
than to individuals, all the responsibilities
of the principal investigator listed above are
the responsibilities of the institution under
the grant, fulfilled on its behalf by the prin-
cipal investigator. In addition, the institu-
tion ‘s responsible for establishing an insti-
tutional biohazards committee’ to: (1) Ad-
vise the institution on policies, (ii) create
and maintain a central reference file and
Hbrary of catalogs, books, articles, newslet-
ters, and other communications as a source
of advice and reference regarding, for exam-
ple, the availability and quality of the safety
equipment, the availability and level of bio-
logical containment for various host-vector
systems, suitable training of personnel and
data on the potential biohazards associated
with certain recombinant DNAs, (lili) de-
velop a safety and operations manual for any
P4 facility maintained by the institution
and used in support of recombinant DNA
research, (lv) certify to the NIH on applica-
tions for research support and annually
thereafter, that facilities, procedures, and
practices and the training and expertise of
the personnel involved have been reviewed
and approved by the Institutional biohazards
committee.

The biohazards committee must be suffi-
elently qualified through the experience and
expertise of its membership and the diversity
of its membership to ensure respect for its
advice and counsel. Its membership should
include individuals from the institution or
consultants, selected so as to provide a diver-
sity of disciplines relevant to recombinant
DNA technology, biological safety, and engi-
neering. In addition to possessing the profes-
sional competence necessary to assess and
review specific activities and facilities, the
committee should possess or have available
to it, the competence to determine the ac-
eeptability of its findings in terms-of ap-
plicable laws, regulations, standards of prac-
tices, community attitudes, and health and
environmental considerations, Minutes of the
meetings should be kept and made available
for public inspection. The institution is re~
sponsible for reporting names of and releyant
background information on the members of
its biohazards committee to the NIH.

C. NIH Initial Review Groups (Study Sec-
tions), The NIH Study Sections, in addition

NOTICES

to reviewing the scientific merit of each
grant application involving recombinant
DNA molecules, are responsible for: (i)
Making an independent evaluation of the
real and potential biohazards of the pro-
posed research on the basis of these guide-~
lines, (ii) determining whether the proposed
physical containment safeguards certified by
the institutional biohazards committee are
appropriate for control of these biohazards,
(iii) determining whether the proposed bio-
logical containment safeguards are appro-
priate, (iv) referring to the NIH Recombi-
nant DNA Molecule Program Advisory Com-
mittee or the NIH Office of Recombinant
DNA Activities those problems pertaining to
assessment of biohazards or safeguard deter-
mination that cannot be resolved by the
Study Sections.

The membership of the Study Sections will
be selected in the usual manner. Biological
safety expertise, however, will be available to
the Study Sections for consultation and
guidance. ,

D. NIH Recombinant DNA Molecule Pro-
gram Advisory Committee. The Recombinant
DNA Molecule Program Advisory Committee
advises the Secretary, Department of Health,
Education, and Welfare, the Assistant Secre-
tary for Health, Department of Health, Edu-
cation, and Welfare, and the Director, Na-
tional Institutes of Health, on a program for
the evaluation of potential biological and
ecological hazards of recombinant DNAs
(molecules resulting from different segments
of DNA that have been joined together in
cell-free systems, and which have the capac-
ity to infect and replicate in some host cell,
either autonomously or as an integrated part
of their host's genome), on the development
of procedures which are designed to prevent
the spread of such molecules within human
and other populations, and on guidelines to
be followed by investigators working with
potentially hazardous recombinants.

The NIH Recombinant DNA Molecule Pro-
gram Advisory Committee has responsibility
for: (1) Revising and updating guidelines
to be followed by investigators working with
DNA recombinants, (1i) for the time being,
receiving information on purported EK2 and
EK3 systems and evaluating and certifying
that host-vector systems meet EK2 or EK3
criteria, (if) resolving questions concerning
potential bichazard and adequacy of con-
tainment capability 1f£ NIH staff or NIH In-
Itial Review Group so request, and (iv) re-
viewing and approving large scale experiments
with recombinant DNAs known to make
harmful products (e.g., more than 10 liters
of culture).

E. NIH Staff. NIH Staff has responsibility
for: (i) assuring that no NIH grants or con-
tracts are awarded for DNA recombinant re-
search unless they (a) conform to these
guidelines, (b) have been properly reviewed
and recommended for approval, and (c) in-
clude a properly executed Memorandum of
Understanding and Agreement, (ii) review-
ing and responding to questions or problems
or reports submitted by institutional bio-
hazards committees or principal investiga-
tors, and disseminating findings, as appro-
priate, (ili) receiving and reviewing applica-
tions for approval to lower containment
Jevels when a cloned DNA recombinant de-
rived from a shotgun experiment has been
Yigorously characterized and there is suffi-
cient evidence that it is free of harmful
genes, (iv) referring items covered under (i!)
and (111) above to the NTH Recombinant DNA
Molecule Program Advisory Committee, as
deemed necessary, and (v) performing site
inspections of all P4 physical containment
facilities, engaged in DNA recombinant re-
search, and of other facilities as deemed
necessary.

38159

APPENDIX D
V. FPoorores

1 Biological Safety Cabinets referred to in
this section are classified as Class I, Class II
or Class III cabinets, A Class I cabinet is a
ventilated cabinet for personnel protection
having an inward flow of air away from the
operator, The exhaust air from this cabinet is
filtered through a high efficiency or high ef-
ficiency particulate alr (HEPA) filter before
discharged to the outside atmosphere. Thia
cabinet is used in three operational modes:
(1) with an 8 inch high full width open
front, (2) with an installed front closure
panel (having four eight inch diameter open-
ings) without gloves, and (3) with an in-
stalled front closure panel equipped with
arm length rubber gloves. The face velocity
of the inward flow of air through the full
width open front is 75 feet per minute or
greater. A Class II cabinet is a ventilated
cabinet for personnel and product protection
having an cpen front with inward air flow
for personnel protection, and HEPA filtered
mass recirculated air flow for product protec-
tion. The cabinet exhaust air ts filtered
through a HEPA filter. The face velocity of
the inward flow of air through the full width
open front is 75 feet per minute or greater.
Design and performance specifications for
Class II cabinets have been adopted by the
National Sanitation Foundation, Ann Arbor.
Michigan. A Class III cabinet is a closed
front ventilated cabinet of gas tight construc-
tion which provides the highest level of per-
sonnel protection of all Biohazard Safety
Cabinets. The interior of the cabinet is pro-
tected from contaminants exterior to the
cabtnet. The cabinet is fitted with arm length
rubber gloves and is operated under a nega-
tive pressure of at least 0.5 inches water
gauge. All supply air is filtered through
HEPA filters. Exhaust air is filtered through
HEPA filters or incinerated before being dis-
charged to the outside environment.

? Defined as observable under optimal lab-
oratory conditions by transformation, trans-
duction, phage infection and,/or conjugation
with transfer of phage, plasmid and ‘or chro-
mosomal genetic information.

*The bacteria which constitute Class 2 of
ref. 5 (‘Agents of ordinary potential hazard
.-.”) represent a broad spectrum of etiologic
agents which possess different levels of vir-
ulence and degrees of communicability. We
think it appropriate for our specific purpose
to” further subdivide the agents of Class 2
into those which we believe to be of rela-
tively low pathogenicity and those which are
moderately pathogenic. The several specific
examples given may suffice to illustrate the
principle.

‘The terms “characterized” and “free of
harmful genes” are unavoidably vague. But
in this instance, before containment condi-
tions lower than the ones used to clone the
DNA can be adopted, the Investigator must
obtain approval from the National Institutes
of Health. Such approval would be contin-
gent upon data concerning: (a) The absence
of potentially harmful genes (e.g., sequen-
ces contained in indigenous tumor viruses
or which code for toxic substances), (b)
the relation between the recovered and de-
sired segment (e.g., hybridization and re-
striction endonuclease fragmentation anal-
ysis where applicable), and (c) maintenance
of the biological properties of the vector.

5A DNA preparation is defined as enriched
if the desired DNA represents at least 99%
(w/w) of the total DNA itn, the preparation.
The reason for lowering the containment
level when this degree of enrichment has
been obtained is based on the fact that the
total number of clones that must be ex~
amined to obtain the desired clone is

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
38460

markedly reduced. Thus, the probability of
cloning a harmful gene could, for example, be
reduced by more that 10°-fold when a non-
repetitive gene from mammals was being
sought. Furthermore, the level of purity spe-
cified here makes it easier to establish that
the desired DNA does not contain harmful
genes.

*The DNA preparation 1s defined as puri-
fled if the desired DNA represents at least
99 percent (w/w) of the total DNA in the
preparation, provided that it was verified by
more than one procedure.

7In special circumstances, in consultation
with the NIH Office of Recombinant DNA Ac-
tivities, an area biohazards committee may
be formed, composed of members from the
institution and/or other organizations be-
yond its own staff, as an alternative when
additional expertise outside the institution
is needed for the indicated reviews.

V. REFERENCES

1. Berg, P., D. Baltimore, H. W. Boyer, S. N.
Cohen, R. W. Davis, D. S. Hogness, D. Na-
thangs, R. O. Roblin, J. D. Watson, S. Weiss-
man, and N. D. Zinder (19'74). Potential Bio-
hazards of Recombinant DNA Molecules. Sci-
ence 185, 303.

2, Advisory Board for the Research Coun-
cils. Report of a Working Party on the Ez-
perimental Manipulation of the Genetic
Composition of Micro-Organisms. Presented
to Parliament by the Secretary of State for
Education and Science by Command of Her
Majesty. January, 1975. London: Her Maj-
esty’s Stationery Office, 1975.

3, Berg, P., D. Baitimore, S. Brenner, R. O.
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4. Laboratory Safety at the Center for Dis-
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No. CDC 75-8118.

5. Classification of Etiologic Agents on the
Basis of Hazard. (4th Edition, July, 1974).
U.S. Department of Health, Education and
Welfare. Public Health Service. Center for
Disease Control, Office of Biosafety, Atlanta,
Georgia 30333.

6. National Cancer Institute Safety Stand-
ards for Research Involving Oncogenic Vir-
uses (Oct., 1974). U.S. Department of Health,
Education and Welfare Publication No. (NIH)
75—790.

7. National Institutes of Health Biohazards
Safety Guide (1974), U.S. Department of
Health, Education and Welfare. Public Health
Service, National Institutes of Health. U.S
Government Printing Office Stock No. 1740-
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8. Biohazards in Biological Research (1973).
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9. Handbook of Laboratory Safety (1971;
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10. Bodily, H. L. (1970). General Adminis-
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16. Kuehne, R. W. (1973). Biological Con-
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16. Runkle, R. 8. and G. B. Phillips. (1969).
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17. Chatigny, M. A. and D.I. Clinger (1969).
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33. R. Curtiss II, personal communica-
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In Vivo Transmission of Drug Resistance
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Radding (1969). Nonessential Functions of
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(1971). Prophage Insertion and Exciston. In
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483-503.

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44. J. S. Parkinson as cited (p. 8) by Her-
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tion to Lambda. In: The Bacteriophage }.
A. D. Hershey, ed. Cold Spring Harbor Lake-
tatory, New York.

VIT. MEMBERS oF THE RECOMBINANT DNA
MoLECULE PROGRAM ADVISORY COMMITTES

CHARMAN

STETTEN, DeWitt, Jr., M.D., PR.D., Deputy
Director for Science, National Institistes
of Health.

VICE CHAIRMAN

JACOBS, Leon, Ph.D., Associate Director for
Collaborative Research, National Institutes
of Health.

ADELBERG, Edward A., Ph.D., Professor, De-
partment of Human Genetics, Schoo) of
Medicine, Yale University,

CHU, Ernest H. Y. Ph.D., Professor, De-
partment of Human Genetics, Medical
School, University of Michigan.

CURTISS, Roy, II, Ph.D., Professor. De-
partment of Microbtology, School of Medi-
cine, University of Alabama.

DARNELL, James E., Jr.. M.D., Professcr,
Department of Molecular Cell Biology,
Rockefeller University.

HELINSKI, Donald R., Ph.D., Professor, De-
partment of Biology, University of Cali-
fornia, San Diego.

HOGNESS, David S., Ph.D., Professor. De-
partment of Biochemistry, Stanford Uni-
versity.

KUTTER, Elizabeth M., Ph.D., Member cf
the Faculty in Biophysics, The Evergreen
State College.

LITTLEFIELD, John W., M.D., Professor &
Chairman, Department of Pediatrics, Chii-
dren’s Medical & Surgical Center, Johns
Hopkins Hospital.

REDFORD, Emmette S., Ph.D., LL.D., Ashbea
Smith Professor of Government and Pub-
lic Affatra, Lyndon B. Johnsons Schooi of
Public Affairs, University of Texas at Ats-
tin .

FEDERAL REGISTER, VOL. 47, NO. 176—THURSDAY, SEPTEMBER 9, 1976
ROWE, Wallace P., MD. Chief, Laboratory
of. Viral Diseases, National Institute of Al-
lergy & Infectious Diseases, National In-
stitutes of Health. .

SETLOW, Jane K., Ph.D., Biologist, Brook-
haven National Laboratory.

SPIZIZEN, John, Ph.D., Member and Chair-
man, Department of Microbiology, Scripps
Clinic & Research Foundation.

SB2YBALSKI, Waclaw, D.Sc., Professor of
Oncology, McArdle Laboratory, Univer-
sity of Wisconsin.

THOMAS, Charles A., Jr., Ph.D., Professor,
Department of Biological Chemistry, Har-
vard Medical School.

EXECUTIVE SECRETARY

GARTLAND, William J., Jr, Ph.D., Health
Scientist Administrator, National Institute
of General Medical Sciences, National In-
stitutes of Health.

LIAISON REPRESENTATIVES

HEDRICH, Richard, Ph.D., Coordination Pro-
gram of Sclence Technology & Human
Value, National Endowment for the Hu-
manities.

LEWIS, Herman W., Ph.D., Division of Bio-
logical and Medical Sciences, National Sci-
ence Foundation.

NIGHTINGALE, Elena O., Ph.D., Assembly of
Life Sciences, National Academy of
Sciences.

SHEPHERD, George R., Ph.D., Division of Bio-
medical and Environmental Research, En-
ergy Research and Development Adminis-
tration.

APPENDIX A TO APPENDIX D

STATEMENT ON THE USE OF BACILLUS SUBTILIS
IN RECOMBINANT MOLECULE TECHNOLOGY

Unquestionably, Escherichia colt 1s the
most well characterized unicellular organism.
Years of basic research have enabled investi-
gators to develop a well characterized genetic
map, to obtain detailed knowledge of viru-
lent and temperate bacteriophages, and to
explore the physiology, genetics, and regula-
tion of plasmids. More recently, the develop-
ment of DNA-mediated transformation has
permitted exogenous fragments or molecules
of DNA to be incorporated into the genome
or to reside as self-replicating units. The dis-
covery of transformation of Bacillus subtilis
by Spizizen (1) stimulated the development
of an alternative model system. The purpose
of this report is to summarize the current
status of this genetic system and to describe
the actual and potential vectors and vehicles
available for recombinant molecule technol-
ogy.

A. Current knowledge of the chromosomal
architecture and mechanisms of genetic ex-
change in B. subtilis. Two mechanisms of
genetic exchange have been utilized to estab-
lish the Linkage map of B, subtilis, DNA-
mediated transformation (capable of trans-
ferring approximately 1 percent of the gen-
ome) and transduction with bacteriophage
PBSI (capable of transferring 5-8 percent of
the chromosome). Recent detailed genetic
studies with PBSI by Lepesant-Kejzlorova
et al. (2) have resulted in the development
of a circular genetic map for this organism.
The current edition of the map (3) contains
196 loci. Biophysical analyses have estab-
lished that the chromosome is circular (4)
and replicates bidirectionally (5).

Transformation with purified fragments of
DNA is a highly efficient process in B. subtilis
with frequencies of 1 to 4 percent usually
attained for any auxotrophic or antibiotic
resistance markers. Frequencies of approxt-
mately 10 percent transformation can be
achieved with DNA prepared from gently
lysed L-forms or protoplasts (6). These large
fragments of DNA are readily incorporated
by the recipient cell. Generalized transduc-

~ NOTICES

tion occurs with bacteriophages SP10 (7),
PBS1 (8), and SPP1 (9), while a low fre-
quency of specialized transduction has been
reported with bacteriophage ¢105 (10).

Although transformation its most efficient
in homologous crosses (B. subtilis into B.
subtilis), it has also been possible to ex-
change DNA among closely related species
(11). The most extensively studied members
of the B. subtilis genospecies include B.
lichenijormis, B. pumilus, B. amylolique-
faciens, and B. globigti (refer to reference 12
for a review and references 13-15 for exam-
ples of this heterologous exchange). This ex-
change occurs even though there is a sur-
prisingly wide discrepancy between DNA-—
DNA hybridization among these organisms
(16). Even though the frequency of trans-
formation is low in the heterologous cross
[e.g., B. amyloliquefaciens (donor)/B sub-
tilis (recipient)], the newly acquired DNA
from B. amyloliquefaciens in the B. subtilis
packground can be readily transferred at
high efficiencies to other recipient strains of
B. subtilis (14). Therefore, the extremely
high frequency of transformation permits
the recognition and selection of rare events.

B. Current and potential vectors for re-
combinant molecule experiments. Lovett and
coworkers have recently described crypti
plasmids in B. pumilus (17) and B,. subtilis
(18). Of these organisms, B. subtilis ATCC
7003 appears to be the most useful since it
earries one to two copies of a plasmid with
@ molecular weight of 46 x 10°. This strain
is also closely related to B. subtilis 168. An-
other strain of B. subtilis (ATCC 15841) con-
tains 16 copies of a plasmid with a molecular
weight of 4.6 x 10%. Currently it is not known
whether genetic markers can be readily in-
troduced into these plasmids. To date it has
not been possible to readily stabilize plas-
mids derived from B. pumilus in B. subtilis
even with heavy selective pressure (P. Lovett,
personal communication).

Two temperate bacteriophages are under
development as vectors in B. subtilis, ¢3T
and SPO2, Lysogeny of thymine auxotrophs
(strains carrying thyA thyB) by bacterio-
phage $3T results tn “conversion” to a Thy+
phenotype. The attachment site for this
bacteriophage and the bacteriophage gene
for thymidylate synthetase (thyP) map be-
tween the bacterial thyA and thyB loci in
the terminal region of the chromosome of
B, subtilis (19).’The viral genome is readily
cleaved by the site-specific endonuclease,

. Bam 1 (20), to produce & fragments (one

of which carries the thyP gene). The thyP
carrying gene can be integrated into the
bacterial genome in the absence of the intact
viral genome. Because deletions are available
that include the tiyP region, it is theoreti-
cally possible to introduce thyP at many
sites on the chromosome. The thyP gene
can be readily purified for insertion into
Plasmids or utilized as a scaffold to integrate
other heterologous DNA into the chromo-
some of B. subtilis. Alternatively, it is pos-
sible to purify fragments of the chromosome
by gel electrophoresis (21, 22), for insertion
into bacteriophage #8T or SPO2. At present,
unfortunately, only the former carries a
selective marker, 1.e., the gene for thymidyi-
ate synthetase, thyP.

C. Development of vehicles. B. subtilis 1s
a Gram-positive sporulating rod that usually
inhabits soil. Although it can exist on
cutaneous surfaces of man (23) and experi-
mental animals, it rarely produces disease.
To develop a suitable vehicle it is imperative
to have @ host that is asporogenic. The most
appropriate deletion mutation is deletion 29
(cit D). In addition to a deficiency in
sporulation this mutant rapidly lyses when
it has reached the end of its growth cycle.
Presumably this is due to the failure to
inactivate one of the autolytic enzymes (24).

88461

Through the introduction of a D-alanine
requirement (34 ug/ml) it is possible to
block transport of compounds that are
transported by active transport (25,26). The
further introduction of thymine auwxotrophy
(defects in the thyA thyB loci) will enable
the strain to survive only with a plasmid
vector carrying the purified thyP gene from
bacteriophage $3T or a defective bacterio-
phage #3T carrying the thyP gene but at-
tached to the chromosome at an alternative
site (due to the presence of deletion 29 in
the host). We have recently isolated tem-
perature-sensitive thyP mutants. If we can
isolate a temperature-dependent lysogen
that will grow only at 48°C it should be
possible to make an unusual vehicle.

D. Site-specific endonucleases. Recently
two restriction modification systems have
been observed between B. subtilis 168 and
other bacilli. Trautner et al. have isolated
an effective system that inhibits infection of
the R strain of B. subtilis by bacteriophage
SPPI propagated on B. subtilis 168 (27). The
site-specific nuclease recognizes the sequence
GGCC.

CCGG .

Young, Radnay, and Wilson observed a
restriction modificaiton system between
B. amyloliquefaciens and B. subtilis 168 (28).
The endonuclease from B. amyloliquefaciens
(20) recognizes the sequence GGTAC (29).

CCTAGG

More recently, two additional enzymes have
been isolated from B. globigii (30). The recog-
nition sequence is not known,

E. Advantages and liabilities of the B. sub-
tilis system—a. Advantages. 1. B. subtilis ts
nonpathogenic. Asporogenic deletion mu-
tants are available to preclude the problem of
persistence through sporulation.

2. The circular chromosomal map is well
defined. At least 196 loci have been posi-
tioned.

3. The organism is commercially important
in the fermentation industry.

4. Large numbers of organisms can be dis-
posed of readily with minimal environmental
impact.

5. Unlike E. co/i, it lacks endotoxin in the
cell wall. Therefore the cells can be used as
@ single cell protein source.

6. The frequency of transformation is very
high, facilitating the detection of rare events.

7. A unique bacteriophage, 43T, exists that
carries a gene that can be readily purified for
“scaffolding” experiments.

b. Disadvantages. 1. The Enowledge of
genetics and physiology of plasmids and
viruses 1s primitive compared with £. colt.

2. High-frequency, specialized transduc-
tion is not available as a means of gene
enrichment.

Based on its promise, it seems appropriate,
and not chauvinistic, to urge development of
this system,

Prepared by:

Dr. Frank Young,
University of Rochester.

REFERENCES

1. Spizizen, J. 1958. Transformation of bio-
chemically deficient strains of Bacillus sub-
tilis by deoxyribonucleate. Proc. Nat. Acad.
Sci. U.S.A. 44:1072-1078.

2. Lepesant-Kejzlarova, J., J. A. Lepesant,
J. Walle, A. Billault, and R. Dedonder. 1975.
Revision of the linkage map of Bacillus sub-
tilis 168: indications for circularity of the
chromosome. J. Bacteriol. 121:823-834.

3. Young, F. E. and G@ A. Wilson. 19°78.
Chromosomal map of Bacillus subtilis p-
596-614. In P, Gerhardt, R. N. Costilow, and
H. L. Sadoff (ed.), Spores VI. American So-
slety for Microbiology, Washington, D.C.

4. Wake, R. G. 1974. Termination of Ba-
etlius subtilis chromosome replication as

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38462

visualized by autoradiography. J. Mo). Biol.
86 5223-231.

5. Harford, N. 1975. Bidirectional chromo-
some replication in Bacillus subtilis. J. Bac-
teriol. 121:835-847.

6. Bettinger, G. E. and F. E. Young. 1975.
Transformation of Bacillus subtilis: Trans-
forming ability of deoxyribonucleic acid in
lysates of L-forms or protoplasts. J. Bacter-
iol. 122:987-993.

7. Thorne, C. B. 1962, Transduction in
Bacillus subtilis. J, Bacteriol. 83:106-111.

8. Takahashi, I. 1961. Genetic transduc-
tion in Bacillus subtilis. Blochem. Biophys.
Res, Commun. 5:171-175.

9. Yasbin, R. E. and F. E. Young. 1974.
Transduction in Bacillus subtilis by bac-
teriophage SPPI. J. Virol. 14: 1343-1348.

10. Shapiro, J. A., D. H. Dean and H. O.
Halvorson. 1974. Low-frequency specialized
transduction with Bacillus subtilis bacterio-
phage $105. Virology 62:393-403.

11. Marmur, J., E. Seaman, and J. Levine.
1963, Interspecific transformation in Bacil-
lus. J. Bacteriol. 85:461-467,.

12, Young, F. E. and G. A. Wilson. 1972,
Genetics of Bacillus subtilis and other
gram-positive sporulating bacilli, p. 77-106.
in H. O. Halvorson, R. Hanson, and L. L.
Campbell (ed.), Spores V. American Society
for Microbiology, Washington, D.C.

13. Chilton, M. D., and B. J. McCarthy.
1969. Genetic and base sequence homologies
in bacilli. Genetics 62:697-710.

14. Wilson, G. A. and FP. E. Young. 1972.
Intergenotic transformation of the Bacillus
subtilis genospecies. J. Bacteriol. 111:705-
716.

15. Yamaguchi, K., Y. Nagata, and B.
Maruo, 1974. Genetic control of the rate
of an amylase synthesis in Bacillus subtilis.
J. Bacteriol. 119:410-415.

16. Lovett, P. S., and F. E. Young. 1969.
Identification of Bacillus subtilis NRRL B-
3275 as a strain of Bacillus pumilus. J. Bac-
teriol. 100:658-661.

17. Lovett, P. S, and M. G. Bramuccl.
1975, Plasmid deoxyribonucleic acid in
Bacillus subtilis and Bacillus pumilus. J.
Bacteriol. 124:484-490,

18. Lovett, P. S. 1973. Plasmid in B. pumi-
qus and the enhanced sporulation of plasmid
negative variants. J. Bactertol. 115:291-298.

19. Young, F. E., M. T. Williams, and G. A.
Wilson. Genetics of Bacilius subtilis. In D.
Schlessinger (ed.) Microblology 1976, in
press.

20. Wilson, G.
Isolation of a

A. and F. EB. Young. 1975.
sSequence-specific endonu-
clease (Bam 1) from Bacillus amyiolique-
jaciens H. J. Mol, Biol. 97:123-125.

21. Brown, L. Recombination analysis
with purified endonuclease fragments in the
RNA polymerase region of Bacillus subtilis.
In D. Schlessinger (ed.), Microbiology 1976,
in press,

22. Harris-Warrick, R. M., Y, Elkana, 8S. D.
Ehriich, and J. Lederberg. 1975. Electro-
phoretic separation of Bucillus subtilis
genes. Proc. Nat. Acad. Sel. U.S.A. 72:2207-
2211.

23. Kloos, W. F., and M. 8. Musselwhite.
1975. Distribution and persistence of Sta-
phylococcus and Micrococcus species and
other aerobic bacteria on human skin. Ap-
plied Micro. 30:381-395.

24, Brown, W. C., and F. EB. Young. 1970.
Dynamic interactions between cell wall
polymers, extracellular proteases and auto-
lytic enzymes. Biochem. Biophys. Res,
Commun. 33:564-568.

25, Clark, V. L, and FP. BE. Young. 1974.
Active transport of D-alanine and related
amino acids by whole cells of Bacillus sub-
tis. J. Bacteriol. 120:1085-1092,

26. Clark, V. L. and FP. E. Young. Active
transport in celia of B. subtilis 168: Loss of

NOTICES

endogenously energized transport in auxoe-
trophs deprived of D-alanine or glycerol.
Submitted to J. Bacteriol.

27. Trautner, T. A., B. Pawlek, S. Bron,
and C, Anagnostopoulos. 1974. Restriction
and modification in B. subtilis: biologic as-
pects. Mol. Gen, Genet. 131:181-191.

28. Young, F. E., E. Radnay, and @. A
Wilson. Manuscript in preparation.

29. Wilson, G. A. and F. E. Young. Unpub-
Ushed data.

380. Wilson, G. A., R. Roberts, and F. E.
Young. Unpublished data.

31, Wilson, G. A. and F. E. Young. Re-
striction and modification in bacilli. In D.
Schlessinger (ed.), Microbiology 1976, iu
press.

APPENDIX B TO APPENDIX D

POLYOMA AND SV40 VIRUS

Polyoma virus is a virus of mice, and in-
fection of wild mouse populations is a com-
mon events, for the virus has often been
isolated from a high proportion of healthy
adult animals, both wild and laboratory
bred, of many colonies (Gross, L., Proc. Soc,
Exp. Biol. 88, 362-368, 1955; Rowe, W. P.
Bact. Rev. 25, 18-31, 1961). As far as is known
the virus almost never causes a disease in
these animals. However, when large quanti-
ties of the virus are inoculated into newborn
or suckling mice or hamsters, a variety of
solid tumors is induced (Gross, L., Oncogenic
Viruses, Second Edition, Pergamon Press,
NY).

Polyoma virus grows lytically in mouse
cells in tissue culture. Thus mouse celis in
eulture are probably transformed only by
virus particles that contain certain kinds of
defective geonmes. Cells of other rodent spe-
cies, however, can be transformed by poly-
oma virus particles that contain complete
genomes (Folk, W., J. Virol., 11, 424-431,
1973). The virus does not replicate to a
significant extent in human cells in tissue
culture (Eddy, B.E., Virol. Monogr., 7, 1-114,
1969; Pollack, R. E., Salas, J., Wang R. Ku-
sano T., and Green, H., J. Cell Physiol. 77,
117-120, 1971). The resistance of the cells
seems to be a consequence of the failure of
the virus to absorb or uncoat. However even
when naked viral DNA is introduced into the
cells only an abortive cycle of replication en-
sues; early viral proteins are made, there is
induction of cellular DNA synthesis, but
no expression of late viral proteins is de-
tectable (Gruen, R. Grassmann, M. and
Grassmann, A. Virology, 58, 290-293, 1974).

There is no evidence that polyoma virus
can infect humans (Hartley, J., Huebner, R.,
Parker, J. and Rowe, W. P., unpublished
data). Thus no antibodies to the virus have
been detected in people living in buildings
that are infested with virus-infected mice,
nor in faboratory workers who have been ex-
posed to the virus for a number of years.

At most, a small segment of polyoma virus
DNA shows weak homology with a portion of
the late region of SV40 DNA (Ferguson, J.
and Davis, R. W., J. Mol. Biol., 94, 135-150,
1975). However, there appears to be no
genetic interaction between the two viruses
and there is no immunological cross-reaction
between the gene products of the two viruses.

SV40 causes perisitent but apparently
harmiess infections of the kidneys of vir-
tually all adult rhesus monkeys (Hsiung,
G. D., Bact. Revs. 32, 185-205, 1968), it causes
tumors when injected into newborn ham-
sters (Girardi, A. J., Sweet, B. H., Slotnick,
Vv. B. and Hillemann, M. R., Proc. Soc. Exp.
Biol. Med., 105, 420-427, 1964) and trans-
forms cells of several mammalian species
(including human). SV40 ts able to infect
humans since antibodies to the virus are
found in a small proportion of the human
population (Shah, E. V., Goverdhan, M. K.
and Ozer, H. l., Am. J. Epid. 93, 291-298,

 

1976} and serum conversions have been
noted in many laboratory personnel who
have been exposed to the virus (Horvath,
L. B., Acta Microbiol. Acta Sci. Hung. 12,
201-206, 1965).

isolations of SV40 have been reported from
humans, twice from patients suffering from
the rare demyelinating disease, progressive
multifocal leukoencephalopathy (Weiner, L.,
Herndon, R., Narayon, O., Johnson, R. T,
Shah, K., Rubinstein L. G. Prezozisi T. J. and
Conley, F. K., New England J. Med 286, 385-
390, 1972) and apparently from a tumor of a
person with metastatic melanoma (Sorianc,
F., Shelburne, C, E. and Gokcen, M., Nature.
249, 421-424, 1974). In other studies a non-
structural antigen characteristic of papova-
viruses, T antigen, has been detected in the
nuciei of cells cultured from 2 meningiomas.
while another SV40-specific antigen, U
antigen, has been found in the cells of
a third tumor of the same type (Weiss, A. F..
Portman, R., Fisher, H., Simon, J. and Zang,
K. D., Proc, Nat. Acad, Sci, USA 72, 609-613.
1975}. Purthermore new papovaviruses have
heen isolated from the brains of patients with
PML (JC virus-Padgett, B. L., Walker, D. L..
zuRhein, G. M., Eckroade, R. I. and Desse}.
B. H., Lancet 1, 1257-1260, 1971), from the
urine of a patient carrying a renal allograit
(BK virus-Gardner, S, D., Field, A. M., Cole-
man, D. V. and Hulme, B. Lancet 1, 1253-1257,
1971) and from a reticulum cell sarcoma and
the urine of patients with the sex-linked
recessive disorder, Wiskott-Aldrich syndrome
(Takemoto, K, K., Rabson, A. S., Mullarkey,
M. F., Blaese, R. M. Garon, C. F. and Nelson.
D. J., Nat. Cancer Inst., 53, 1205-1207, 1974)
AN of these viruses which are distributed
widely throughout human populations share
antigenic and biological properties with &V
40; the virus particles are identical in size
and architecture (Madeley, C. R., In Virus
Morphology, Churchill-Livingstone, London.
134-135, 1972); the non-structural intrace!-
lular T antigen, which appears to be coded by
the A gene of SV40 cross reacts extensively
with antigens found in cells infected ct
transformed by BK or JC viruses: both Ic
and BK viruses induce tumors in newborn
hamsters (Walter, D. L., Padgett, B. L., Zu-
Rhein, B. M., Albert, A. E. and Marsh, R. F..
Science 181. 674-676, 1978: Shah, K. V.,
Daniel, R. W. and Strandberg, J., J. Nat, Can-
cer Inst. 54, 945-950, 1975); BK virus causes
transformation of hamster cells in culture
(Major, E. D., and DiMayorca, G., Proc. Nat.
Acad. Sel. US 70, 3210-3212, 1973: Portolani,
M., Barbanti, A., Brodano, G. and LaPlaca.,
M.J., Virol, 15, 420-422, 1975) and ig able to
complement the growth of certain tempera-
ture-sensitive mutants of SV40 (Mason, D. H
and Takemoto, K, K., submitted for publica-
tion).

FURTHER WORK

At present, a potential eukaryotic vecvor
of choice is polyoma virus. And while avail-
able information indicates that it fulfils
aH the necessary criteria, we recommend
that the following subjects be further in-
vestigated:

1. The molecular mechanism of resistance
of human celis to the virus.

2. The extent of homology between poly-
oma virus DNA and the DNAs of human

papovaviruses.

3. The ability of human papovaviruses ta
complement defective polyoma virne
genomes.

Report of a Working Group Consisting of:

Dr. Bernard Fields, Harvard University
School of Medicine.

Dr. Thomas J. Eelly, Jr., Johns Hopkins
University School of Medicine.

Dr. Andrew Lewis, National Institute of Aij-
lergy and Infectious Diseases.

Dr. Malcolm Martin, National Institute of

FEDERAL REGISTER, VOL. 41, NO. 176-—--THURSDAY, SEPTEMBER 9. 1976
Allergy and Infectious Diseases.

Dr. Robert Martin, National Institute of
Arthritis, Metabolism,.and Digestive Dis-
eases.

Dr. Elmer Pfefferkorn, Dartmouth Medical
School.

Dr. Wallace P. Rowe, National Institute of
Allergy and Infectious Diseases.

Dr. Aaron Shatkin, Roche Institute of
Molecular Biology.

Dr. Maxine Singer, National Cancer Insti-
tute.

Rapporteur: Dr. Joe Sambrook, Cold Spring
Harbor Laboratory.

APPENDIX C TO APPENDIX D

SUMMARY OF THE WORKSHOP ON THE DESIGN
AND TESTING OF SAFER PROKARYOTIC VEHICLES
AND BACTERIAL HOSTS FOR RESEARCH ON RE~
COMBINANT DNA MOLECULES

Torrey Pines Inn, La Jolla, California

The development of techniques for the
cloning of DNA from both prokaryotic and
eukaryotic organisms in bacteria has had
great impact on research in biology and
medicine and promises extraordinary social
benefits. The biohazards involved in the use
of this technology in many instances are
very difficult to assess. For this reason codes
of practice are being formulated in the
United States and other countries for the
conduct of those experiments that present
@ potential biohazard. One of the require-
ments for conducting certain cloning experi-
ments is the use of safer vector (bacterio-
phage or plasmid) -host systems, i.e., vector-
bacterium systems that have restricted ca-
pacity to survive outside of controlled condi-
tions in the laboratory. Approximately sixty
scientists from the United States and several
foreign countries participated in a workshop
on the Design and Testing of Safer Prokaryo-
tic Vehicles and Bacterial Hosts for Research
on Recombinant DNA Molecules at La Jolla,
California, on December 1 to 3, 1975. The
workshop was sponsored by the Research
Resources Branch of the National Institute
of Allergy and Infectious Diseases. The pur-
poses of the meeting were the exchange of
recent data on the development of safer
prokaryotic host-vector systems, devising
methods of testing the level of containment
provided by these systems and exploring the
various directions that future research should
take in the construction of safer bacterial
systems for the cloning of foreign DNA.

The first session of the workshop, chaired
by W. Szybalski (University of Wisconsin),
was devoted to bacterlophage vectors. Szy-
balski outlined the main safety features of
the two-component, phage-bacterlal system,
in which the host bacteria offer the safety
feature of not carrying the cloned DNA, and
the phage vectors cannot be propagated in
the absence of an appropriate host. There
are two primary escape routes for the clones
of foreign DNA carried by the phage vector:
(1) Establishment of a stable prophage or
plasmid in the laboratory host used for phage
propagation, and subsequent escape of this
self replicating lysogen or carrier system, and
(2) escape of the phage vector which carries
the cloned DNA and its subsequent produc-
tive encounter with a suitable host in the
natural environment. The general consensus
was that to ensure safety, both routes should
be blocked by appropriate genetic modifica-
tions. For phage i, route (1) can be blocked
by phage mutations that interfere with lyso-
genization (att-, int-, cI-, cIII-, vir) and
plasmid formation (Nt, ninR, vS, rie, cl7;
Ots, crots), and by mutations on the Escheri-
chia coli host that affect these processes
(attB-, dncAts) and host survival. Route (2),
(which is of low probability since \ phages
do not survive well in natural environments
(no AcI phage was recovered after ingestion
of 10*-10" particles), are killed by desicea-

NOTICES

tion, and have a low chance to encounter a
naturally sensitive host) can be blocked
further by the following phage modifications:
(a) Mutations which result in extreme insta-
bility of the infectious phage particles under
all conditions other than those specially de-
signed for phage propagation in the labora-
tory (e.g., high concentrations of putrescine
or some other compound), or (b) employing
phage vectors in which the tail genes are
deleted and which permit propagation of
only the DNA-packed heads; only under lab-
oratory conditions could such heads be made
transiently infectious by rejoining them
with separately prepared tails. The high in-
stability of the phage would minimize the
possibility of transfer of the cloned genes
into receptive bacteria found in nature.
Moreover, the propagation of the phage can
be blocked by many conditional mutations,
which would be designed to block any sec-
ondary route of escape, mainly depending on
transfer of the cloned DNA into another
phage or bacterial host. It was recommended
further that the vector be designed in such
@ manner as to permit easy insertion and
monitoring of the foreign DNA and rapid
assay of the safety features and give a high
yield of cloned DNA (not less than 104 mole-
cules per ml). There also was general agree-
Ment that host-phage systems other than
E, coli should be considered, especially those
restricted to very rare and unusual environ-
ments. Also, plasmids derived from phage
vectors and which give very high DNA yields
while exhibiting safety features, e.g., \dvcrots,
should be considered as vehicles for cloned
DNA.

Szybalski and S. Brenner (Cambridge Unl-
versity) stressed that research on recombi-
nant DNA molecules may lend itself to
very simple and inexpensive mechanical con-
tainment, eg. a small sealed glove box,
since all the vectors that carry such re-
combinant molecules possibly can be both
created and destroyed in such a box, while
development of special methods might per-
mit study of many properties of the recom-
binant DNA, without ever removing it from
the box.

These safety features were reflected in
the subsequent presentations. F. Blattner
and W. ‘Williams (Universiy of Wisconsin)
described four specially constructed \-¢80
phages which incorporate many of these
safety features, and which they named
Charon phages, for the mythical boatman of
the river Styx. Some of these highly con-
tained phages give yields of over 10" parti-
cles/ml. R. Davis, J. Cameron and K. Struhl
(Stanford University) found that \ phages
that carry foreign DNA never grow as well
as the parental vector, which would select
against their survival in nature. They also
reported that some eukaryotic genes could
be expressed in £. coli, partially compensat-
ing for deficiencies in the histidine pathway
or in polA or lig functions. These investi-
gators surveyed over 1000 strains of E. coli
isolated in the natural environment and
did not find a single strain that could sup-
port propagation of the dvir vector.

V. Bode (Kansas State University) dis-
cussed the possibility of growing tail-free
4 heads. Such heads, which are packed with
DNA, are very fragile, unless stored in 0.1 M
putrescine buffer. Head yields close to 10"/ml
could easily be attained and, when required,
heads could be quantitatively rejoined with
separately supplied tails under special lab-
oratory conditions. W. Arber, D, Scandella
and J. Elliott (University of Basel) described
bacterial host mutants that permit efficient
infection only by phages with a full com-
plement of DNA. This permits selecting for
vectors that carry long fragments of foreign
DNA.

K. Matsubara, T. Mukai and Y. Takagi
(University of Osaka and Kyushu Univer-

38468

sity), and G. Hobom and P. Phillippsen
(University of Freiburg and Stanford Univer-
sity) described various defective i plasmids
(Adv) that could be used as efficient vectors.
Matsubara has shown that temperature-
sensitive cro mutations permit obtaining be-
tween 1000 and 3000 cloned molecules per
cell and at the same time result in killing
of the carrier cells at body temperature. The
mutations Ots and Pts were also evaluated
as safety features. Phillippsen described
many new Adv plasmids created by cutting
A DNA with HindIII and BamlI restriction
endonucleases followed by ligation. The final
talk by F. Young, G. Wilson and M. Williams
(University of Rochester) summarized the
progress on the development of safer
Bacillus subtilis host mutants and phages,
especially ¢3, as vectors. New restriction
nucleases, Bgl-1 and Bgl-2, were also de-
scribed.

The morning session on bacteriophage
vectors was followed by a session on plasmid
vectors that was chaired by D. Helinski (Uni-
versity of California, San Diego). Helinski
presented the following properties as highly
desirable characteristics of a safer plasmid
vehicle: (a) Non-conjugative; (b) non-
mobilizable or poorly mobilizable by a con-
Jugative plasmid; (c) possesses little or no
extraneous genetic information; (d)} poorly
recombines or does not recombine with the
chromosome of the host cell; (e) provides
no selective advantage to the host cell or the
selective property is conditional; and (f)
possesses mutations that restrict its mainte-
nance to a specific host, prevent replication
at mammalian body temperature and/or
provide with the capability of killing any
cell to which it might be transmitted other
than the host cell. V. Hershfield (University
of California, San Diego) described the prop-
erties of a variety of derivatives of the ColE1
derivatives, ColEl-trp, constructed in col-
laboration with C. Yanofsky and N. Franklin
(Stanford University) provides the means to
use the tryptophan genes of E. coli as a se-
lective marker in transformation with re-
combinant DNA in situations where it is de-
sirable to avoid antibiotic resistance genes.
In addition, Hershfield described collabora-
tive work with H. Boyer that resulted in the
development of a mini-ColE1 plasmid and
derivatives of this plasmid (mini-ColE1-kan
and mini-ColEl-irp) as cloning vehicles.
Finally, she described the temperature-sensi-
tivity properties of trp and kan derivatives
of a temperature-sensitive replication mu-
tant of ColE1 isolated by J. Collins (Molecu-
lar Biology Institute, Stockhelm) and hybrid

‘ ColE1 plasmids carrying the EcoRI generated

Cts fragment of bacteriophage \-trp61.

J. Carbon (University of California, Santa
Barbara) described a replica plating method
that greatly facilitates the detection of E. coli
clones bearing ColE1l plasmids. The proce-
dure, which utilizes the F, plasmid to pro-
mote the transfer of a hybrid ColEl plasmid
to @ suitable auxotrophic recipient, was suc-
cessful in identifying clones bearing hybrid
Plasmids carrying a number of different re-
gions of the £. coli chromosome. The con-
tributions of A. J. Clark and collaborators
(University of California, Berkeley) were rel-
evant to the problem of the mobilization and
subsequent transfer of non-conjugative
plasmids carrying foreign DNA of a poten-
tially hazardous nature. Clark described the
variations in transmission frequencies be-
tween the nonconjugative plasmids pSc10t,
PML31, pSC138 and a number of pSC101 hy-
brids containing various EcoRI fragments of
F when the conjugal transfer of these
plasmids was promoted by several different
conjugative plasmids.

I. C. Gunsalus and collaborators (Univer-
sity of Iilinols) and A. Chakrabarty (General
Electric Research and Development Center)

FEDERAL REGISTER, VOL. 41, NO. 176-—-THURSDAY, SEPTEMBER 9, 1976
38164

described the properties of a variety of plas-
mids isolated from Pseudomonas putida.
These contributions were followed by a dis~
eussion on the merits of developing plasmid-
host systems involving Pseudomonas strains
that naturally exhibit unusual growth re-
quirements. Similar studies with plasmids
isolated from Bacillus megaterium by EB.
Carlton (University of Georgia) from B. sub-
tilis by P. Lovett (University of Maryland)
and other naturally occurring Bacillus
species by W. Goebel and K. Bernhard
{Microbiology Institute, Wurzburg) were dis-
cussed and their further development as
plasmid-host cloning systems was explored.
It was clear from these presentations that
considerable progress has meen made re-
cently in the identification and characteriza-
tion of a variety of plasmid elements that oc-
cur naturally in Pseudomonas and Bacillus
species, Several of the plasmids described
show considerable promise as plasmid cloning
systems involving a host other than E. coli.

A third session on the ecology and epi-
demiology of vector-host systems was chaired
by S. Falkow (University of Washington).
This workshop emerged, in part, from ex-
pressed fears that microorganisms contain-
ing cloned fragments of foreign DNA may
potentially pose a threat to health or dis-
rupt the normal ecological chain in some
manner. Consequently, this session was de-
voted to a review of currently available in-
formation on the ecology and epidemiology
of E. colt and related bacterial species since
it was recognized that Z. coli K-12 would be
the prokaryotic host most commonly em-
ployed in the cloning of DNA molecules in
the immediate future. F. @rskov (Escherichia
Reference Center, Copenhagen) reviewed the
state of E. coli serotyping and what has been
learned about the distribution of E. coli types
in health and disease. Only certain E. coli
types are generally recognized as good colo-
nizers of the human gut and such strains
come from a handful of the 160 well defined
O (Hpopolysaccharide) antigen types and in~
variably possess EK (acidic polysaccharide
capsule) antigens. Some serotypes apparently
have become disseminated worldwide and
possibly represent the proliferation of a bac-
terial clone because of, as yet unknown,
selective pressures. In contrast, E. coli K-12
has no detectable O or K antigens and is
considered to be rough. This may account,
at least in part, for its demonstrate poor
ability to colonize the human or animal gut.
However, R. Freter (University of Michigan)
pointed out that we still remain largely
ignorant of the factors which control in-
testinal FE. coli populations. Freter also noted
that while adherence to the mucosal surface
of the small intestine is important in the
pathogenesis of FE. coli diarrheal disease, the
‘normal’ long-lasting symbiotic relationship
between a mammalian host and bacterium js
established in the cecum and colon. It jis in
these locations that factors come into play to
determine whether an E£. coli strain passing
through the intestine will become success-
fully implanted or whether it will be quickly
eliminated in the feces. The factors control-
ling implantation include competition for
substrates, inhibitors and the physiological
state of the organism when it reaches the
large bowel. For example, ingested E. coli
previously grown under usual laboratory
conditions fare poorly while cells of the same
strain ‘pre-adapted’ in Eh, pH, etec., often
colonize well. Freter has developed a con-
tinuous flow culture model which may be
useful in studying the mechanisms of im-
plantation. Falkow reviewed the pathogenic-
ity of E. coli. B. coli causes diarrheal disease
either by direct tnvasion of the bowel epi-
thelium or by elaboration of enterotoxin(s).
While invasive E. coli appear to owe their
pathogenicity to a constellation of at least

NOTICES

five unlinked chromosomal gene clusters,
toxigenic E. coli species generally owe their
pathogenicity to the possession of two
species, Ent and K. The introduction of Ent
and K plasmids may be sufficient to convert
@ normal wild-type E. coli into a strain now
capable of causing overt clinical disease.
However, the introduction of these plasmids
into E. coli K-12 sublines had no discernible
effect on their ablity to cause disease, al-
though the K-12 strains could now better
colonize calves. Despite the observation that
E, coli K-12 did not appear to offer a signifi-
eant hazard as a potential enteric pathogen
even when it possessed well-defined deter-
Tainants of pathogenicity it was emphasized
by @rskov, Freter and Falkow that E. coli
K-12 strains carrying recombinant DNA
molecules could still act as effective genetic
donors in vivo and still posed a significant
problem requiring control. E. Geldreich (U.S.
Environmental Protection Agency, Cincin-
nati, Ohio) discussed the possible outcomes
of the release of EF. coli containing recom-
binant DNA molecules into the aquatic en-
vironment and concluded that total reliance
cannot be placed. on sewage treatment and
the natural self-purification capacity of re-
ceiving waters to limit potential hazards.
While these are realistic barriers to the dis-
semination Of E. coli and associated fecal
organisms via the water route, they are not
infallible because of technological Hmita-
tions, improper operational practices and
system overloading. Finally, M. Starr (Uni-
versity of California, Davis) described the
numerous genera of gram-negative bacteria
found naturally occurring in the soil and on
plants. He stated. that most of these orga-
nisms do not appear to be a reasonable alter-
native to EF. coli K-12 as a host for recom-
binant DNA molecules. Indeed, Starr pointed
out that since such genera as Erwinia, Rhiz-~
obium and Agrobacterium are known to con-
jJugate with £. coli, the potential dissemina-
tion of recombinant DNA molecule includes a
greater spectrum of microorganisms than
just enteric species.

The fourth session of the workshop,
chaired by R. Curtiss III (University of Ala-
bama), was concerned with the construction
of safer bacterial hosts for DNA cloning. The
goals in constructing safer host strains enu-
merated at the beginning of the session in-
cluded introduction of mutations that
would: (a) Preclude colonization in normal
ecological niches; (b) preclude cell wall bio-
synthesis except in specially defined media;
(c) cause degradation of genetic information
in normal ecological niches; (d) cause vec-
tors to be host-dependent; (e) minimize
transmission of recombinant DNA to other
strains in normal ecological niches; (f) in-
crease usefulness for recombinant DNA
molecule research; and (g) permit moni-
toring.

Most of the progress in developing safer
hosts has been achieved with £. coli K-12,
although F. Young described a B. subtilis
strain with a deletion for sporulation genes
which readily undergoes autolysis. The
strain also has defects in genes for purine
and TTP biosynthesis and a mutation con-
ferring a D-alanine requirement can be in-
troduced to cause cell wall biosynthesis to
be defective. This strain may be defective in
transformation, however, and therefore
might useful only with a phage vector
which has yet to be developed and/or dis-
covered.

A. I. Bukhari (Cold Spring Harbor Labora-
tory) described the use of the dapD8 muta-
tion in £. coli K-12 to block cell wall bio-
synthesis and another non-reverting muta-
tion which causes sensitivity to bile salts
and detergents, The dapD8 allele is the most
stable dap point mutation known, although
it does revert at frequencies of 10-3 to 10-*.

The mutation conferring bile salts sensivity
was obtained after Mu-1 infection of an Hir
strain and, although exhibiting the theoret-
ically useful properties of ease of DNA isola-
tion and inability to survive in the intestinal
tract, might be due to Mu insertion which
would compromise its use for safe strain
construction,

Curtiss reported on the work performed by
him and his coworkers in constructing and
testing numerous sirains with different mu-
tations, Survival of strains in vivo was tested
by feeding rats 10" cells in milk by stomach
tube. Apur mutations did not reduce strain
titers in feces whereas AthyA,; AthyA drm;
and AthyA dra mutations gave 10?-fold, 10?-
fold and 10'-fold reductions, respectively, in
strain titers in feces. Strains with AthyA
rautations also exhibited thymineless death
in in vitro tests. Since strains with dapDs
allele can revert to Dap’, strains were con-
structed with both dupD8 and AbioH-asd
mutations. These strains have not been ob-
served to revert to Dap* but can survive
passage through the rat intestine and in
growth media lacking diaminopimelic acid
but containing NaCl and 0.5% usable car-
bon sources. This survival was due to the
production of the mucopolysaccharide, col-
anic acid, which permits many of the cells
to grow and survive as spheroplasts. A Agal-
cht" mutation (also deletes att, bio and
uvrB genes) was introduced which blocks
colanic acid biosynthesis and leads to no de-
tectable survivors in media lacking diamino-
pimelic acid or following passage through the
rat intestine. The dapD8 AbioH-asd Agal-
cht strains are more readily lysed, trans-
form at higher frequencies and are conju-
gation-defective in matings with donors
possessing conjugative plasmids in the
P, W and O incompatability groups but
Con’ as recipients for F, I and T group
plasmids when compared to the dap* gal+
parent strain. Strains with endéA muta-
tions were also observed to exhibit in-
creased transformation frequencies. Attempts
to introduce temperature-sensitive polA
alleles- into strains to block replication
of ColE] cloning vectors at elevated temper-
atures and to cause DNA degradation at ele-
vated temperatures in the presence of recA
and Athy alleles often do not have the same
properties in the constructed strains as in
the strains in which the allele was original-
ly induced, Many mutations causing a Con-
phenotype have been investigated, but many
of these revert and/or do not exhibit a Con-~
phenotype in matings with donors possess-
ing conjugative plasmids of the incompati-
bility groups commonly found in enteric
microorganisms. Some Con- mutants exhibit
increased sensitivity to bile salts; thus, the
mutant described by Bukhari may also ex-
hibit a Con- phenotype. All of the strains
constructed by the Curtiss group are SulI+
and most have mutations abolishing restric-
tion alone or both restriction and modifica-
tion, Thus, sufficient information is now
known to construct a usable safer E. coli
K-12 host. Curtiss and collaborators are now
introducing AthyA and dna mutations inte
their dapD8& AbioH-asd Agal-chl'-uvrB nAsr
nalA* (for ease in monitoring) Su \* #80"
strain to accomplish this objective.

The final session involved a general dis-
cussion of some of the major points raised

* previously in the workshop. There was gen-

eral agreement at this session that both
plasmid-host and phage-host systems have
been developed that should meet the criteria
of an EK2 system specified by the National
Institutes of Health guidelines for research
on recombinant DNA molecules. Additional
testing is required to confirm the EK2 prop-
erties of these available systems, but it is
anticipated that these vector-host systems
will meet these tests.

FEDERAL REGISTER, VOL. 417, NO. 176—THURSDAY, SEPTEMBER 9, 1976
Dr. Donald R. Helinski, University of Cali-
fornia, San Diego.
Dr. Stanley Falkow, University of Washing-
ton.
Dr. Roy Curtiss IIT, University of Alabama.
Dr. Waclaw Szybalski, University of Wiscon-
sin,
APPENDIX D TO
APPENDIX D

SUPPLEMENTARY INFORMATION ON PHYSICAL
CONTAINMENT

CONTENTS

I. Biological safety cabinets.
Table I.
II. Universal biohazard warning symbol.

III. Laboratory techniques for biohazard

control.

. Pipetting.

. Syringes and needles.

. Opening culture plates, tubes, bot-

tles, and ampoules.

. Centrifuging.

. High-speed centrifuges.

. Blenders, ultrasonic disintegrators,

colloid mills, ball mills, Jet mills,
’ grinders, mortar and pestle.
G. Miscellaneous precautions and rec-
ommendations.

. Personal hygiene, habits, and practices.

V. Care and use of laboratory animals.

A. Care and handling.

B. Cages housing infected animals.

C. General guidelines that apply to

animal room maintenance.

D. Necropsy rules for infected animals.

Decontamination and disposal,

. Introduction. ,

. Decontamination methods.

. Laboratory spills.

Disposal.

Characteristics of chemical decon-
taminants in common use in labo-
ratory operations.

Properties of some common decon-
taminants.

. Vapors and gases.

. Residual action of decontaminants.

. Selecting chemical decontaminants

for research on recombinant DNA
molecules.
Table II. __

. Housekeeping.

A. Introduction.

B. Floor care.

Cc. Dry sweeping.

D. Vacuum cleaning.

E. Selection of a cleaning solution.

F. Wet mopping—two-bucket method.

@. Alternative floor cleaning method

for animal care areas and areas
with monolithic floors.

Clean-up of biohazardous spills.

A. Biohazardous spill in a biological

safety cabinet.

B. Biohazard spill outside a biological

safety cabinet.

Cc, Radioactive biohazard spill outside a

biological safety cabinet.

A secondary reservoir and filtration
apparatus for vacuum systems.

X. Packaging and shipping.

A. Introduction.

B. Packaging of
materials.

C. Labeling of packages containing re-
combinant DNA materials.

D. Additional shipping requirements
and Hmitations for recombinant
DNA materials.

SHO QWpP

VI.

HOOD

Homo wl

VIII.

recombinant DNA

NOTICES
Table III.
Table Iv.
XI. Training aids, materials and courses.
A. Slide-tape cassettes.
' B. Films.
Cc, Courses,
XII. Outline of a safety and operation

manual for a P4 facility.
References.

I. BrotocicaL SAFETY CABINETS

Biological Safety Cabinets suitable for
confining operations involving recombinant
DNA molecules are described below:

1. Class I. A ventilated cabinet for person-
nel protection only, with an unrecirculated
inward flow of air away from the operator.
The exhaust air from this cabinet may be
filtered through a high-efficiency or high-
efficiency particulate air (HEPA) filter be-
fore being discharged to the outside atmos-
phere. This cabinet is suitable for research
work with the Center for Disease Control
(CDC) classes of etiologic agents 1, 2 and
3 where no product protection is required.
This cabinet may be used in three opera-
tional modes: (i) With an eight-inch high,
full-width open front; (ii) with an installed
front closure panel (having four, eight-inch
diameter openings) without gloves; and (ili)
with an installed front closure panel
equipped with arm length rubber gloves. See
Table I for ventilation requirements, agent
use limitations, and minimum performance
requirements.

2. Class II. A ventilated cabinet for per-
sonnel and product protection having an
open front with inward air flow for per-
sonnel protection, and HEPA-filtered re-
circulated mass air flow for product protec-
tion. The cabinet exhaust air is filtered
through a HEPA filter. Two models of this
cabinet are available, Type 1 and Type 2.

(i) Type 1. The Type 1 recirculates ap-
proximately 70 percent of the air. The ex-
haust air from this cabinet may discharge

38165

into the laboratory or be diverted out of the
laboratory. This cabinet is suitable for CDC
classes of etiologic agents 1, 2, and 3. Vapors
or gases which are hazardous from a4 toxic,
radioactive, or flammability standpoint
should not be used in this cabinet because of
the high quantity of recirculated air.

(li) Type 2. The Type 2 cabinet recirculates
approximately 30 percent of the air. The ex-
haust air from this cabinet is normally
ducted out of the laboratory through a HEPA
filter and, occasionally, an activated charcoal
filter depending on the operation. The cab-
inet may be used with gases or vapors that
are hazardous from a toxic, radioactive, or
flammability standpoint. However, any con-
sideration of use of such materials should
be evaluated carefully from the standpoint
of build-up to dangerous levels and problems
of decontamination of the cabinet. See Table
I for ventilation requirements, agent use lim-
itations, and minimum performance require-
ments.

3. Class III. A closed front ventilated cab-
inet of gastight construction providing total
protection for personnel and product from
contaminants exterior to the cabinet. The
cabinet is operated under a negative pres-
sure of at least 0.5 inches water gauge. All
supply air is HEPA-filtered. Exhaust air is
HEPA-filtered or incinerated to protect the
environment. This cabinet, fitted with arm
length rubber gloves, provides the highest
containment of these three classes of cab-
inets and is utilized for all activities involv-
ing high risk agents (ie, CDC etiologic
agents, class 4). See Table I for ventilation
requirements, agent use limitations, and
minimum performance requirements.

The integrity of any cabinet depends on
initial and periodic evaluation to meet estab-
lished performance tests. Table I outlines the
minimum performance required to assure
that the cabinets will provide protection of
‘personnel and the environment.

 

 

 

 

 

 

 

 

TABLE I
BIOLOGICAL SAFETY CABINETS
BAPETY PERFORMANCE REQUIREMENTS AND SPECIFICATIONS
cure 1976
CABINE? USE CLASSIPICATION | PERFORMANCE REQUI
met = on TTY EXHAUST AIR (CEM)® © LEAK TIGHTNESS «= EXHAUST.
(linear feet PILTER
_ — per minute) "hood 6*hood . EFFICIENCY
zB
Class £ PI-P3 2-3 75 200 300 Not applicable 99,974 s
g
2
Glass It, Type 2 Pi-73 1-3 B 260 400 Gas tights >
Leak pate < 99.978 e
1x10°° cc/sec o
et 2%wg pressure oe
Glass IT, Type 2 PIP} 1-3 300 250 *% Pressure tights
No air/scap 99.976
bubble at 2*wg
Pressure
Class 11r et 4 Mot applicable a a Ges tights
bate ee < 9.570
Sie

at Seg Fe pressure

 

“a - Foc work with recombinant DA nolecul
b ~ Center for Disease Control (YS Public E Health Service)e
© ~ Cr#=cubic feet per minute.

@ = Based on one volume of alr Change each 3 minutes, In the absence of unusual

heat or moisture that would cequice more aig changes,

FEDERAL REGISTER, VOL. 41, NO. 176——THURSDAY, SEPTEMBER 9, 1976
38166

Ti. UNIVERSAL BIOHAZARD WARNING SYMBOL (1)

The biological hazard warning symbol
(biohazard symbol) specified herein shall be
used to signify the actual or potential pres-
ence of a bichazard and to identify equip-
ment, containers, rooms, materials, experi-
mental animals or combinations thereof
which contain or are contaminated with
viable hazardous agents.

The biohazard symbol shall be designed
and proportioned as illustrated here:

 

 

 

 

 

 

i

 

 

[vimensron [alelciolelr lala]
Hes dstebiena

The symbol shall be as prominent as prac-
tical, and of a size consistent with the size
of the equipment or material to which it is
afixed, provided the proportions shown
above are maintained, and, in any case, that
the symbol can be easily seen from as many
directions as possible,

Except when circumstances do not permit,
the symbol shall be oriented with one of the
three open circles pointed up and the other
two forming a base.

The symbol color shall be a fluorescent
orange or orange-red color.* Background
color is optional as long as there is sufficient
eontrast for the symbol! to be clearly defined.

o

 

Revised 3-39-66
wPay-Clo™ Fire Crange of Lhe Switzer Ercthers, Inc, ia efted am an exerpley
Sot an endursement,

The biohazard symbol shall be used or
displayed only to signify the actual or
potential presence of biological hazard.

Appropriate wording may be used in as-
sociation with the symbol to indicate the
nature or identity of the hazard, name of
individual responsible for its control, pre-
cautionary information, etc. but never
should this information be superimposed on
the symbol. (See next page)

NOTICES

 

    

ADMITTANCE 10 AUTHORIZED PERSONNEL ONLY
Hazard identity:
Responsible Investigator:

 

In cose of emergency call:

 

 

_. Home phone __

Daytime phone

 

Authorizotion for entrance must be obtained from
the Responsible Investigator named above,
III, LABORATORY TECHNIQUES FOR BIOHAZARD
CONTROL

A. Pipetting. 1. No infectious or toxic ma-
terials should be pipetted by mouth (2, 3,4).

2. No infectious mixtures should be pre-
pared by bubbling expiratory air through 4
liquid with a pipette (2,3, 4).

3. No infectious material should be blown
out of pipettes (2, 3,4).

4. Pipettes used for the pipetting of in-
fectious or toxic materials should be plugged
with cotton (2, 3,4).

5. Contaminated pipettes should be placed
horizontally in a pan containing enough
suitable disinfectant to allow complete im-
mersion (2,3,4). They should not be placed
vertically in a cyclinder.

6. The pan and pipettes should be auto-
claved as a unit and replaced by a clean pan
with fresh disinfectant (2,3, 4).

7. Infectious material should not be mixed
by alternate suction and expulsion through
a pipette (2,3, 4).

8. Mark-to-mark pipettes are preferable
to other types, as they do not require ex-
pulsion of the last drop (5).

9. Discharge should be as close as pos-
sible to the fluid or agar level, or the con-
tents should be allowed to run down the
wall of the tube or bottle whenever possible—
not dropped from a height (5).

10. A disinfectant-wetted towel over the
immediate work surface is useful in some
cases to minimize the splash from accidental
droppage (9).

B. Syringes and Needles (9). 1. To lessen
the chance of accidental injection, aerosol
production or spills, avoid unnecessary use of
the syringe and needle. For instance:

(i) Use the needle for parenteral injec-
tions but use a blunt needie or a cannula on
the syringe for oral or intranasal inocula-
tions.

(ii) Do not use a syringe and needle as a
substitute for a pipette In making dilutions
of dangerous fluids.

2. Use the syringe and needle in a Biologi-
cal Safety Cabinet only and avoid quick and
unnecessary movements of the hand holding
the syringe. -

3. Examine glass syringes for chips and
cracks, and needles for barbs and plugs.

Note: This should be done prior to steril-
ization before use.

4. Use needle-locking {Luer-Lok® type)
syringes oniy, and be sure that the needie is
locked securely into the barrel. A disposable
syringe-needle unit (where the needle is an
integral part of the unit) is preferred.

5. Wear surgical or other type rubber gloves
for all manipulations with needles and
syringes.

6. Fill the syringe carefully to minimize
air bubbles and frothing of the inoculum.

7. Expel excess air, liquid and bubbles
from a syringe vertically into a cotton pledget
moistened with the proper disinfectant, or
into a small bottle of sterile cotton.

8. Do not use the syringe to expel force-
fully a stream of infectious fluid into an open
vial or tube for the purpose of mixing. Mix-
ing with a syringe is condoned only if the
tip of the needle is held below the surface
of the fluid in the tube.

9. If syringes are filled from test tubes,
take care not to contaminate the hub of the
needle, as this may result in transfer of in-
fectious material to the fingers.

10. When removing a syringe and needle
from a rubber-stoppered bottle, wrap the
needle and stopper in a cotton pletget mois-
tened with the proper disinfectant. If there
is danger of the disinfectant contaminating ~
sensitive experiments, a sterile dry pledget
may be used and discarded immediately into
disinfectant solution.

11. Inoculate animals with the hand
“behind” the needle to avoid punctures.

12. Be sure the animal is properly re-
strained prior to the inoculation, and be on
the alert for any unexpected movements of
the animal,

13. Before and after injection of an animal,
swab the site of injection with a disinfectant.

14, Discard syringes into a pan of disin-
fectant without removing the needle. The
syringe first may be filled with disinfectant
by immersing the needle and slowly with-
drawing the plunger, and finally removing
the plunger and placing it separately into the
disinfectant. The filling action clears the
needle and dilutes the contents of the
syringe. Autoclave syringes and needles in
the pan of disinfectant.

15. Use separate pans of disinfectant for
disposable and nondisposable syringes and
needles to eliminate a sorting problem in the
service area.

16. Do not discard syringes and needies
into pans containing pipettes or other glass-
ware that must be sorted out from the
syringes and needles.

C. Opening Culture Plates, Tubes, Bottles,
and Ampoules. 1. Plates, tubes and bottles of
fungi may release spores in large nur bers
when opened. Such cultures should be ma-
nipulated in a Biological Safety Cabinet
(6,15).

2. In the absence of definite accidents or
cbvious spillage, it is not certain that open-
ing of plates, tubes and bottles of other
microorganisms has caused laboratory in-
fection. However, it is probable that among
the highly infective agents, some infections
have occurred by this means and are repre-
sented in the 80% for which no known act
or accident is ascribable (3).

3. Water of syneresis in petri dish cul-
tures is usually infected and forms a film
between the rim and Hd of the inverted

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
plate. Aerosols are dispersed when this film
is broken by opening the plate. Vented plas-
tic petri dishes where the lid touches the
rim at only three points are less likely to
offer this hazard (8,19).

4. The risk may also be minimized by us-
ing properly dried plates, but even these
(when incubated anaerobically) are likely to
be wet after removal from an anaerboci jar,
Filter papers fitted into the lids reduce, but
do not prevent, dispersal. If plates are
obviously wet they should be opened in the
Biological Safety Cabinet (8).

5. Less obvious is the release of aerosols
when screw-capped bottles or plugged tubes
are opened. This happens when a film of in-
fected liquid which may collect between the
rim and the liner is broken during removal
of the closure (8).

6. Dried, infected culture material may
also collect at or near the rim or neck of
culture tubes and may be disposed into the
air when disturbed (18). Containers of dry
powdered hazardous materials, (e.g., Class 3
fungal agents in the spore phase of growth)
should be opened only in a Biological Safety
Cabinet (6, 14).

7, When the neck of an ampoule contain-
ing Hquid is broken after nicking with 4 file,
the snapping action creates aerosols. The
following methods have been recommended:

(i) After nicking the ampoule with a file,
wrap the ampoule in disinfectant-wetted
cotton before breaking. Wear gloves (2).

(ii) The bottom of the ampoule should be
held in several layers of tissue paper to pro-
tect the hands, and a file mark made at the
neck. A hot glass rod should be carefully
applied to the mark. The glass will crack, al-
lowing air to enter the ampoule and equalize
the pressures. After a few seconds the am-
poule should be wrapped in a few layers of
tissue and broken along the crack. The tis-
sues and ampoule neck can then be discarded
into disinfectant, and the contents of the
ampoule removed with a syringe. If the am-~-
poule contains dried cultures, about 0.5 cm?
of broth should be added slowly to avoid
blowing dried material out. The contents
may then be mixed without bubbling and
withdrawn into a culture tube (8).

(iii) The researcher uses an intense, but
tiny, gas-oxygen flame and heats the tip of
the hard glass ampoule until the expanding
internal air pressure blows a bubble. After
allowing this to cool, he breaks the bubble
while holding it in a large low temperature
flame: this immediately incinerates any in-
fectious dust which may come from the am-
poule when the glass is broken (16). Prelim-
inary practice with a simulant ampoule of
the same type actually in use is necessary to
develop a technique that will not cause ex-
plosion of the ampoule.

(iv) A simple device has been recommend -
ed consisting of a sleeve of rubber tubing
into which the ampoule is inserted before it
is broken (17,18).

D. Centrifuging. 1. A safety centrifuge cab-
inet or safety centrifuge cup (3,7,8,14,22)
may be used to house or safeguard all cen-
trifuging of infectioug substances. When
bench type centrifuges are used in a Bio-
logical Safety Cabinet, the glove panel should
be in place with the glove ports covered. The
“centrifuge operation creates air currents that
may cause escape of agent from an open cab-
inét (2,3,4,13).

2. In some situations, in the absence of O-
ring cap sealed trunnion cups, specimens can
be enclosed in sealed plastic bags before cen-
trifugation (12).

3. Before centrifuging, inspect tubes for
cracks, inspect the inside of the trunnion
cup for rough walls caused by erosion or ad-
hering matter, and carefully remove bits of

glass from the rubber cushion (4,10).

NOTICES

4. A germicidal solution should be added
between the tube and trunnion cup to disin-
fect the materials in case of accidental
breakage. This practice also provides an ex~-
cellent cushion against shocks that might
otherwise break the tube (4,10).

5. Avoid decanting centrifuge tubes. If
you must do so, afterwards wipe of the outer
rim with a disinfectant; otherwise the in-
fectious fluid will spin off as an aerosol
(4, 10).

‘ g. Avoid filling the tube to the point that
the rim, cap or cotton plug ever becomes wet
with culture (4, 10).

7. Screw caps, or caps which fit over the
rim outside the centrifuge tube are safer
than plug-in closures. Some fluid usually
collects betwen a plug-in closure and the
rim of the tube. Even screw-capped bottles
are not without risk, however; if the rim is
soiled some fluid will escape-down the out-
side of the tube. Screw-capped bottles may
jam in the bucket, and removing them is
hazardous. Propping such bottles higher in
the bucket with additional rubber buffers
is mechanically unsound (8).

8. Kitchen foil is often used to cap centri-
fuge tubes. This creates more risk then the
screw cap. Foil caps often become detached
in handling and centrifuging (8).

9. The balancing of buckets is often mis-
managed. Gare must be taken to ensure that
matched sets of trunnios, buckets and
plastic inserts do not become mixed. If the
components are not inscribed with their
weights by the manufacturer, colored
stains can be applied to avoid confusion.
When the tubes are balanced, the buckets,
trunnions and inserts should be included
in the procedure; and care must be taken
to ensure that the centers of gravity of the
tubes are equidistant from the axis of rota-
tion. To illustrate the importance of this,
two identical tubes containing 20g of mer-
cury and 20g of water respectively will bal-
ance perfectly on the scales; but their
performance in motion is totally different,
leading to violent vibration with all its at-
tendant hazards (5).

10. Fill and open centrifuge tubes or
trunnion cups in a Biological Safety Cabinet
(10).

E, High-Speed Centrifuges (22), 1. In high-
speed centrifuges the bowl is connected to
a vacuum pump. If there is a breakage or
accidental dispersion of infected particles the
pump and the oil in it will become con-
taminated. A high efficiency filter should be
placed between the centrifuge and the
pump (8).

2. High speed rotor heads are prone to
metal fatigue, and where there is a chance
that they may be used on re than one
machine each rotor should be accompanied
by its own log book indicating the number of
hours run at top or de-rated speeds. Failure
to observe this precaution can result In
dangerous and expensive disintegration. Fre-
quent inspection, cleaning and drying are
important to ensure absence of corrosion or
other traumata which may lead to creeping
cracks, Rubber O-rings and tube closures
must be examined for deterioration and be
kept lubricated with the material recom-
mended by the makers. Where tubes of dif-
ferent materials are provided (e.g., celluloid,
polypropylene, stainless steel), care must be
taken that the tube closures designed
specifically for the type of tube in use are
employed. These caps are often similar in
appearance, but are prone to leakage if ap-
plied to tubes of the wrong material. When
properly designed tubes and rotors are well
maintained and handled, leaking should
neyer occur (5).

3. Cleaning and disinfection of tubes,
rotors and other components requires con-

38167 ©

siderable care. It is unfortunate that no
single process is suitable for all items, and
the various manufacturers’ recommendations
must be followed meticulously if fatigue, .
distortion and corrosion are to be avoided.
This is not the place to catalogue recom-
mended methods, but one less well appre-
ciated fact is worthy of mention. Celluloid
(cellulose nitrate) centrifuge tubes are not
only highly inflammable and prone to
shrinkage with age and distortion on boiling,
but can behave as high explosive in an auto-
clave (5). Large-scale zonal cenirifugation
requires special attention (11).

F. Blenders, ultrasonic disintegrators,
colloid mills, ball mills, jet mills, grinders,
motar and pestle. All these devices release
considerable aerosols during their operation.
For maximum protection to the operator
during the blending of infectious materials,
the following practices should be observed:

1. Operate blending and cell-disruption
and grinding equipment in a Biological
Safety Cabinet (9).

2. Use safety blenders designed to prevent
leakage from the rotor bearing at the bottom
of the bowl (9).

3. In the absence of a leak-proof rotor, in-
spect the rotor bearing at the bottom of the
blender bowl for leakage prior to operation.
Test it in a preliminary run with sterile
saline or methylene blue solution prior to
use with infected material (9).

4, Sterilize the device and residual infec-
tious contents promptly after use. Use a
towel moistened with disinfectant over the
top of the blender (9).

5. Glass blender bowls are undesirable for
use with infectious material because of po-
tential breakage. If used, they should be cov-
ered with a polypropylene jar to prevent
dispersal of glass (8).

6. A new machine, the Colworth Sto-
macker (England), in which material ts
homogenized in a plastic bag in a closed con-
tainer, would appear to be safer than some
of the other blenders (8).

7. A heat-sealed flexible plastic film en~
closure for a grinder or blender can be used,
put it must be opened in a Biological Safety
Cabinet (7).

8. Blender bowls sometimes require sup~
plemental cooling to prevent destruction of
the bearings and to minimize thermal efforts
on the product (7).

9. Before opening the safety blender bowl,
permit the blender to rest for at least one
minute to allow settling of the aerosol cloud.

10. Clinical or other laboratories handling
human blood should be aware of the aerosols
produced by the microhaematocrit centri-
fuge, the autoanalyzer stirrer, and the mico-
tonometer, inasmuch as it seems that sir-
borne transmission of infectious hepatitis
may occur in the laboratory (20).

G. Miscellaneous precautions and recom-
mendations. 1, Water baths and Warburg
baths used to inactivate, incubate, or test in-
fectious substances should contain a disin-
fectant. For cold water baths, 70 percent
propylene glycol is recomemnded (4, 10).

2. Deepfreeze, Hquid nitrogen, and dry ice
chests and refrigerators should be checked
and cleaned out periodically to remove any
proken ampoules, tubes, etc., containing in-
fectious material, and decontaminated. Use
rubber gloves and respiratory protection dur-
ing this cleaning. All infectious or toxic
roaterial stored in refrigerators or deep-
freezes should be properly labelled. Security
measures should be commensurate with the
hazards (4,10,21).

3. Freeze-dried culture ampoules should al-
ways be opened in a Biological Safety Cabi-
net. The ampoule should be wrapped In a
disinfectant-soaked swab before breaking it
open to minimize the risk of cutting the
hands, and to a lesser extent of releasing

FEDERAL REGISTER, VOL. 41, NO. 176—-THURSDAY, SEPTEMBER 9, 1976
* 88468

aerosol of dried material. Whenever possible,
ampoules should be filled with dry nitrogen
after freeze-drying, thus avoiding implosion
that may occur during the sealing as well
as opening of evacuated ampoules. The whole
process of freeze-drying itself should be per-
formed in a Biological Safety Cabinet. Filtra-
tion of the effluent air from the vacuum
pump is desirable either up (preferably), or
down stream of the pump (5).

4, Ensure that all virulent fluid cultures or
viable powdered infectious materials in glass
vessels are transported, incubated, and stored
in easily handled, nonbreakable leak-proof
containers that are large enough to contain
all the fluid or powder in case of leakage or
breakage cf the glass vessel (4,10).

5. All inoculated petri plates or other in-
oculated solid media should be transported
and incubated in leak-proof pans or leak-
proof containers (4,10).

6. Care must be exercised in the use of
membrane filters to obtain sterile filtrates
of infectious materials. Because of the fra-
gility of the membrane and other factors,
such filtrates cannot be handled as nonin-
fectious until culture or other tests have
proved their sterility (4,10).

7. Shaking machines should be examined
carefully for potential breakage of flasks or
other containers being shaken. Screw capped
durable plastic or heavy walled glass flasks
should be used. These should be securely
fastened to the shaker platform. An addi-
tional precaution would be to enclose the
flask in a plastic bag with or without an
absorbent material.

8. No person should work alone on an ex-
tremely hazardous operation (4,10).

IV, PERSONAL HYGIENE, HABITS, AND PRACTICES

Personal hygienic practices in the labora-
tory are directed, in most part, toward the
prevention of occupationally acquired phys-
ical injury or disease. To a less obvious ex-
tent, they can raise the quality of the lab-
oratory work by reducing the possibilities for
contamination of experimental materials.
The reasons for many of the recommended
precautions and practices are obvious, but,
in some instances, amplification will permit
a better review of the applicability to any
one specific laboratory.

Consequently, what might be forbidden in
one laboratory might be only discouraged
in another, and be permissible in a third.
Nevertheless, adherence to safe practices that
become habitual, even when seemingly not
essential, provides a margin of safety in sit-
uations where the hazard is unrecognized.
The history of occupational injury is replete
with examples of hazards unrecognized until
too late. The following guidelines, recom-
mendations, and comments are presented
with this in mind:

1. Food, candy, gum, and beverages for
human consumption will be stored and con-
sumed only outside the laboratory (5, 10).

2, Foot-operated drinking fountains should
be the sole source of water for drinking by
human occupants of the laboratory (27).

3. Smoking is not permitted in the lab-
oratory or animal quarters. Cigarettes, pipes,
and tobacco will be Kept only in clean areas
(5, 10, 26).

4, Shaving and brushing of iceth are net

permitted in the laboratory. Razors, tooth-
brushes, toiletry supplies, and cosmetics are
permissible only in clean change rooms or
other clean areas, and should never be used
until after showering or thorough washing
of the face and hands (27).

5. A beard may be undesirable in the lab-
oratory in the presence of actual or potenttal
airborne contamination, because it retains
particulate contamination more persistently
than clean-shaven skin. A clean-shaven face
js essential to the adequate facial fit of a

NOTICES

face mask cr respirator when the work re-
quires respiratory protection (10,27,31).

6. Develop the habit of keeping hands
away from mouth, nose, eyes, face, and hair.
This may prevent self-inoc tion (10,27).

7. For product protection, persons with
long hair should wear a suitable hair net
or head cover that can be decontaminated.
This has Iong been a requirement in hos-
pital operating rooms and in the manufac-
ture of biological pharmaceutical products.
A head cover also w srotect the hair from
fluid splashes, from swinging into Bunsen
flames and petri dishes, and will reduce facial
contamination caused by habitual repetitive
manual adjustment of the hair (5).

8. Long-flowing hair and loose-flapping
clothing are dangerous in the presence of
open flame or moving machinery. Rings and
wrist watches also are a mechanical hazard
during operation of some types of machines
(5,10)...

9. Contact lenses do not provide eye protec-
tion, The capillary space between the con-
tact lenses and the cornea may trap any ma-
terial present on the surface of the eye.
Caustic chemicals trapped in this space can-
not be washed off the surface of the cornea.
If the material in the eye is painful or the
contact lens is displaced, muscle spasms will
make it very difficult, if not impossible, to
remove the lens. For this reason, contact
lenses must not be worn by persons exposed
to caustic chemicals unless safety glasses
with side shields, goggles, or plastic face
masks are also worn to provide full protec-
tion. It is the respofiSibility of supervisors to
identify employees who wear contact lenses
(25, 26).

10. Personal items, such as coats, hats,
storm rubbers or overshoes, umbrellas,
purses, etc., do not belong in the laboratory.
These articles should be kept elsewhere (25).

11. Plants, cut flowers, an aquarium, and
pets of any kind are undesirable sources of
yeast, molds, and other potential microbial
contaminants of biological experimental ma-
terials (25).

12. Books and journals returnable to the
institutional library should be used only in
the clean areas a8 much as possible (10,27).

138. When change rooms with showers are
provided, the employer should furnish skin
lotion (27).

14. When employees are subject to poten-
tial occupational infection, the shower and/
or face/hand-washing facilities should be
provided with germicidal soap (8,27).

15. Personal cloth handkerchiefs should
not be used in the laboratory. Cleansing tis-
sue should be available instead.

16. Hand washing for personal protec-
tion:

(i) This should be done promptly after
removing protective gloves. Tests show it is
not unusual for microbial or chemical con-
tamination to be present despite use of
gloves, due to unrecognized small holes,
abrasions, tears, or entry at the wrist.

(ii) Throughout the dey, at intervals
dictated by the nature of the work, the
hands should be washed. Presence cf a wrist
watch discourages adequate washing of the
wrist (10,25).

(Gili) Hands should be washed after re-
moving solled protective clothing, before
leaving the laboratory area, before eating,
and before smoking. The provision of hand
cream by the employer encourages these prac-
tices (5,8,10).

(iv) A disinfectant wash or dip may be
desirable in some cases, but Its use must not
be carried to the point of causing roughen-
ing, desiccation or sensitization of the sKin.

17. Anyone with a fresh or healing cut,
abrasion, or skin lesion should not work with
infective material unless the injured area is

completely protected (8.245).

 

oR

 

 

 
 
 

 

18. Persons vaccinated for smallpox may be
shedders of vaccinia virus during the phase
of cutaneous reaction. Therefore, vaccinatlon
requires permission of the appropriate super-
visor, because two weeks’ absence may be
necessary before returning to work with nor-
mal cell cultures or with susceptible animals,
especially the normal mouse colony (25).

i9. The surgeon’s mask of gauze or filter
paper is of little value for personal respira-
tory protection (29). It is designed to prevent
escape of droplets from the nose cr mouth
(28G). If biohazards demand respiratory
protection, then nothing but a full face res-
pirator or ventilated hood will suffice. A half-
mask respirator does not protect the eyes,
which are an unevaluated avenue of infec-
tion through the conjuctiva and the naso-
lacrimal duct (5.8).

20, Nonspecific contamination by environ-
mental organisms from humans, animals,
equipment, containers for specimens cr sup-
plies, and outside air is a complication that
may affect or invalidate the results of an
experiment. The human sources of this con-
tamination are evaluated as follows:

(i) Sneezing, coughing and talking (23A,
24A). Sneezing, variously reported to gen-
erate as many as 32,000 or 1,000,000 droplets
below 100 microns in diameter; coughing,
which produces fewer and larger droplets;
and talking, which has been reported to aver-
age only 250 droplets when speaking 100
words, show great differences between per-
sons in regard to the number of microorga-
nisms aerosolized. As a general rule, it may
be said that these actions by normal healthy
persons may play a less important role in
transmission of airborne infection to humans
or experimental materials than does libera-
tion of microorganisms from human skin.

(il) Dispersal of bacteria from human skin.
There is a tremendous variation in the num-
ber of bacteria shed from the skin by a
clothed subject. For instance, in one study,
the number varied from 6,000 to 60,000 per
minute (23C). These bacteria were released
on skin scales which were of a_ size
that could penetrate the coarse fabric used
for the laboratory and surgical clothing in
the test (23D). Dispersal of skin bacteria
was several times greater from below the
waist than from upper parts of the body
(24D), Effective reduction is accomplished by
use of closely-woven or impervious clothing
fitted tightly at the neck, wrists, and ankles
to prevent the clothing from acting as a
bellows that disperses air carrying skin scales
laden with bacteria (23B). Such clothing
sometimes is too warm to work in. It was
found that a significant reduction in disper-
sal of bacteria occurred with the wearing of
close-fitting and closely-woven underpants
beneath the usual laboratory clothing (23D).
The purpose of this summary is to alert
laboratory personnel to the existence of this
source of contamination (9).

(ili!) Prolific dispersal of bacteria occurs
from infected abrasions, small pustules,
bolls, and skin disease (23F, 24B). Washing
the lesions with germicidal soap will greatly
decrease the number of organisms on the
skin and dispersal into the air. Healthy nasal
earriers who generate aerosolized staphy-
lococci usually can be identified by the pres-
ence of heavy contamination of their fin-
gers, face, and hair (23E). This point may
be useful in investigating the sourcerof
staphylococcal contamination of ceil lines,

(iv) Footwear. In moderate and high risk
situations, shoes reserved for only laboratory
use have been recommended as @ precau-
tion against transporting spilled infectious
agents outside the laboratory. However, in
experiments during which reduction of po-
tential contamination of experimental mate-
rials is important, laboratory-only shoes can
reduce the microbial load brought into the

FEDERAL REGISTER, VOL. 41, NO. 176-—-THURSDAY, SEPTEMBER 9, 1976
laboratory each day by street shoes. Shoes
are efficient transporters. In one study, there
were 4 to 850 times as many bacterla per
square centimeter on the laboratory foot-
wear as on the floor itself (80).

Vv. CARE AND USE OF LABORATORY ANIMALS
(10,32-37)

A. Care and handling. 1, Special attention
must be given to the humane treatment of
all laboratory animals in accordance with the
Animal Welfare Act of 1970. The implement-
ing rules and regulations appear in the Code
of Federal Regulations (CFR) Title 9, Chap-
ter I, Subchapter A, Parts 1, 2, 3. Recom-
mended provisions and practices that meet
the requirements of the Act have been pub-
lished by the U.S. Public Health Service (32).

2. There are specific minimum require-
ments (33) concerning the caging, feeding,
watering, and. sanitation for dogs, cats,
guinea pigs, hamsters, rabbits, and nonhu-
man primates. To meet these requirements,
the animal room supervisor must have a
copy of 9 CFR Chapter I, Subchapter A,
Parts 1, 2, 3.

8. Each laboratory should establish proce-
dures to ensure the use of animals that are
free of diseases prejudicial to the proposed
experiments and free from carriers of disease
or vectors, such as ectoparasites, which en-
danger other experimental animals or per-
sonnel (10).

B. Cages housing infected animals (10).
1, Careful handling procedures should be
employed to minimize the dissemination of
dust from cage refuse and animals.

2. Cages should be sterilized by autoclav-
ing. Refuse, bowls and watering devices
should remain in the cage during steriliza-
tion.

3. All watering devices should be of the
“non-drip” type.

4. Cages should be examined each morn-
ing and at each feeding time so that dead
animals can be removed.

5. Heavy gloves should be worn when feed-
ing, watering, handling, or removing in-
fected animals. Bare hands should NEVER
be placed in the cage to move any object
therein.

6. When animals are to be injected with
biohazardous material, the animal caretaker
should wear protective gloves and the labora-
tory workers should wear surgeons gloves.
Animals should be properly restrained to
avoid accidents that might result in dissem-
inating biohazardous material, as well as to
prevent injury to the animal and to per-
sonnel.

7. Animals exposed to bichazardous aerosols
should be housed in ventilated cages, in gas-
tight cabinet systems, or in rooms designed
for protection of personnel by use of venti-
lated suits.

8. Animals inoculated by means other than
by aerosols should be housed in equipment
suitable for the level of risk involved.

9. Infected animals to be transferred be-
tween buildings should be placed in venti-
lated cages or other aerosol-proof containers.

10. The oversize canine teeth of large
monkeys present a particular biting hazard;
these are important in the potential trans-
mission of naturally-occurring, and very
dangerous, monkey virus infections. Such
teeth should be blunted or surgically re-
moved by a veterinarian.

11. Presently available epidemiological evi-
dence indicates that infectious hepatitis may
be transmitted from non-human primates
(typically chimpanzees) to man. Newly im-
ported animals may be naturally infected
with this disease, and persons in close con-
tact with such animals may become infected.
After six months residence in this country,
chimpanzees apparently no longer transmit
the disease. A record should be maintained

NOTICES

for each newly imported animal. A sign
should be posted at rooms housing these ani-
mals to warn that the animals are potentlally
infectious.

C. General Guidelines that Apply to Animal
Room Maintenance (10). 1. Doors to animal
rooms should be kept closed at all times ex-
cept for necessary entrance and exit.

2. Unauthorized persons should not be per-
mitted to enter animal rooms.

8. A container of disinfectant should be
kept in each animal room for disinfecting
gloves and hands, and for general decon-
tamination, even though no infectious ani-
mals are present. Hands, fioors, walls, and
cage racks should be washed with an ap-
proved disinfectant at the recommended
strength as frequently as the supervisor
directs.

4, Floor drains in animal rooms, as well

‘as floor drains throughout the building

should be flooded with water or disinfectant
periodically to prevent backup of sewer gases.

5. Shavings or other refuse on floors should
not be washed down the floor drain because
such refuse clogs the sewer lines.

6. An insect and rodent control program
should be maintained in all animal rooms
and in animal food storage areas.

7. Special care should be taken to prevent
live animals, especially mice, from finding
their way into disposable trash.

D. Necropsy rules for infected animals
(10). 1. Necropsy of infected animals should
be carried out by trained personnel in Bio-
logical Safety Cabinets with the hinged glass
panel down. The glove port panel with or
without attached gloves, and a respirator
should be used at the discretion of the su-
pervisor.

2. Surgeons gowns should be worn over
laboratory clothing during necropsies.

3. Rubber gloves should be worn when per-
forming necropsies.

4, The fur of the animal should be wetted
with a suitable disinfectant.

5. Small animals should be pinned down
or fastened on wood or metal in a metal
tray.

6. Upon completion of necropsy, all poten-
tially biohazardous material should be placed
in suitable containers and sterilized imme-
diately.

7. Contaminated instruments should be
placed in a horizontal bath containing a
suitable disinfectant.

8. The inside of the Biological Safety
Cabinets and other potentially contaminated
surfaces should be disinfected with a suit-
able germicide.

8. Grossly contaminated rubber gloves
should be cleaned in disinfectant before re-
moval from the hands, preparatory to sterili-
zation,

10. Dead animals: should be placed in
proper leak-proof containers, autoclaved and
properly tagged before being placed outside
for removal and incineration.

VI. DECONTAMINATION AND DISPOSAL
(7, 10, 38-42)

A. Introduction. Available data on the
efficacy of various decontaminants for etio-
logic agents indicate that no major surprises
will be forthcoming regarding the suscepti-
bility of organisms containing recombinant
DNA molecules. In the absence of adequate
information, tests to determine the efficacy
of candidate decontaminants should be con-
ducted with the specific agent of interest.
The goal of decontamination is not only the
protection of personnel and the environment
from exposure to infectious agents, but also
the prevention of contamination of experi-
mental materials by & variable, persistent,
and unwanted background of microorga-
nisms. This additional factor should be con-
sidered in selecting decontamination mate-
rials and methods.

38469

B. Decontamination Methods. Physical and
chemical means of decontamination fall into
four main categories: Heat; Liquid De¢on-
taminants; Vapors and Gases; and UV Radia-
tion.

1. Heat. The application of heat, either
moist or dry, is recommended as the most
effective method of sterilization. Steam at
121 C under pressure in the autoclave is the
most convenient method of rapidly achiev-
ing sterllity. Dry heat at 160 to 170 C for
periods of 2 to 4 hours Is suitable for destruc-
tion of viable agents on impermeable non-
organic material Buch as glass, but 1s not
reliable in even shallow layers of organic or
inorganic material that can act as insulation.
Incineration is another use of heat in the
decontamination of microorganisms and also
serves as an efficient means for disposal.

2, Liquid Decontaminants. In general, the
liquid decontaminants find their most prac-
tical use in surface decontamination and, at
sufficient concentration, as decontaminants
of quid wastes for final disposal in sanitary
sewer systems, There are many misconcep-
tions concerning the use of liquid decontami-
nants. This is due largely to a characteristic
capacity of such liquids to perform dra-
matically in the test tube and to fail miser-
ably in a practical situation. Such failures
often occur because proper consideration was
not given to such factors as temperature,
time of contact, pH, concentration, and the
presence and state of dispersion, penetrability
and reactivity of organic material at the site
of application. Small variations in the above
factors may make large differences in effec-
tiveness of decontmination. For this reason,
even when used under highly favorable con-
ditions, complete reliance should not be
placed on Nquid decontaminants when the
end result must be sterility.

There are many Hquid decontaminants
available under a wide variety of trade
names, In general, these can be categurized
as halogens, acids or alkalies, heavy metal
salts, quaternary ammonium compounds,
phenolic compounds, aldehydes, ketones,
alcohols and amines. Unfortunately, the more
active the decontaminant the more likely it
is that the decontaminant will possess un-
desirable characteristics, such as the posses-
sion of corrosive properties. None is equally
useful or effective under all conditions,

3. Vapors and Gases. A variety of vapors
and gases possess decontamination proper-
ties. The most useful of these are formalde-
hyde and ethylene oxide. When these can be
employed in closed systems and under con-
trolied conditions of temperature and hu-
midity, excellent decontamination can result.
Vapor and gas decontaminants are primartly
useful tn decontaminating: (1) Biological
Safety Cabinets and associated effluent air-
handling systems and air filters; (ii) bulky
or stationary equipment that resists pentra-
tion by liquid surface decontaminants; (ii!)
instruments and optics that might be dam-
aged by other decontamination methods; and
(iv) rooms and buildings and associated altr~
handling systems. i

4. Radiation. The usefulness of ultraviolet
(UV) irradiation as a decontaminant is
limited by its low penetrating power. No in-
formation is available regarding the effective-
ness of UV irradiation for decontaminating
microorganisms containing recombinant
DNA molecules. Dependence on UV must be
based on the results of experiments imitating
particular anticipated environmental condi-
tions and applications. Ultraviolet Ught ta
generally of limited application and is’ pri-
marily useful tn air locks and animal hold-
ing areas for controlling low levels of air-
borne contaminants.

No one procedure or material will solve all
decontamination problems. The only method
of assuring the efficacy of selected method-
ologies Is to critically examine the resulta

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
38470

obtained in practical tests with the micro-
organism(s) of interest.

C. Laboratory spilis. A troublesome prob-
lem that may occur in the laboratory is the
decontamination of an overt biological spill
The occurrence of a spill poses less of a prob-
lem if it occurs in a Biological Safety Cab-
inet provided splattering to the outside of
the cabinet does not occur, Direct applica-
tion of concentrated liquid decontaminant
and a thorough wipe down of the internal
surfaces of such cabinetry will usually be ef-
fective for decontaminating the work zone
but gaseous decontaminants would be re-
quired to rid the interior sections of the
cabinet of contaminants. Each researcher
must realize that in the event of an overt ac-
cident, research materials such as tissue cul-
tures, media, and animals within such cabi-
nets may well be lost to the experiment.

The greater problem arises if the incident
occurs in the open laboratory. All laboratory
protocols should be designed to prevent such
occurrences. The first action in the event of
an overt laboratory spill is evacuation of the
affected area to minimize the exposure of
personnel involved. Next, the spill area must
be isolated to prevent exposure of personnel
and experimental materials beyond those in-
volved in the immediate area of the spill. The
procedures adopted must be rapidly effec-
tive and must not create additional aerosol
or foster mechanical transfer of materials
to unaffected areas. Personnel carrying out
the procedures must be provided with pro-
tective clothing and equipment, including
respiratory protection. Consideration must
be given to the safe disposal of all materials
and liquids resulting from cleanup proce-
dures. Reentry of personnel to the area
should be avoided until it can be reasonably
established that the area has been effectively
decontaminated. Further specific details are
provided in Section VIII.

D. Disposal. Decontamination and disposal
in infectious disease laboratories are closely
interrelated acts in which decontamination
constitutes the introductory phase of dis-
posal. All materials and equipment used in
research on recombinant DNA molecules will
ultimately be disposed of; however, in the
sense of daily use, only a portion of these
will require actual removal from the labora-
tory complex or on-site destruction. The re-
mainder will be recycled for use either with-
in the same laboratory or in other labora-
tories that may or may not engage in DNA
recombinant research. Examples of the latter
that immediately come to mind are: Re-
usable laboratory glassware, instruments
used in necropsy of infected animals, and
laboratory clothing. Disposal should there-
fore be interpreted in the broadest sense of
the word, rather than in the restrictive sense
of dealing solely with a destructive process.

The principal questions to be answered
prior to disposal of any objects or matertals
from laboratories dealing with potentially
infectious microorganisms or animal tissues
are:

1. Have the objects or materials been effec-
tively decontaminated by an approved proce-
dure?

2. If not, have the objects or materials
been packaged in an approved manner for
immediate on-site incineration or transfer
to another laboratory?

3. Does disposal of the decontaminated
objects or materials involve any additional
potential hazards, biological or otherwise, to
personnel either: (i) Those carrying out the
immediate disposal procedures or (11) Those
who might come into contact with the
objects or materials outside the laboratory
complex?

Laboratory materials requiring disposal
will normally occur as liquid, solid, and
animal room wastes. The volume of these

NOTICES

can become a major problem when there
is the requirement that all wastes be de-
contaminated prior to disposal. It is most
evident that a significant portion of this
problem can be eliminated if the kinds of
materials initially entering the laboratory
are reduced. In any case, and wherever pos-
sible, materials not essential to the research
should be retained in the nonresearch areas
for disposal by conventional methods. Ex-
amples are the packaging materials in which
goods are delivered, disposable carton-cages
for transport of animals, and large carboys
or tanks of fluids which can be left outside
and drawn from as required. Reduction of
this bulk will free autoclaves and other de-
contamination and disposal processes within
the laboratory for the more yapid and effi-
cient handling of materials known to be
contaminated.

Inevitably, disposal of materials raises the
question, ‘‘“How can we be sure that the ma-
terials have been treated adequately to as-
sure that their disposal does not constitute
a hazard?” In the small laboratory, the prob-
lem is often solved by requiring that each
investigator decontaminate all contaminated
materials not of immediate use at the end of
each day and place them in suitable con-
tainers for routine disposal. In larger labora-
tories where the mass of materials for dis-
posal becomes much greater and sterilization
and decontamination bottlenecks occur, ma-
terials handling and disposal will likely be
the chore of personnel not engaged in the
actual research. In either situation, a case
can be made for establishing a positive
method of designating the state of materials
to be disposed of. This may consist of a tag-
ging system stating that the materials are
either sterile or contaminated.

Disposal of materials from the laboratory
and animal holding areas will be required for
research projects ranging In size from an in-
dividual researcher to those involving large
numbers of researchers of many disciplines.
Procedures and facilities to accomplish this
will range from the simplest to the most
elaborate. The primary consideration in any
of these is to dispel the notion that labora-
tory wastes can be disposed of in the same
manner and with as little thought as house-
hold wastes. Selection and enforcement of
safe procedures for disposal. of laboratory
materials are of no less importance than the
consideration given to any other methodol-
ogy for the accomplishment of research
objectives.

Materials of dissimilar nature will be com-
mon in laboratorles studying recombinant
DNA molecules. Examples are combinations
of common flammable solvents, chemical car-
cinogens, radioactive isotopes, and concen-
trated viruses or nucleic acids. These may re-
quire input from a number of disciplines in
arriving at the most practical approach for
their decontamination.

E. Characteristics of chemical decontami-
nants in common use in laboratory opera-
tions. Every person actively working with
viable miroorganisms, no matter how remote
the field of specialization, will, from time to
time, find it necessary to decontaminate by
chemical methods work areas and materials,
equipment, and specialized instruments.
Chemical decontamination is necessary be-
cause the use of pressurized steam, the most
rapid and reliable method of sterilization,
is not normally feasible for decontaminating
large spaces, surfaces, and stationary equip-
ment. Moreover, high temperatures and
moisture often damage delicate instruments,
particularly those having compléx optical
and electronic components.

Chemicals with decontaminant properties
are, for the most part, available as powders,
crystals, and liquid concentrates. These may
be added to tap water for application as sur-

face decontaminants, and some, when added
in sufficient quantity, find use as decontam-
inants of bulk MNquid wastes. Chemical de-
contaminants that are gaseous at room tem-
peratures are useful as space-penetrating
decontaminants. Others become gases at rea-
sonably elevated temperatures and can act
as either aqueous surface or gaseous space-
penetrating decontaminants.

Inactivatiqgn of microorganisms by chem-
ical decontaminants may occur in one or
more of the following ways: (1) Coagula-
tion and denaturation of protein, (2) Lysis,
{3} Binding to enzymes, or inactivation of
an essential enzyme by either oxidation,
binding, or destruction of enzyme substrate.

The relative resistance to the action of
chemical decontaminants can be substan-
tially altered by such factors as: Concentra-
tion of active ingredient, duration of con-
tact, pH, temperature, humidity and pres-
ence of extrinsic organic matter. Depending
upon how these factors are manipulated,
the degree of success achieved with chemi-
cal decontaminants may range from mini-
mal inactivation of target microorganisms
to an indicated sterility within the limits of
sensitivity of the assay systems employed.

There are dozens of contaminants avail-
able under a wide variety of trade names,
In general, these decontaminants can be
classified as halogens, acids or alkalies, heavy
metal salts, quaternary ammonium com-
pounds, phenolic compounds, aldehydes, ke-
tones, alcohols, and amines. Unfortuately,
the more active the decontaminant the
more likely it will possess undesirable char-
acteristics. For example, peracetic acid is a
fast-acting, universal decontaminant. How-
ever, in the concentrated state it is a hazard-
ous compound that can readily decompose
with explosive violence. When diluted for
use, it has a short half-life, produces strong,
pungent, irritating odors, and is extremely
corrosive to metals. Nevertheless, it is such
an outstanding decontaminant that it is
commonly used in germ-free animal studies
despite these undersirable characteristics.

The halogens are probably the second most
active group of decontaminants. Chlorine,
jodine, bromine, and fluorine will rapidly
kill bacterial spores, viruses, rickettsiae, and
fungi. These decontaminants are effective
over a wide range of temperatures. In fact,
chlorine has been shown to be effective at
—40 F. (On the other hand, phenols and
formaldehyde have high temperature coeffi-
cients). The halogens have several undesir-
able features. They readily combine with
protein, so that an excess of the halogen
must be used if proteins are present. Also,
the halogens are relatively unstable so that
fresh solutions must be prepared at frequent
intervals. Finally, the halogens corrode
metals. A number of manufacturerg of de-
contaminants have treated the halogens to
remove some of the undesirable features. For
example, sodium hypochlorite reacts with p-
toluenesulfonamide to form Chloramine T,
and jodine reacts with certain surface-active
agents to form the popular iodophors. These
“tamed” halogens are stable, non-toxic,
odorless, and relatively mnencorrosive to
metals. However, the halogens are highly
reactive elements, and, because they are
reactive they are good germicides. When 4@
haolgen acts as a decontaminant, free halo-
gen is the effective agent. Raising the pH or
combining the halogen with other com-
pounds to decrease the corrosive effect will
also decrease the germicidal power. A trade-
off situation occurs.

Ineffectiveness of a decontaminant is
due primarily to the failure of the de-
contaminant to contact the microorga-
nisms rather than failure of the decon-
taminant to act.-If one places an item in

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976

 
a liquid decontaminant, one can see that
the item is covered with tiny bubbles.
Of course, the area under the bubbles
is dry, and microorganisms in these dry
areas will not be affected’ by the decon-
taminant. Also, if there are spots of
grease, rust or dirt on the object, micro-
organisms under these protective coat-
ings will not be contacted by the decon-
taminant. Scrubbing an item when im-
mersed in a decontaminant is helpful,
and a decontaminant should have, and
most do have, incorporated surface-
active agents.

F, Properties of some common decon-
taminants—1. Alcohol. Ethyl or iso-
propyl alcohol in a concentration of 70—
80 percent by weight is often used. Al-
cohols denature proteins and are some-
what slow in their germicidal action.
However, they are effective decontami-
nants against lipid-containing viruses.

2. Ether and Chloroform. 'These com-
pounds are not ordinarily used as decon-
taminants, but they do demonstrate the
fact that lipid-containing viruses are
inactivated by these organic solvents,
whereas non-lipid-containing viruses
are quite resistant.

3. Formaldehyde. Formaldehyde for
use as a decontaminant is usually mar-
keted as a solution of about 37 percent
concentration referred to as formalin
or as a solid polymerized compound
called paraformaldehyde. Formaldehyde
in a concentration of 5 percent active
ingredient is an effective liquid decon-
taminant. It loses considerable activity
at refrigeration temperatures and the
pungent, irritating odors make formal-
dehyde solutions difficult to use in the
laboratory. Formaldehyde vapor gener~
ated from formaldehyde solution is an
effective space decontaminant for decon-
taminating rooms or buildings, but in
the vapor state with water it tends to
polymerize out on surfaces to form para-
formaldehyde, which is persistent and
unpleasant. Formaldehyde gas can be
liberated by heating paraformaldehyde
to depolymerize it. In the absence of
high moisture content in the air, formal-
dehyde released in the gaseous state
forms less polymerized residues on sur-
faces and less time is required to clear
treated areas of fumes than formalde-
hyde released in the vapor state.

4. Phenol. Phenol itself is not often used
as a decontaminant. The odor is somewhat
unpleasant and a sticky, gummy residue
remains on treated surfaces. This is espe-
clally true during steam sterilization. Al-
though phenol itself may not be tn wide-
spread use, phenol homologs and phenolic
compounds are basic to a number of popular
decontaminants. The phenolic compounds
are effective decontaminants against some
viruses, rickettsiae, fungl and vegetative bac-
teria, The phenolics are not effective in ordi-
nary usage against bacterial spores.

5. Quaternary Ammonium Compounds or
Quats. After 30 years of testing and use,
there is still a considerable controversy about
the efficacy of the Quats as decontaminants,
These cationic detergents are strongly sur-
face-active and are effective against lipid-
containing viruses. The Quats will attach to
protein so that dilute solutions of Quats will
quickly lose effectiveness in the presence of
proteins. The Quats tend to clump micro-

NOTICES

organisms and are neutralized by anionic
detergents, such as soap. The Quats have the
advantages of being nontoxic, odorless, non-
staining, noncorrosive to metals, stable, and
inexpensive.

6. Chlorine. This halogen is a universal
decontaminant active against all microor-
ganisms, including bacterial spores. Chlorine
combines with protein and rapidly decreases
in concentration in its presence. Free, avail-
able chlorine is an active element. It is a
strong oxidizing agent, corrosive to metals.
Chlorine solutions will gradually lose
strength so that fresh solutions must be pre-
pared frequently. Sodium hypochloride 1s
usually used as a base for chlorine decon-
taminants. An excellent decontaminant can
be prepared from household or laundry
bleach. These bleaches usually contain 5.26
percent available chlorine or 52,500 ppm, If
one dilutes them 1 to 100, the solution will
contain 625 ppm of available chlorine, and,
if a nonionic detergent such as Naccanol is
added in a concentration of about 0.7 per-
cent, a very good decontaminant is created.

7. Iodine. The characteristics of chlorine
and iodine are similar. One of the most
popular groups of decontaminants used in
the laboratory is the iodophors, and Wes-
codyne is perhaps the most popular. The
range of dilution of Wescodyne recommended
by the manufacturer is 1 oz. in 5 gal. of water
giving 25 ppm of available iodine to 3 oz. in
5 gal. giving 75 ppm. At 75 ppm, the con-
centration of free iodine is .0075 percent. This
small amount can be rapidly taken up by any
extraneous protein present. Clean surfaces
or clear water can be effectively treated by
75 ppm available iodine, but difficulties may
be experienced if any appreciable amount of
protein is present. For bacterial spores, a
dilution of 1 to 40 giving 750 ppm is recom-
mended by the manufacturer. For washing
the hands, it is recommended that Wescodyne
be diluted 1 to 10 or 10 percent in 50 percent
ethyl alcohol (a reasonably good decon-
taminant itself) which will give 1,600 ppm of
available iodine, at which concentration rela-
tively rapid inactivation of any and all micro-
organisms will occur.

G. Vapors and gases. The use of formalde-
hyde as a vapor or gas has already been dis-
cussed. Other chemical decontaminants
which have been used this way included
ethylene oxide, peracetic acid, beta-propliolac-
tone (BPL), methyl bromide, and ethylene
amine. When these can be used in closed
systems and under controlled conditions of
temperature and humidity, excellent decon-
tamination can be otbained. Residues from
ethylene oxide must be removed by aeration;
but otherwise tt is convenient to use,
versatile, and noncorrosive. Paracetic acid ta
corrosive for metals and rubber. BPL in the
vapor form acts rapidly against bacteria,
rickettsiae, and viruses. It has a half-life of
3.5 hours when mixed with water, is easily
neutralized with water, and lends itself to
removal by aeration. The National Institutes
of Health does not recommend BPL as a
decontaminant because it has been identified
as a suspect carcinogen.

H. Residual action of decontaminants. As
noted in the preceding discussion of decon-
taminant properties, many of the chemical
decontaminants often have residual proper-
ties that may be considered a desirable fea-
ture in terms of aiding in the control of
background contamination. One is cautioned,
however, to consider residual properties care-
Tully. Ethylene oxide used to sterilize labora-
tory shoes can leave residues which cause
skin irritation. Animal cell cultures, as well
as viruses of interest, are also inhibited or
inactivated by decontaminants persisting af-
ter routine cleaning procedures. Therefore,
reusable items that are routinely held in

liquid decontaminant prior to autoclaving

38471

and cleaning should recelve particular atten-
tion in rinse cycles. Similarly, during gen-
eral area decontamination with gases or va-
pors, it may be necessary to protect new and
used clean items by removing them from the
area or by enclosing them in gastight bags
or by insuring adequate aeration following
decontamination.

I. Selecting chemical decontaminants for
research on recombinant DNA molecules. No
single chemical decontaminant or method
will be effective or practical for all situations
in which decontamination is required. Selec-
tion of chemical decontaminants and proce-
dures must be preceded by practical consid-
eration of the purposes for the decontamina-
tion and the interacting factors that will ul-
timately determine how that purpose is to
be achieved. Selection of any given procedure
will be influenced by the information derived
from answers‘to the following questions:

1. What is the target microorganism (s) ?

2. What decontaminants in what form are
Known to, or can be expected to, inactivate
the target microorganism (s) ?

3. What degree of inactivation is required?

4. In what menstruum is the microorga-
nism suspended; i.e., simple or complex, on
solid or porous surfaces, and/or airborne?

5. What is the highest concentration of
cells anticipated to be encountered?

6. Can the decontaminant either as an
aqueous solution, a vapor, or a gas reason-
ably be expected to contact the microorga-
nisms, and can effective duration of contact
be maintained?

7. What restrictions apply with respect to
compatibility of materials?

8. Does the anticipated use situation re-
quire immediate availability of an effective
concentration of the decontaminant or will
sufficient time be available for preparation
of the working concentration shortly before
its anticipated use?

The primary target of decontamination in
the infectious disease laboratory is the
microorganism under active investigation.
Laboratory preparations oor infectious
agents usually have titers grossly in excess
of those normally observed in nature. The
decontamination of these high-titer ma-
terlais presents certain problems, Mainte-
nance systems for bacteria or viruses are
specifically selected to preserve viability of
the agent. Agar, proteinaceous nutrients,
and cellular materials can be extremely ef-
fective in physically retarding or chemically
binding active moieties of chemical decon-
taminants. Such interferences with the de-
sired action of decontaminants may require
the use of decontaminant concentrations
and contact times in excess of those shown
to be effective in the test tube. Similarly, a
major portion of decontaminant contact
time required to achleve a given level of
agent inactivation may be expended in in-
activating a relatively small number of the
more resistant members of the population.
The current state of the art provides little
information on which to predict the prob-
able virulence of these survivors. These
problems are, however, common to all po-
tentially pathogenic agents and must always
be considered in selecting decontaminants
and procedures for thelr use.

Microorganisms exhibit a range of resist-
ance to chemical decontaminants. In terms
of practical decontamination, most vegeta-
tive bacteria, fungi and lipid-containing vi-
ruses, are relatively susceptible to chemical
decontamination. The non-lipid-containing
viruses and bacteria with a waxy coating
such as tubercle bacillus occupy a mid-range
of resistance. Spore forms are the most re-
sistant.

A decontaminant selected on the basis of
its effectiveness against microorganisms on*
any range of the resistance scale will be ef-

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
38472

fective against microorganisms lower on the
scale. Therefore, if decontaminants that ef-
fectively control spore forms are selected for
routine laboratory decontamination, it can
be assumed that any other microorganisms
generated by laboratory operations, even in
high concentrations, would also be inacti-
vated.

An additional area that must be considered
and for which there is little definitive infor-
mation available is the “inactivation” of
nucleic acids. Nucleic acids often have better
survival characteristics under adverse con-
ditions than do the intact virions and cells
from which they were derived. Strong oxi-
dizers, strong acids and bases, and either
gaseous or aqueous formaldehyde should re-
act readily with nucleic acids, Their ability

NOTICES

to destroy the nucleic acid being studied,
however, should be confirmed in the experi-
menter’s laboratory. Because of innate dif-
ferences in the chemistry of RNA and DNA
the effectiveness of a decontaminant for one
cannot be extrapolated to the other. For ex-
ample, RNA molecules are susceptible to mild
alkaline hydrolysis by virtue of the free hy-
droxyl group in the 2’ position, whereas DNA
molecules are not susceptible to mild alka-
line hydrolysis.

Table II summarizes pertinent characteris-
tics and potential applications for several
categories of chemical decontaminants most
likely to be used in the biological laboratory.
Practical concentrations and contact times
that may differ markedly from the recom-
mendations of manufacturers of proprietary

products are suggested. It has been assumed
that micoorganisms will be afforded a high
degree of potential protection by organic
menstruums, It has not been assumed that
@ sterile state will result from application of
the indicated concentrations and contact
times. It should be emphasized that these
data are only indicative of efficacy under
artificial test conditions. The efficacy of any
of the decontaminants should be conclu-
sively determined by individual investigators.
It is readily evident that each of the de-
contaminants has a range of advantages and
disadvantages as well as 8 range of potentia!
for inactivation of a diverse microfilor. Equal-
ly evident is the need for compromise as an
alternative to maintaining a veritable “drug
store” of decontaminants.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

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jo Rulers Le microecope and cane:

VII. HOUSEKEEPING

A. Introduction. Well-defined housekeep-
ing procedures and schedules are essential
in reducing the risks of working with etio-
logic agents and in protecting the integrity
of the research program. This is particularly
true in the biological laboratory operating
under less than total containment concepts
and in all areas used for the housing of ani-
mals, whether or not they have been inten-~
tionally infected. A well-conceived and well-
executed housekeeping program limits physi-
eal clutter that could distract the attention
and interfere with the activities of labora-
tory personnel at a critical moment in a po-
tentially hazardous procedure, provides a
work area that will not in itself be a source
of physical injury or contamination, and pro-
vides an area that promotes the efficient use
of decontaminants in the event of the In-
advertent release of a harmful agent. Less
immediately evident are the benefits of es-
tablishing, among personnel of widely vary-
ing levels of education, an appreciation of
the nature and sources of biological con~
tamination. ;

Housekeeping is an omnibus term that can
be interpreted as broadly or as narrowly as
one chooses. It can be seen that many of
the procedures found under special headings,
such as decontamination, disposal, and ani-
mal care, are, in reality, specific instructions
for safely accomplishing otherwise routine
housekeeping chores. In these safety sug-
gestions for research on recombinant DNA
molecules, it has been elected to address
specifically only tasks of a janitorial nature
under the subject of housekeeping.

FEDERAL REGISTER, VOL. 41,

The objectives of*houseKeeping in the bio-
logical laboratory are to:

1. Provide an orderly work area conducive
to the accomplishment of the research
program.

2. Provide work areas devoid of physical
hazards.

8. Provide a clean work area with back-
ground contamination ideally held to a zero
level but more realistically to a level such
that extraordinary measures in sterile tech-
niques are not required to maintain integ-
rity of the biological systems being.
researched.

4. Prevent the accumulation of materials
from current and past experiments that con-
stitute a hazard to laboratory personnel.

5. Prevent the creation of aerosols of haz-
ardous materials as a result of the housekeep-
ing procedures used.

Procedures developed in the ares of house-
Keeping should be based on the highest level
of risk to which the personnel and integrity
of the experiments will be subject. Such an
approach avolds the confusion of multiple

‘practices and retraining of personnel. The

primary function, then, of routine house-
Keeping procedures is to prevent the accumu-
lation of organic debris that (i) may harbor
microorganisms that are a potential threat
to the integrity of the biological systems un-
der investigation, (ii) may enhance the sur-~
vival of microorganisms inadvertently re-
leased in experimental procedures, (ili) may
retard penetration of decontaminants, (iv)
may be transferable from one area to an-
other on clothing and shoes, (v) may, with
sufficient buildup, become a bichazard as

@ consequence of secondary aerosolization by
personnel and air movement, and (vi) may
cause allergenic sensitization of personnel,
e.g., to animal danders.

Housekeeping in animal care units has
the same primary function as that stated
for the laboratory and should, in addition,
be as meticulously carried out in quarantine
and conditioning areas as in areas used to
house experimentally infected animals. No
other areas in the laboratory have the con-
stant potential for creation of significant
quantities of contaminated organic debris
than do animal care facilities,

In all laboratories, efforts to achieve total
decontamination and to conduct a major
cleanup of the biological complex are nor-
mally undertaken at relatively long time in-
tervals. Routine housekeeping must be relied
on to provide a work area free of significant
sources of background contamination. The
provision of such a work area is not simply a
matter of indicating in a general way what
has to be done, who will do it, and how
often. The supervisor must view each task
critically in terms of the potential biohazard
involved, decide on a detailed procedure for
its accomplishment, and provide instructions
to laboratory personnel in a manner that
minimizes the opportunity for misunder-
standing.

The following checklist outlines a portion
of the items requiring critical review by the
laboratory supervisor. It is not intended to
be complete but is presented as an example
of the detailed manner in which housekeep-
ing in the biological laboratory complex must
be viewed.

NO. 176-——THURSDAY, SEPTEMBER 9, 1976
Administration Areas

Aisles

Animal Food Storage

Animal Bedding Storage
Biological Safety Cabinets
Bench Tops and Other Work Surfaces
Ceilings

Change Rooms

Cleaning Solution Disposal
Cages and Cage Racks

Dr¥ Ice Chests

Deep Freeze Chests

Entry and Exit Ways
Equipment Storage

Floors

Glassware

General Laboratory Equipment Cleanup
Hallways

Incubators

Instruments

Insect and Rodent Control
Light Fixtures

Mechanical Equipment Areas
Mops

Pipes—Wall and Celling Hung
Refrigerators

Showers

Supply Storage

UV Lamps

Vacuum Cleaners

Waste Accumulations

Waste Water Disposal

Others

Housekeeping in the laboratory is one of
the avenues that leads to accomplishing the
research program safely. It is important that
housekeeping tasks be assigned to personnel
who are knowledgeable of the research pro-
gram and special hazards of the research en~
vironment. The recommended approach to
housekeeping i8 the assignment of house-
keeping tasks to the research teams on an in-
dividual basis for their immediate work areas
and on a cooperative basis for areas of com-
mon usage. Similarly, animal caretaker per~
sonnel should be responsible for housekeep-
ing in animal care areas. The laboratory su-
pervisor must determine the frequency with
which the individual and cooperative house-
keeping chores need be accomplished. He
should provide schedules and perform fre-
quent inspection to assure compliance. This
approach assures that research work fiow
patterns will not be interrupted by an alien
cleanup crew, delicate laboratory equipment
will be handled only by those most Knowl-
edgeable of its particular requirements, and
the location of concentrated biological prep-
arations and contaminated equipment used
in their preparation and application wlll be
known.

B. Floor care. Avoidance of dry sweeping
and dusting will reduce the formation of
nonspecific environmental aerosols. Wet mop-
ping or vacuum cleaning with a high-efii-
ciency particulate air (HEPA) filter on the
exhaust is recommended.

Careful consideration must be given to de-
sign and quality in the selection of cleaning
equipment and materials and in their use to
prevent the substitution of one hazard for
another.

In the absence of overt hazardous spills,
the cleaning process commonly will consist
of an initial vacuuming to remove all gross
particulate matter and a follow-up wet
mopping with a solution of chemical de-
contaminant containing a detergent. De-
pending on the nature of the surfaces to be
cleaned and availability of floor drains, re-
moval of residual cleaning solutions can be
accomplished by a number of methods.
Among these are: Pickup with a partially
dry mop, pickup with a wet vacuum that
has an adequately filtered exhaust, or remov-
al to a convenient floor drain by use of a
floor squeegee.

After cleaning up a spill of Infected mate-
ral, the residual solution should not be

NOTICES

discharged to a sanitary sewer until it has
been autoclaved or given further chemical
treatment, such as by the addition of sodi-
um hypochlorite sufficient to provide a final
concentration of 500 ppm chlorine. Most
household bleaches are marketed with a
chlorine content of 5.25%. These in a final
dilution of 1:100, yield 525 ppm of available
chlorine. After allowing a contact time of 15
minutes, these solutions may be flushed
down any available drain. Chlorine solutions
in these high concentrations may be too
corrosive for general application to floors
and equipment. In any event, if solutions
are used in this way, after the contact time
the area should be rinsed with water.

C. Dry sweeping.— While it is recommended
that dry sweeping be minimized, this may be
the only method available or practicable
under certain circumstances. In such cases,
sweeping compounds used with push brooms
and dry-dust mop heads treated to suppress
aerosolization of dust should be used.

Sweeping compounds available from the
usual janitorial supply firms fall in three
categories:

Wax-based compounds used on vinyl floors
and waxed floor coverings.

Oil-based compounds for concrete floors.

Oll-based compounds with abrasives (such
ag sand) to achieve a dry scouring action
where much soll is present.

Dry-dust mop heads can be purchased as
treated disposabie units or as reusable, wash-
able heads that must be treated with appro-
ptiate sprays or by other means to improve
their dust-capturing property.

D. Vacuum cleaning. In the absence of a
HEPA filter on the exhaust, the usual wet and
dry industrial-type vacuum cleaner is a
potent aerosol generator. The HEPA-filtered
exhaust used in conjunction with a well-
sealed vacuum unit, however, can negate
this factor because of its ability to pass large
volumes of exhaust air while retaining par-
ticles with a minimum effictency of 99:97 per-
cent. Wet and dry units incorporating a
HEPA filter on the exhaust are available from
@ number of manufacturers.

There are no particular requirements with
respect to the manner in which the dry
vacuuming is accomplished other than te
emphasize that the objective js to remove all
debris and particulate matter. The manufac-
turer’s directions adequately detail the fre-
quency of bag changes, filter changes, and
mechanical adjustments.

Dry material vacuum-collected during these
floor-cleaning activities is potentially con-
taminated, but the nature of the risk is
probably greater to the experiment than to
the experimenter. It 1s wise to effect bag and
filter changes and to clean out collection
tanks in a manner that will avoid or mini-
mize acrosolizing the contents of the vacuum
cleaner.

A vacuum machine that collects debris in
a disposable bag is preferable to machines
that collect the major debris in a tank and
on an exposed primary filter. Even though it
may serve as a primary filter, the disposable
bag must be removed with caution. A bel-
lows effect may pump dust out of the bag
if its intake opening is not sealed before
moving it to a plastic bag for transfer out of
the area. In any event, the outer surface of
the disposable bag will probably bear some
dust contamination, which also may occur
on inner surfaces of the machine.

To avold contaminating experimental ma-
terlals, the emptying of vacuum collection
tanks and changing of bags and filters are
best done away from the immediate labora-
tory area, for example, in a small area that
can be easily cleaned afterwards. The use of
heavy rubber gloves is recommended when
removing wastes from tanks in case broken
glass ia present. After making the filter

changes, all external surfaces of the imme-

38173

diate work area and the equipment should
be wiped with a cloth moistened in decon-
taminant, The operator might plan for a
change of laboratory clothing afterwards so
as to minimize carrying contamination into
other areas of the laboratory.

Avoid use of dry vacuum cleaning equip-
ment in work with high risk agents in the
open laboratory. Should it be necessary to
use it, it ils recommended that gaseous ster-
ilization may be used to minimize aerosoli-
zation of microorganisms before waste is
emptied from the vacuum container. Be-
cause complete penetration of sterilizing
gases into the collected dry dust may be a
problem, all wastes should be placed in a
plastic bag, which then is tightly closed and
incinerated or disposed of in an approved
manner.

When dry vacuum cleaning equipment
has been used within a gastight safety cab-
inet system, it can be treated in an attached
double-door carboxyclave (an autoclave
equipped with an ethylene oxide gas sterill-
zation system) to allow for removal and
emptying of the collection tank.

If a wet vacuum is to be used for pickup
of the detergent-germicide solution from
the ficor, the manufacturer's recommenda-
tions on filter life should be followed. In
addition, the operation of the vacuum
should be closely observed for evidence of
operating changes indicating restricted air-
flow or, conversely, increased flow indicating
filter failure. Liquids collected in the vac-
uum cleaner after floor mopping will con-
tain decontaminant materials. These liquids
may be poured down a convenient floor
drain, except in the case of cleanup wastes
from an overt spill. The collected Hquid
should then be autoclaved or treated with
chlorine solution before disposal.

Provisions should be made for regular de-
contamination of the entire vacuum clean-
er with formaldehyde gas or vapor, or ethyl-
ene oxide. This should be done after use
if the vacuum is used in any manner for
cleanup of overt spills of infectious material,

E. Selection of a cleaning solution. The
selection of a detergent-decontaminant com-
bination for routine cleaning of the labora-
tory complex should be based on the require-
ments of the area of greatest potential for
contamination by the widest spectrum of
microorganisms. With rare exception, this
will be identified as the animal holding area
and the expected microorganisms may well
include fungi, viruses, and the vegetative
and spore forms of bacteria. A decontaminat-
ing solution for such a range of microorga-
nisms would, however, be expensive and ex-
cessively corrosive for routine use. Except
in those rare instances where it can be as-
sumed that pathogenic spores are being shed
by laboratory animals, the risks from the
spores are more likely to affect the experi-
ments than the personnel. The spores tend
to be associated with organic debris from
bedding and food, thus offering potential
for removal or at least a large initial reduc-
tion in their numbers by vacuum cleaning. A
wide range of cleaning solutions that are
mildly sporicidal, reasonably residual, and
are not destructive to the physical plant are
available. Phenol derivatives in combination
with a detergent have these characteristica
and have been selected for routine use in a
number of research facilities. There are num-
erous detergent-phenolic combinations avail-
able on the market. The phenols are one type
of a broad spectrum of biocidal substances
that inclade the mercurials, quaternary
ammonium compounds, chlorine compounds,
todophores, alcohols, formaldehyde, glutaral-
dehyde, and combinations of alcohol with
either iodine or formaldehyde. These have
been discussed in Section VI.

The laboratory supervisor should make a
selection from those types most readily avall-

FEDERAL REGISTER, VOL. 41, NO. 176-——THURSDAY, SEPTEMBER 9, 1976
38474

able which meet the general criteria of effec-
tiveness, residual properties, and low corro-
siveness.

F. Wet mopping—two-bucket method. Wet
mopping of floors in laboratory and animal
care areas is, from a safety standpoint, most
conveniently and efficiently accomplished
using a two-bucket system. The principal
feature of such a system is that fresh deter-
gent-decontaminant solution is always ap-
plied to the floor from one bucket, while all
spent cleaning solution wrung from the mop
ig collected in the second bucket. Compact
dolly-mounted double-bucket units with
foot-operated wringers are available from
most janitorial supply houses. A freshly
Jaundered mop head of the cotton string
type should be used daily. This requires that
@ mop with removable head be provided as
opposed to a fixed-head type. In practice, the
mop is saturated with fresh solution, very
lightly wrung into the second pucket and
applied to the floor using a figure eight mio-
tion of the mop head. After every four or five
strokes, the mop head is turned over and the
process continued until an area of approxi-
mately 100 ft? has been covered. After allow-
ing a contact time of five minutes, the solu-
tion is removed with either a wet vacuum
cleaner with HEPA-filtered exhaust or with
the wrung-out mop. The mopping is con-
tinued in 100 ft? increments until the total
ficor area has been covered. Floor-cleaning
procedures are most effectively completed
after the majority of the work force has de-
parted and should progress from areas of
jeast potential contamination to those of
greatest potential. Before a mop head ts sent
to @ laundry, it should be autoclaved. Spent
cleaning fluids are disposed of by flushing
down the drain.

If the cleanup follows an overt spill of in-
fectious material, the spent cleaning solution,
after removal from the floor, should be auto-~
elaved or treated with chlorine solution.
Chlorine (as household bleach) should be
added to give 500 ppm and held for a contact
time of 15 minutes before dumping in the
sanitary sewer.

G. Alternative floor cleaning method for
animal care areas and areas with monolithic
floors. The absence of permanently placed
laboratory benches and fixed equipment,
coupled with the mobility of modern cage
racks, makes possible alternate floor-cleaning
procedures in animal care facilities. As in all
considerations of methodologies in biomedi-
cal laboratory facilities, it is necessary to
assess the compatibility of procedures and
facilities from the hazard point of view. The
alternative floor-cleaning procedure to be
discussed requires that floors are completely
sealed or of monolithic construction so that
Hquid leakage to adjacent areas does not
occur end that floor drains or wet vacuum
cleaners are available.

Subsequent to the removal of all debris by
ary vacuum, move the cage racks to one side
of the room. Cover the floor of the remaining
cleared portion of the room with detergent-
decontaminant solution applied at a rate of
approximately one gallon per 144 ft from a
one-galion tank sprayer, using a setting on
the nozzle which will cause the solution to
flow on and not create a spray, The nozzle is
pleced close to the floor. Allow a fifteen-
minute contact period; then push the clean-
ing solution to the floor drain with a large
floor squeegee or pick ft up with a wet Vac-
uum. Allow the floor to air dry; move the
cage racks into the cleaned area, and repeat
the process for the remaining floor area. Floor
drains in these areas should be rim-flush, et
Jeast six inches in diameter, and fitted with
a sereen or porous trap bucket to catch large
debris that escapes the initial dry cleaning.
Such screens and baskets should be emptied
after treatment with a decontaminant. If
space utilization does not require frequent
floor washdown, pour a half-gallon of deter-

NOTICES

gent-decontaminant solution into the draln
each week to keep the trap in the waste line
filled against backup of sewer gases.

VII. CLEAN-UP OF BIOHAZARDOUS SPILLS
(3, 8, 10)

A. Biokacardous spill in a biological safety
cabinet. Chemical decontamination proce-
dures should be initiated at once while the
cabinet continues to operate to prevent
escape of contaminants from the cabinet.

1. Spray or wipe walle, work surfaces, and
equipment with a 2 percent solition of an
jiodophor-decontaminant (Wescodyne or
equivalent). A decontaminant detergent has
the advantage of detergent activity, which is
important because extraneous organic sub-
stances frequently interfere with the reaction
petween the microorganisms and the active
agent of the decontaminant. Operator should
wear gloves during this procedure.

2. Flood the top work surface tray, and, if
a Class II cabinet, the drain pans and catch
basins below the work surface, with a decon-
taminant and allow to stand 10-15 minutes.

3. Remove excess decontaminant from the
tray by wiping with a sponge or cloth soaked
in a decontaminant. For Class II cabinets,
drain the tray into the cabinet base, lift out
tray and removable exhaust grille work, and
wipe off top and bottom (underside) surfaces
with a sponge or cloth soaked in a decon-
taminant. Then replace in position and drain
recontaminant from cabinet base into ap-
propriate container and autoclave according
to standard procedures, Gloves, cloth or
sponge should be discarded in an autoclave
pan and autociaved.

B. Biohazard spill outside a bdiological
sayety cabinet. 1. Hold your breath, leave the
room immediately, and close the door.

2. Warn others not to enter the contaml-
nated area.

3, Remove and put into a container con-
taminated garments for autoclaving and
thoroughly wash hands and face.

4, Wait 30 minutes to allow dissipation of
aerosols created by the spill.

83. Put on a long-sleeve gown, mask, and
rubber gloves before reentering the room.
(For a high risk agent, & jumpsuit with
tight-fitting wrists and use of a respirator
should be considered).

6. Pour a decontaminant solution (5%
jodophor or 5% hypochlorite are recom-
mended) around the spill and allow to flow
into the spill. Paper towels soaked with the
decontaminant may be used to cover the
area. To minimize aerosolization, avoid pour-
ing the decontaminant solution directly onto
the spill.

7, Let stand 20 minutes to allow an ade-
quate contact time.

8. Using an autoclavable dust pan and
squeege, transfer all contaminated materials
(paper towels, glass, liquid, gloves, etc.)
into a deep autoclave pan. Cover the pan
with aluminum foil or other suitable cover
and autoclave according to standard direc-
tions.

9. The dust pan and squeegee should be
placed in an autoclavable bag and auto-
claved according to standard directions. Con-
tact of reusable items with non autoclavable
plastic bags should be avoided—separation of
the plastic after autoclaving can be very diffi-
cult.

C. Radioactive biohazard spill outside a
Diological safety cabinet. In the event that a
pichazardous spill! also involves a radiation
hazard, the clean-up procedure may have te
be modified, depending on an evaluation of
the risk assessment of relative biological and
radiological hazard.

Laboratories handling radioactive sub-
stances nust have the services of a designated
radiation protection officer available for con-
eultation.

The following procedure indicates suggest-
ed variations from the biohazard spill pro-

eodure (above) that should be considered
when a radioactive biohazard spill occurs out-
side a Biological Safety Cabinet.1

1, Holding your breath, leave the room Im-
mediately and close the door.

2. Warn others not to enter the contami-
rated area.

3. Remove and put in ea container con-
taminated garments for autoclaving and
thoroughly wash hands and face.

4. Wait thirty minutes to allow dissipa-
tion of aerosols created by the spill.

*Pefore clean-up procedures begin, a radta-
tion protection officer should survey the spill
for external radiation hazard to determine
the relative degree of risk,

5. Put on a long-sleeve gown, mask, and
rubber gloves before reentering the room.
(For a high risk agent, a Jumpsuit with tight-
fitting sleeves and a respirator should be ccn-
sidered).

6. Pour a deconiaminant solution (5% lodc-
phor or 5 hypochlorite are recommended)
aronnd the spill and allow to flow into the
spill. Paper towels soaked with the decon-
taminant may be used to cover the area. Tc
minimize aerosolization, avoid pouring the
decontaminant solution directly onto the
spill.

7. Let stand 20 minutes to allow adequaie
disinfectant contact time.

8. tin most cases, the spill will involve
nC or +H, which present no external hazard.
However, if more energetic beta or gamma
emitters ere involved, care must be taken to
prevent hand and body radiation exposure.
The radiation protection officer must make
this determination before the clean-up opera-
tion is begun.

If the radiation protection officer approves,
the bio-hazard-handling procedure may be-
gin: Using an autoclavable dust pan and
squeegee, transfer all contaminated materials
(paper towels, glass, liquid, gloves, etc.) into
a deep autoclave pan. Cover the pan with
aluminum foil or other suitable cover and
autoclave according to standard directions.

*If the radiation protection officer deter-
mines thafiradioactive vapors may be re-
leased and thereby contaminate the auto-
clave, the material must not be autoclaved,
In that case, sufficient decontaminant solu-
tion to immerse the contents should be added
to the wastes container. The cover should be
sealed with waterproof tape, and the con-
tainer stored and handled for disposal as
radioactive waste. Radioactive and biohazara
warning symbols should be affixed to the
waste container. As a general rule, autociar-
ing should be avoided.

9. If autoclaving has been approved, the
aust pan and squeegee should be placed in an
autoclavable bag and autoclaved according
to standard directions. Contact of reusable
items with plastic bags should be avoided—
separation of the plastic after autoclaving
can be very difficult.

*A final radioactive survey should be made
of the spill area, dust pan, and squeegee with
a Geiger counter, or a smear should be taken
and counted in a liquid scintillation counter.

IX. A SECONDARY RESERVOIR AND FILTRATION
APPARATUS FOR VACUUM SYSTEMS

The aspiration of tissue culture media
from monolayer cultures and of superna-
tants from centrifuged samples into collec-
tion vessels or reservoirs is a common pro-
cedure in many laboratories. To prevent the
accidental contamination by aerosols or
fluids of house vacuum systems or labora-
tory pumps, some investigators have in-
stalled side arm flasks containing cotton,
sulfuric acid or decontaminant between the
reservoir and the vacuum line, Cotton it
not completely effective as a filtering agent,

 

3 Changes in procedures have been starred
and italicized.

FEDERAL REGISTER, VOL. 43, NO. 176-—THURSDAY, SEPTEMBER 9, 1976
sulfuric acid will corrode pipes, and con-
taminants may lose their inactivating abil-
ity upon standing. The introduction of a
cartridge-type filter that is moisture resist-
ant and has a rated capacity to remove
particles 350 mm (0.35u) or larger in size
provides an effective barrier to virus
aerosols.

The secondary reservolr and filtration ap-
paratus can be assembled from readily avail-
able units as shown in Figure 1. A length
of plastic tubing 4% inch I.D x ‘ig inch
wall is attached at one end of the reservoir
and at the other end to the lower arm of
a filtration and media storage flask. These
fiasks vary in capacity from 250 to 4000 mi,
the choice of flask depending on available
space and amount of fluid that could be
accidentally aspirated. A second tube ‘of
the same dimensions js attached from the
upper arm of the flask to the inlet port of
the disposable filter assembly. The third
tube is attached from the filter assembly to
a@ vacuum source. The tubes are securely
held to the filter by fittings supplied with
the filter and the other tubing connections
can be secured by worm drive hose clamps.

Ideally the flask should be placed higher
than the reservoir of collection vessel. If fluid
is accidentally drawn into the flask, the
Hquid can drain back into the resérvoir by
gravity if the connection at the vacuum line
is broken. This prevents the loss of fluid
which the investigator needs to retain.

Should the flask be used only for the re-
covery and storage of waste fluids, then the
addition of a few grams of Dow Corning
Antifoam A to the flask will reduce violent
foaming of fluids aspirated into it. Such
fluids can be decontaminated by introducing
into the reservoir a final 5% concentration
of an iodophor or other appropriate decon-
taminant, holding for 30 minutes and drain-
ing as above.

If the filter becomes contaminated or re-
quires changing, the filter and flask can be
safely removed by clamping the line between
filter and vacuum source. The filter and flask
should be autoclaved before the filter is
discarded. A new filter can then be installed

and the assembly replaced.

APPENDIY O, Page 9-79

Ree +
@ SECONDARY REDSAVOR Ang TATHaT om APPA tOT ee

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B Depceeme fear etteeity CUetpor OFk, Pom Tontz

Bet Carre cies Wren, ak Colsieng NA MEVECOMMAR acs

 

th
eure ee a

Ficeure
X. PACKAGING AND SHIPPING

A, Introduction. Federal regulations and
carrier tariffs have been promulgated to
ensure the safe transport of hazardous bio~

FEDERAL REGISTER, VOL. 41, NO.

NOTICES

logical materials. The NIH Guidelines specify
that all DNA recombinant materials will be
packaged and shipped in containers that
meet the requirements of these regulations
and carrier tariffs. In addition when any por-
tion of the recombinant DNA material is
derived from an etiologic agent Hsted in
paragraph (c) of 42 CFR 72,25 (which 1s
included at the end of this section, page
D-85) the labeling requirements in these reg-
ulations and carrier tariffs shall apply.

B. Packaging of recombinant DNA mate-
rials. 1. Volume less than 50 ml. Material
shall be placed in a securely closed, water-
tight container [primary container (iest
tube, vial, etc.)] which shall be enclosed in
@ second, durable watertight container
(secondary container). Several primary con-
tainers may be enclosed in a single secondary
container, if the total volume of all the
primary containers so enclosed does not ex-
ceed 50 ml. The space at the top, bottom,
and sides between the primary and secondary
containers shall contain sufficient nonpar-
ticulate absorbent material to alsorb the
entire contents of the primary container (s)
in case of breakage or leakage. Each set of
primary and secondary containers shall then
be enclosed in an outer shipping container
constructed of corrugated fiberboard, card-
board, wood, or other material of equivalent
strength.

If dry ice is used as a refrigerant, it must
be placed outside the secondary container(s).
er(s).

Descriptions of this packaging method are
given in Table III.

2. Volumes of 50 ml or Greater. Material
shall be placed in a securely closed, water-
tight container (primary container) which
shall be enclosed in a second, durable water-
tight container (secondary container). Single
primary containers shall not contain more
than 500 ml. of material. However, two or
more primary containers whose combined
volumes do not exceed 500 ml. may be placed
in a single secondary container. The space
at the top, bottom, and sides between the
primary and secondary containers shall con-
tain sufficient non-particulate absorbent ma-
terial to absorb the entire contents of tha
primary container(s) in case of breakage or
leakage. Each set of primary and secondary
containers shall then be enclosed in an outer
shipping container constructed of corrugated
fiberboard, cardboard, wood, or other ma-
terial of equivalent strength. A shock absorb-
ent material, in volume at least equal to that
of the absorbent material between the pri-
mary and secondary containers, shall be
placed at the top, bottom, and sides between
the secondary container and the outer ship-
ping container. Not more than eight sec-
ondary shipping containers may be enclosed
in a single outer shipping container. (The
maximum amount of materials which may

38475

be enclosed within a single outer shipping
container should not exceed 4,000 ml).

If dry ice is used as a refrigerant, it must
be placed outside the secondary container(s).
If dry ice is used between the secondary con-
tainer and the outer shipping container, the
shock absorbent material shall be placed so
that the secondary container does not become
loose inside the outer shipping container as
the dry ice sublimates.

Descriptions of packages which comply
with the regulations of the Department of
Transportation (DOT) are given in Table IV.

C. Labeling of packages containing re-
combinant DNA naterials. 1. Materials which
do not contain any portion of an etiologic
agent listed in paragraph (c) of 42 CFR 72.25.

Material data forms, letters, and other
information identifying or describing the
material should be placed around the out-
side of the secondary container. Place only
the address label on the outer shipping con-
tainer.

DO NOT USE THE LABEL FOR ETIOLOGIC
AGENTS/BIOMEDICAL MATERIAL.

2. Materials which contain any portion of
an etiologic agent Hsted in paragraph (c)
of 42 CFR 72.25,

Material data forms, letters, and other
information identifying or describing the
material should be placed around the outside
of the secondary container. In addition to
the address label, the label for Etiologic
Agents/Biomedical Material must be affixed
to the outer shipping container. This label is
described in paragraph (c)(4) of 42 CFR
72.25.

3. Materials which contain any portion
of a plant pest (plant pathogens) which are
so defined by the Department of Agriculture
(USDA).

Material data forms, letters, and other in-
formation identifying or describing the ma-
terial should be placed around the outside
of the secondary container. In addition to
the address label, the shipping labels fur-
nished by the USDA as part of the General,
Courtesy, or Special Permits required for re-
search with and shipment of such agents
shall be affixed to the outer shipping
container.

D. Additional shipipng requirements and
limitations for recombinant DNA mate-
rials —1, Domestic Transportation. Civil
Aeronautics Board Rule No. 82 (Air Trans-
port Association Restricted Articles Tariff
6-D) requires that a Shipper's Certificate,
depicted below, be completed and affixed to
all shipments which bear the ETIOLOGIC
AGENT/BIOMEDICAL MATERIALS label re-
quired under the provisions of the Inter-
state Quarantine regulations (42 CFR
72.25(c)). The Certificate must be com-
pleted in duplicate and affixed to the outer
shipping container.

This Is to certify that the contents of this consignment are properly classified. described by pronee
shipping name and are packed. marked and labelled and are in proper condition for carriage by nig
according to all applicable carrier and government regulations. {For international shisments add
“and to the JATA Restricted Articles Regulutions’J This cons.anment {ts within the limitations
Drescribed for: PASSENGER AIRCRAFTICARGO ONLY {cross out nonapplicabie).

 

 

 

 

 

 

 

 

 

 

Number of Specify Each Article Separately : . Net Quantity
Packages (Proper Shipping Name) Classification per Package
ETIOLOGIC AGENT, n.0.8. ETIO. AG,
Shipper: Dete .
Bignsture of Shipped

176—THURSDAY, SEPTEMBER 9, 1976
38476

Shipments of recombinarit DNA Materiala
exceeding 50 ml in volume and containing
any portion of an etiologic agent listed in
paragraph (c) of 42 CFR 72.25 are restricted,
by DOT regulations, to transport by sargo
only aircraft. When the volume of a single
primary container exceeds the 60 ml limita-
tion, this restriction must be indicated on the
Shipper’s Certificate by crossing out “Passen-
ger Aircraft”.

When dry ice is used as 8 refrigerant an
“ORA—Group A—DRY ICE LABEL” should
be affixed to the outer shipping container.

NOTICES

The amount of dry ice used and the date
packed should be designated on the label.

2, International Transportation.—in addi-
tion to the packaging and labeling require-
ments of the regulations previously cited, in-
ternational shipments of recombinant DNA
materials in which any portion of the mate-
yial is derived from an etiologic agent listed
in paragraph (c) of 42 CFR 72.25 must have
one or more of the following documents—
depending on the country of destination:

(1) Parcel Post Customs Declaration (PS
2966) tag.

APPENDIX D, Pace D-83

TABLE TIL

(2) Parce) Post Customs Declaration (PS
2966—A) label. -

(3) International Parcel Post—Instructions
Given by Sender (POD 2922) label.

(4) Dispatch note (POD 2972) tag

(5) “Violet Label”

(6) Shipper’s Certificate specified In the
current International Air Transport Associa-
tion Teriff. Individual country requirements
are listed in “International Postage Rates and

_ Fees” (USPO Publication 51).

Description of Packager for Material tn Volume lest then 50 wl,

Wolune Primary
Gut) Container
35 Sealed viel(s) or onali
mex. glace test tube, ecrew cept
@¥ stopper, taped
x One 20 x 150 on teat tubs,
Ci Gaped* etopper cr multiple
Joes wal) viale
» Plastic’ screw cap* bottle
on or Pyrex glass with ekire
less zubdbey stopper |
so Multiple watercight viale
or or * tubes, taped stoppers
Jess

fhe Qlexibiliey of the pleetic bottle requires that # atopper ox screw cap
flat-sided preacription bottle fa too fragile for use, For air transport,

and all ecrew-cepped container of unfrozen Liquid must be
that may result dn leakage past the acrew cap,

place vith wire, tepe, or other means,
at both ende to prevent atmospheric: decompresalon

©0.D, = outside dimenaions.

af Monparticulete absorbent material at

Bi 610 x 708 and 804 x 906 are trade designations for ovtelde dimensions of §-10/16 Inches diemeter x 7-8/16" height, and B-4/26"

ae} Wone required, but with the 306 x 400 cans or larger cane u

df If mteriale are to ba refrigerated,
@hipping container,
Gerben dioxide,

top, bottom and eided that will completely abaork contents

4¢ da recommended that an overpack be use
A leak proof outer container must be used for water ice.

Becondary
Packing Container
af Hetai can 2" diam, x 2"
O.D, metal ecrew cap
al Metal can 2-1/2" diam,
x 6-2/2" high 0,D, screw
fap
s/ Metal can 2-1/2" dian. x
6-1/2" high 0,D. ecrew
cap
al One or more Priction-

weal tin cana b/ 306 x 400 -
or larger

he secured in place by adhesive tape, The
all stoppers, corks, and cape oa primary containers must be secured in
placed in $ or 6 wil polyvinyl tubing heat-sealed

Outer Shipping £/

Packing Container

one Fiberbody; metal screw

Required eaep, top and Letton;
A-1/2" dion, % 7 to
7-1/2" O.De

Rona Fiberbody; metal ecrew

Required gap, top and bottom;
3-1/4" diem, x 7 fo
7-1/2" 0.0,

Rene Fiberbody; metal acrew

Required. cap, top and bottom)
3-1/4" ‘diam, #7 to
7-1/2" OD.

cf Fiberboard box

pousl equivalent-eize glase

of the prinery contelner{e).

x 9-8/16".

ae xuffictent nonparticulate shock-absorbent material to prevent rattling.

d@ to contain the refrigerant and the secured (original) outer
If dry ice ie used the outer tontainer muet permit release of
Interior eupporte must be provided to hold the container(e) in the original position(s) after wet ox dry ice haa Blesipated,

FEDERAL REGISTER, VOL, 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
‘NOTICES 88497
TABLE 1¥
Besediption of Packages for Material {nm Volumes of 50 al or greater

*

. Packing Suter Shipping Container
Yoluse Primary Secondary With Without With Without
(al) Container Packing Container Refrigerant Refrigerant Refrigerant Refrigerant
S81 te Plaatic® or Pyrex af Conatsts of metal con- Styrofoam box shock= cf Piberboard box closely Corrugated fiber=
100 al glass screw cap* tainer & outer container absorbent insulation fitting the atyrofoam board or cardboard
bottle; cubber or speciffed in Table ILL box, taped shut box, taped ahut
ekire rubber
stopper, Cuped®
103 Oria 100 wl plasticA af No. Jerimp seal tin dan Styrofoam box shocks = ef Fiberboard box closely Y¥3C cardboard bax
max, screw cap* narrow 404 x 700 or a 1-gallon absorbent insulation iecing the styrofoam PS) type, 9-3/16
neck bottle or friction-seal tin can, box, taped shut x 9-3/16" x 11-1/6"
Pyrex glase, taped®# 610 x 708, top acldered high 0.0. taped,
er clipped st 4 points bf shue with 3” type
Fad tape
200 Two 100 ml plastic® al No, 3 crimp seal tin can Styrofoam box shock= of Piberboard box closely V3C cardboard box
max, screw cap* bottles 404 x 700 or a I~gallon absorbent {neuiation fitting the styrofoam PS3 type, 9-3/16%
or Pyrex glass, teped friction-seal tin can, box, taped shut x 9-3/16" x L1-1/4"
610 x 708, top soldered high 0.D. taped
or clipped at 4 pointe bf shut with 3" type 2
PS) tape a
ao
250 One 250 wk, plastic& af No, J crimp aeal tin can Styrofoam box shocks ef Fiberboard box closely V3C cardboard box od
max, farrow mouth acrew . 404 x 700 or a 1-~gallon absorbent inauletion fitting the styrofoam PSI type, 9-3/16"
cap* bottle or Pyrex friction-seal tin can, box, taped shut x 9-3/16" x 11-2/46" FF
Blass skirted rubber 610 x 708, top soldered o¢ high 0.D, taped —
stopper, taped@ Clipped at 4 pointe b/ whut with 3" type 3
PS] tape 2
300 Two 250 ol plastict af 2-galion Frictlon-seal tin Styrofoam box shock« gv Fiberbosrd box closely VIC cardboard box zg
max, actew cap* bottles can, 804 x 908, top absorbent ineulatioa Ficting the atycofoem = 22-2/4" x 12-1/4"
oc Pyrex glass bottles, aoldered or clipped at & box, faped shut x 10-3/16" high
taped pointa b/ O.D. taped shut
with 3" wide Psd
gape,
$00 500 ml Pyrex gloss al No, 12 eriap meal tin ean — Styrofoaa box ahock= = ef Fiberboard box closely V3C cardboard box
max, bottle, rubber-shice 603 x 810 2-galion Friction~ absorbent inaulatton Citting the styrofoam = 12-1/4" x 12-174

“tha Flexibility of the plastic bottle requires that a stopper or screw cap be secured in place by adhesive tapes
Yor air trenaport, alk stoppers, corks, and cape om primary containers must be secured ia

flat-eided prescription bottle ts toe fragile for use.
quid must be placed in § or 6 mil polyvinyl tubing heat-sealed

Place with wire, cape, or other meane, and all serew-capped containera of unfrozen 1i

stopper, taped, of
500 mi plastic®
bottle, narrow or
wide pouth, screw
cap*, taped

weal tin can, 804 x 906,
top soldered or clipped at
4 points b/

et both ends to prevent atmospheric decompression that may result tn leakage past the ecrew cap,

0,D. = outeide dimenatona,

dox, taped shut

o/ Nonparticulate absorbent oatecdal at top, bottom and aldes that will completely absorb contents of the primary contalner(a),

xu 10-3/16" high

O.D. taped ahue

with 3“ wide 253
tape. For the fo, 12
can @ cardboard

box fe ok taped

ebut

Tha usual equivalant-alze plese

B/ 610 x 708 and 804 x 908 are trade designations for outatde dimensions of 610/16 inches dismeter x 2-8/16" hetght, and B-A/16" x 9-B/16%.

gf Shock absorbent material, in volume at least
the escondary container and the
not becone loose inside the outer ehipping container ae th.

outer shipping container.

equal to that between tha primary and secondary container(e), at the top,
The shock absorbent material ehall be eo placed that the eecondary contalaer(e) deep
je water ice or dry ice fe diesipsted.

FEDERAL REGISTER, VOL, 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976

bottom, and eldes betvess
38478

NOTICES
APPENDIX D, Page D-85

ATTACHMENT 1

DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE

_ PUBLIC HEALTH SERVICE
CENTER FOR DISEASE CONTROL
ATLANTA, GEORGIA 30333

Telephone: (404) 633-3313, Ext. 3683

TITLE 42—PUBLIC HEALTH

Chapter I~Public Health Service, Department of Health, Education, and Welfare
SUBCHAPTER F-QUARANTINE, INSPECTION; LICENSING

Section 72.25 of Part 72, Title 42, Code
of Federal Regulations, is amended to
greed as follows:

$72.23 Etiologic agents
(a) Definitions. As used in this sec~

lon:

aD An “etiologic agent’ means a vi-+
able microorganism or its toxin which
* gauses, or may cause, human disease.

QA “diagnostic specimen” means
any human or animal material in¢lud-
ang, but not limited to, excreta, secreta.
blood and its components, tissue, and
tissue fluids being shipped for purposes
of diagnosis.

(3) A “biological product” means &
Dlological product prepared and manu-
Yactured in accordance with the provi-
sions of 9 CFR Part 10, Licensed Veteri-
Nary Biological Products, 42 CFR Part 73,
Zicénsed Human Biclogical Products. 21
CFR 130.3, New drugs for investigational
usein humans, 9 CFR Part 103, Biological
Products for Experimental Treatment of
Animals, or 21 CFR 120.3(a), New drugs
for investigational use in animals, and
Which, in accordance with such provi-
aions, may be shipped in interstate traffic,

<b) Transportation; etiolegie agent
minimum packaging requirements, -No
person may knowingly transport or cause
fo be transported ‘in Interstate traffe,
directly or indirectly, any material, in-
eluding but not limited to, diagncstic
Specimens and biological products, con-
taining, or reasonably believed by such
person to contain, an etiologic agent un~

ss such material is packaged to with-
Mand leakage of contents, shocks, pres
gure changes, and other conditions inci-
@ent to ordinary handling in transpor-
tation. .

e) Transportation; etiologic agents
fubject to additional requirements. No
person may knowingly transport or cause
to be transported in interstate traffic,
directly or indirectly, any material, other
than diagnostic specimens and biological
products, containing, or reasonably be-
Heved by such person to contain, one or
more of the following etiologic agents

 

3The requirements of this section are in
Q@ddition to and not fn Heu of any other packs
waging or other requirements for the transe*
portation of ettolovic arenta oy interstate
tramc prescribed by the Departments of
Transportation and other agencies of the
Pederal Government,

PART 72—INTERSTATE QUARANTINE
Subpart C~Shipment of Certain Things

unless such material is packaged In ace
cordance with the requirements specified

_ in paragraph (b) of this section, and
unless, in addition; such material fs pack~
aged and shipped in accordance with the
requirements specified in subparagraphs
(1)~(6) of this paragraph:

* BacreriaL AGENTS

ActinobaciNus—all species,

Arizona hinshawii—all serotypes,

Baciilus anthracis. ~

Bartonelia—all species.

Bordetella—all species,

Borrelia recurrentis, B.vincentt,

Brucella—al) species,

Clostridium botulinum, Cl. chauvoei, Cl. hae«
molyticum, Cl, histolyticum, Cl, novyt, Cb,
septicum, Cl, tetani,

Corynebacterium diphtheriae ©, equl, C, hae«
molyticum, C. pseudotuberculosis, C. pyo-
genes, C. renale.

Diplococeus (Streptococeus) pneumonias,

Erysipelothriz insidtosa,

Escherichia coli, all enteropathogenic Sero-

types.

Francisella (Pasteurella) tularensis.

Haemophilus ducreyi, H. influenzae,

Herellea vaginicola,

Klebdsiella—all species and all serotypes,

Leptespira interrogans—all serotypes,

Listeria—all species,

Mime nolymorpha,

AMorazelia—all species.

Mycobacterium—all species,

Mycoplasma—all species, .

Neisseria gonorrhoeae, N, meningitidis,

Pasteureiia—all species.

Pseudomonas pseudomatlet,

Salmoneiia—all species and all serotypes,

Shigella—all species and all serotypes,

Sphaerophorus necrophorus,

Staphylococcus aureus.

Streptodacillus monilijormis,

Strepincoccus pyogenes.

Treponema careteum, T. pallidum, and T.
pertenue. .
Vibrio fetus, V. comma, including biotype

El Tor, and ¥. parahemolyticus,

Yersenia (Pasteurella) pestis,

FPuncat Acentg

Actinomycetes (including Nocardia spacies,
Actinomyces species and Arachnia propi-+
onica),

Blastomyces dermatitidis,

Coccid‘oides immitis.

Cryptococcus neoformans,

Histoplasma capsulatum,

Paracoccidioides brasiliensis,

Vinat, RICKETTSIAL, ANG CHLAMYDIAL
-AGENTS

Adenoviruses human—all types,
Arboviruses,

Coziella burnetii.

Corsackie A and B viruses~all types,
Cytomegaloviruses,

Dengue virus.

Echoviruses—all types.

Encenhalomyocarditis virus,

Hemorrhagic fever agents, including Crimean
hemorrhagic fever (Congo), Junin, and
Machupo viruses, ‘and others ag yet une
defined.

Hepatitis-associated antigen,

Herpesvirus—ali members,

Infectious bronchitis-like virus,

influenza viruses—all types.

Lassa virus.

Lymphocytic choriomeningitis viTud,

Marburg virus,

Measles virus,

Mumops virus.

Parainfluenza viruses—all types,

Polioviruses~—all types.

Poxviruses—all members.

Psittacosis « Ornithosis = Trachoma-Lymp ioe
granuioma group of agents,

Rabies virus—all strains,

Reoviruses—all types. -

Respiratory syncytial virus,

Rhinoviruses—all types,

Rickettsia~ail species,

Rubella virus.

Simian viruses—all types.

Tick-borne encephalitis virus complex, {ne
cluding Russtan spring-summer encephae
litis, Kyasanur forest disease, Omsk herore
rhagic fever, and Central European encephe
alitis viruses,

Vaccinia virus.

Varicelia virus.

Variota major aud Variola minor viruses.

Vesicular stomatis virus.

Fellow fever virus,

(1) Volume less than 50 mI, Material
shall be placed in a securely closed, wae
tertight container (primary container
dtest tube, vial, ete.)) which shall be en-
closed in a second, durable watertight
container (secondary container), Esve
eral primary containers may be enclosed
in a Single secondary container, if the
total yolume of all the primary contains
ers So enclosed does not exceed 50 ml.
‘The space at the top, bottom, ‘and sices
between the primary and secondary cone
tainers shail contain sufficient nonpore
ticulate absorbent material to absorb tne
entire contents of the primary contains
er(s) in case of breakage or leakage.
Each set of primary and secondary cons
tainers shall then be enclosed in an outer
shipping container constructed of corru+
gated fiberboard, cardboard, wood,, or
other material of equivalent strenrth.

(2) Volume 50 ml. or greater, Pore tr
ing of material in volumes of §9 n°.
moore shall include, in addition, @ six:
absorbent material, in volume at least
equal to that of the absorbent material

   

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
between the primary and secondary con-
tainers, at the top, bottom, and sides be-,
tween the secondary container and the
outer shipping container. Single primary
containers shall not contain more than
500 ml. of material. However, two or more
primary containers whose combined vol-
umes do not exceed 500 ml. may be placed
in a single, secondary container, Not
more than eight secondary shipping con-
tainers may be enclosed in a single outer
shipping container. (The maximum
amount of etiologic agent.which may be
enclosed within a single outer shipping
container shall not exceed 4,000 ml.)

(3) Dry ice. lf dry ice is used as & re-
frigerant, it must be placed outside the
secondary container(s). If dry ice fs used
between the secondary container and the
outer shipping container, the shock ab-
sorbent material shall be so placed that
the secondary container does not become
loose inside the outer shipping container
as the dry ice sublimates,

(4) Labels. The label for Etiologic
Agents/Biomedical Materlal, except for
size and color, must be as shown:

a irelmeciim ct Bey

BIOMEDICAL
MATERIAL

Mee ala Ned
Bre RN Te ved
NOTIFY OIRECTOR COC
er Se ethic}
CREE SLR)

 

(i) The color of material on which the
label is printed must be white and the
symbol and printing in red.

 

NOTICES

APPENDIX D, Page D-86

(ii) The label must be a rectangle
measuring 51 mm. (2 inches) high by
102.5 mm. (4 inches) long.

(iii) The red symbo] measuring 38 mm.
(114 inches) in diameter must be cen-
tered in a white square measuring 51
mm. (2 inches) on each side.

(iv) Type size of the letters of label
shall be as follows:

ETIOLOGIC AGENT.......-.-. 10 pt. rev.
BIOMEDICAL MATERIAL....... 14 pt.
IN CASE OF DAMAGE OR

LEAKAGE Wooo n nen e ene ene 10 pt. rev.
NOTIFY DIRECTOR CDC

ATLANTA, GA...2.-----2-- ae 8 pt. rev.
404 633 5313..------neennen nnn 10 pt. rev,

(5) Damaged packages. Carriers shall
promptly, upon discovery of damage to
the package that indicates damage to the
primary container, isolate the package
and notify the Director, Center for Dis-
ease Control, 1600 Clifton Road NE.,
Atlanta, GA 30333 (telephone (404) 633-
5313), and the sender. ,

(6) Registered mail or equivalent sys-
tem. Transportation of the following
etiologic agents shall be by registered
mail or an equivalent system which ree
quires or provides for sending notifica-
tion to the shipper immediately upon
delivery:

Actinobacillus mallet,

Cocetdioides immitis.

Francisella (Pasteurella) tularensis.

Hemorrhagic fever agents, including, but not
limited to, Crimean hemorrhagic fever

(Congo), Junin, Machupo viruses,
Herpesvirus simiae (B virus),

Histoplasma capsulatum,
Lassa virus.

Marburg virus.
Pseudomonas pseudomaliet,

Tick-borne encephalitis rirus complex, in«
cluding. but not timited to, Russtan spring-
summer encephalitis, Kyasanur forest dise
ease, Omsk hemorrhagte fever, and Central
European encephalitis yiruses, Variola
minor end Variola:major,

Yersenia (Pasteurella) pes?

(d) Notice of deliverz:. failure to re
ceive. When notice‘of delivery of agents
containing, or suspected of containing,
etiologic agents listed in paragraph (c)

. (6) of this section is not received by the

sender within 5 days following antici-
pated delivery of the package, the shipper
shall notify the Director, Center for Dis-
ease Control, 1600 Clifton Road NE.,
Atlanta, GA 30333 (telephone (404)
633-5313).

(e) Requirements; variations. The Ad-
ministrator may approve variations from
the requirements of this section if, upon
review and evaluation, he finds that such
variations provide protection at least
equivalent to that provided by compli-

‘ance with the requirements specified in

this section and makes such findings a
matter of official record.

(Sec. 361, 58 Stat. 703; 42 U.S.C. 264)

‘Pena e)

Effective July 30, 1972

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
38480 NOTICES

 

evant | PACKAGING AND
CULTURE | LABELING OF
aeconsent ! ETIOLOGIC

rACnING \ AGENTS

- FIGURE 2

CAP

SS

SECONDARY
CONTAINER
SPECIMEN
RECORD
HSM 9.203

 

 

PROOF

 

tae
eco”

 

Cap

 

 

 

 

 

 

 

 

 

SHIPPING , ENT
CONTAINER a KING
EA r? AL
LABEL

 

 

 

 

 

 

 

 

 

 

 

ADDRESS on
LABEL ee
° Sie)
- CROSS SECTION
OF PROPER PACKING

The Interstate Quarantine Regulations 142 CFR, Pat 72 26
Etiologic Agents) was revised July 31, 1972 to provide for
packaging and labeling requirements for etiolugie agents tint
certain other materials shipped in interstate traffic,

Figures 1 and 2 diagram the packaging und Jabeling of etra-
fogic agents in volumes of less than 50 ml. in accordance with
the provisions of subparagraph (C) (1) of the cited regulation,
Figure 3 itlustrates the color and size of the tubel, described
in subparagraph (C) (4) of the regulations, which shall be
affixed to all shipments of etiologic agents.

For further information on any provision of this regufatian
contact:
Center for Disease Control
Attn: Biohazards Contral Office
1600 Clifton Road
Atlanta, Georgia 30333

Telephone: 404 6333311

FIGURE 3

ETIOLOGIC AGENTS

gm BIOMEDICAL
MATERIAL

IN CASE OF DAMAGE
Fa . OR LEAKAGE.

te RAL ACL 1

artery Wt? MelseL (1) )

PvE kt ahd

 

FEDERAL REGISTER, VOL. 4), NO. 176—THURSDAY, SEPTEMBER 9, 1976

L$-d S8eg ‘a XIaNgday
NOTICES 38481

 

APPENDIX D, Page 0-38

PACKAGING AND LABELING OF ETIOLOGIC AGENTS

  
 
  
   
   
  
 
 
 
 
  
 
 
  

ABSORBENT PACKING MATERIAL

PRIMARY CONTAINER (Battle, blood buy, eh.]®

*NOTE: Single primary containers may not exceed 500
mi. of material. Two or more primary containers whose
combined volumes do not exceed S00 mi. may be ens
closed in a single, secondary container, The maximum
volume of etiviugic agent which may be enclosed ina
single outer shipping cuntainer shafl nut exceed 4000 ml.

SHOCK ABSORBENT MATERIAL

SECONDARY CONTAINER (Gusketed screwcop
with waterproot? tape or hermetically sealed cun}

OUTER SHIPPING CONTAINER

MAILING LABEL

ETIOLOGIC AGENT LABEL

The Interstate Quarantine Regulations (42 CFR, Part
72.25, Etiologic Agents) was revised July 31, 1972, to
provide for packaging and labeling requirements for etio-
logic agents and certain other materials shipped in inter-
state traffic, The illustration shows acceptable packaging
and labeling of etiologic agents in acenrdunce with sub-
paragraphs {c) (2) and (4) of the cited regulation.

 

 

For further information on any provision of this regulation contact:

Center for Disease Control
Attn: Biohazards Control Office
1600 Clifton Road

Atlanta, Georgia 30333

Telephone: 404-633-3311

FEDERAL REGISTER, VOL. 41, NO. 176—THURSDAY, SEPTEMBER 9, 1976
38482

XI. TRAINING AIDS, MATERIALS AND COURSES

A. Slide-tape cassettes. 1. Assessment of
Risk in the Cancer Virus Laboratory ($10).

2. Effective Use of The Laminar Flow Bio-
logical Safety Cabinet ($10).

3. Formaldehyde Decontamination of
Laminar Flow Biological Safety Cabinets
($10).

4. Certification of Class II (Laminar Flow)
Biological Safety Cabinets ($13).

5. Hazard Control in the Animal Labora-
tory ($10).

6. Basic Principles of Contamination Con-
trol (In preparation).

7. Selection of a Biological Safety Cabinet
(In preparation) .

These slide tape cassettes are available for
purchase from the National Audiovisual Cen-
ter. The price for each is given above after
the title. Send your order prepaid with a
check or money order made payable to Na-
tional Archives Trust Fund and mail to:
Sales Branch, National Audiovisual Center
(GSA), Washington, D.C. 20409.

8. Research Laboratory Safety.

This slide tape cassette, stock number
176.79, is available for $75 from the National
Safety Council, 425 North Michigan Avenue,
Chicago, Illinois 60611.

B. Films. 1. Air Sampling for Microbiologi-
eal Particulates (M—926}.

2. Handling the Laboratory Guinea Pig
(T2618-X).

8. Handling
(T2617-X).

4, Infectious Hazards of Bacteriological
Techniques (M-382).

5. Laboratory Design for Microbiological
Safety (M-1091).

6. Plastic Isolators: New Tools for Medical
Research (M-599).

7. Safe Handling of Laboratory Animals
(M-455).

8. Surface Sampling for Microorganisms
(Rodac Method) (M-924).

9. Surface Sampling for Microorganisms
(Swab Method) (M-925).

These films are available on loan without
charge from: Media Resources Branch, Na-
tional Medical Audiovisual Center (Annex),
Station K, Atlanta, Georgia 30324. The same
films (except 2 and 3) can be rented or
bought from: National Audiovisual Center
(GSA), (Rental Branch)—(Sales Branch),
Washington, D.C. 20409.

C. Courses. 1. Biohazard and Injury Con-
trol in the Biomedical Laboratory. Presented
by the University of Minnesota, School of
Public Health and the National Cancer In-
stitute, Office of Research Safety. Direct in-
quiries to Dr. Donald Vesley, University of
Minnesota, School of Public Health, 1325
Mayo Memorial Building, Minneapolis,
Minnesota 55455. June 22-24, 1976, Los
Angeles, CA; October 26-28, 1976, Boston,
MA; December 7-9, 1976, Bethesda, MD.

2. Biohazard Containment and Control for
Recombinant DNA Molecules. Presented by
the University of Minnesota, School of Pub-
lic Health and the National Cancer Institute,
Office of Research Safety. Direct inquires as
above. September 8-9, 1976, Stanford, CA;
September 21-11, 1976, Cold Spring Harbor,
NY.

3. Safety in Laboratory. Presented by Na-
tional Institute of Occupational Safety and
Health, Division of Training and Manpower
Development, by special arrangement. Robert
A. Taft Laboratories, 4676 Columbia Park-
way, Cincinnati, Ohio 45226.

4. Laboratory Safety Management. Pre-
sented by the Laboratory and Training Divi-
sion, Bureau of Laboratories, Center for Dis-
ease Control, Atlanta, Georgia, September 14-
16, 1976 and September 13-15, 1977.

the Laboratory Mouse

NOTICES

KIL, OUTLINE OF A SAFETY AND OPERATION
MANUAL FOR A P4 FACILITY

A. Purpose.

B. Policy.

C. Responsibility and Authority. 1, Man-
agement.

2. Supervisor.

3. Each Employee.

4, Facility Safety Officer.

5. Biohazard Safety Committee.

D. Racility Assignment Procedures.

E. Reporting of Major and Minor Accidenis
and Injuries, Exposure to Tozie or Infec-
tious Materials, Unsafe Conditions and Prop-
erty Damages, and Rendering First-Atd.

F. General Laboratory Safety. 1. Fire.

2. Equipment.

3. Physical.

4, Chemical.

5. Radiological,

G. Safety Procedures Associated with Bio-
hazard Activities of the Laboratory. 1. Per-
sonnel Practices. .

2. Operational Practices.

H. Medical Surveillance.

I. Facility Operations. 1. Personnel Access
Procedures.

2. Access Procedures for Equipment Mate-
rials and Supplies.

3. Maintenance and Support.

4. Zorie Classification.

5. Facility Monitoring Procedures.

6. Housekeeping.

J. Others. 1. Packaging and Shipment of
Biohazardous Materials.

2. Emergency Procedures.

3. Insect and Rodent Control.

4. Orientation and Training.

Appendix D was prepared by a Working
Group Consisting of: W. Emmett Barkley
(Chairman), National Cancer Institute,
NIH; Manuel S. Barbeito, National Cancer
Insitute, NIH; Everett Hanel, Jr., Frederick
Cancer Research Center; George S. Michael-
sen, School of Public Health, University of
Minnesota; Vinson R. Oviatt, Division of
Research Services, NIH; Warren V. Powell,
Division of Research Services; NIH; John
Richardson, Center for Disease Control;
James F. Sullivan, National Animal Disease
Laboratories; and Arnold G. Wedum, Fred-
erick Cancer Research Center.

REFERENCES

1. American National Standards Institute.
1968. Z 35.1.

2. Wedum, A. G. 1964. Laboratory safety
in research with infectious aerosols. Public
Health Rep. 79:619-633.

3. Phillips, G. B. 1965. Microbiological haz-
ards in the laboratory. J. Chem. Education.
Part One—Control, 42:A43-A48. Part Two—
Prevention, 42:A117—A130.

4, Hellman, A. 1969. Biohazard control and
containment in oncogenic virus research.
USPHS, NIH, NCI. :

5. Darlow, H. M. 1969. Safety in the micro-
biological laboratory. In J. R. Norris and D.
W. Ribbons (eds.). Methods in Microbiology.
Academic Press, New York, pp. 169-204.

6. Chatigny, M. A. and Clinger, D. I. 1969.
Contamination control in microbiology. In
R. L. Dimmick and A. B. Akers (eds.). AD
Introduction to Experimental Aerobiology.
John Wiley & Sons, New York, pp. 194-263.

7. Chitigny, M. 1961. Protection against in-
fection in the microbiological laboratory, de-
vices and procedures. In W. W. Umbreit (ed.).
Advances in Applied Microbiology No. 3. Aca-
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8. Collins, C. H., Hartley, E. G., and Piis-
worth, R. 1974. The prevention of laboratory-
acquired infection. Public Health Laboratory
Service Monograph Series No. 6. Her Majesty's
Stationery Office, London.

9. Anon. 1968. Good Laboratory Practices
Manual. National Cancer Institute, Be-
thesda, MD.

10. U.S. Public Health Service, 1974. NIH
Biohazards Safety Guide. GPO Stock #1740—
00383. Supt. Documents, U.S. Government
Printing Office, Washington, D.C. 20402.
($3.85)

11. Baldwin, C. L., Lemp, J. F., and Barbe-
ito, M. S. 1975. Biohazards assessment in
large-scale zonal centrifugation. Appl. Mi-
crobiol. 29:484—490.

12. Morris, 6. A. and Everall, P. H. 1972.
Safe disposal of air discharged from centri-
fuges. J. Clin. Path. 25:742.

13. Hall, C. V. 1975. A biological safety
centrifuge. Health Lab. Sci. 12:104-106.

14. Reitman, M. and Wedum, A. G. 1966.
Microbiological safety. Public Health Rep.
71:659-665.

15. Hanel, E. and Kruse, R. H. 1967. Lab-
oratory -acquired mycoses. Misc. Publ. 28, AD-
665376. Frederick Cancer Research Center,
P. O. Box B, Frederick, MD.

16. Smadel, J. BE. 1951. The hazard of ac-
quiring virus and rickettsial diseases in the
laboratory. Amer. J. Public Health 41:788-
7195.

17. Heckly, R. J. Quoted in A. H. Harris
and M. B. Coleman (eds.). 1963. Laboratory
infections and accidents. In Diagnostic Pro-
cedures and Reagents. Amer. Public Health
Assoc., Inc., New York.

18. Grieff, D. 1969. Safe procedure for open-
ing evacuated glass ampoules containing
dried pathogens. Appl. Microbiol. 18:130.

19. Harney, R. W. S., Price T. H., and Joyn-
son, D. H. M. 1976. Observations on environ-
mental contamination in a microbiological
laboratory. J. Hyg. Camb. 76:91-96.

20. Rutter, D. A. and Evans, C. G. T. 1972.
Aerosol hazards from some clinical labora-
tory apparatus. Brit. Med. J. 1:594-597.

21. Godber, G. 1975. Report of the working
party on the laboratory use Of dangerous
pathogens. Cmnd 6054, ISBN 0 10 160540, 40
pp. Her Majesty's Stationery Office, London.

22. Iodine, L. S. (ed.). 1973. Centrifuge Bio-
hazard Proceeding of a Cancer Research
Safety Symposium, Frederick Cancer Re-
search Center, Frederick, Md.

23. Hers, J. F. Ph. and Winkler, K. C. 1973.
Airborne transmission and airborne infec-
tion. John Wiley & Sons, New York: A. Page
415, B. Page 425, C. Page 429, D. Page 434,
EB. Pages 436-437, F. Page 461, G. Page 494.

24, Lidwell, O. M. 1967. Take-off of bacte-
ria and viruses, pp. 116-137. In P. H. Gregory
and J. L. Monteith (eds.). 17th Symposium
of the Society for General Microbiology,
Cambridge Univ. Press, London: A. Page 17,
B. Page 128, C. Pages~127-128, D. Page 130.

25. Lennette, E. H., et al., 1974. Laboratory
Safety regulations, viral and rickettsial dis-
ease laboratory. California State Department
of Health, Berkeley, CA.

26. U.S. Public Health Service. 1975. Lab
safety at the Center for Disease Control.
DHEW Publication No. CDC 76-8118. USPHS,
CDC, Atlanta, GA.

27. U.S. Army 1969, Safety regulations,

microblological, chemical and industrial
safety. FDR 385-1. Fort Detrick, Frederick,
MD.

28. Lidwell, O. M., Brock, B., Shooter, R. A.
Cooke, E. M., and Thomas, G. E. 1975, IV.
Airborne dispersal of Staphylococcus aureus
and its nasal acquisition by patients.

29. Guyton, H. G. and Decker, H. M. 1963
Respiratory protection provided by five new
contagion masks, Appl. Microbiol. 11:66-68.

30. Braymen, D. T., Songere, J. R., and Sul-
livan, J. F. 1974. Effectiveness of footwear
decontamination methods for preventing the
spread of infectious agents. Lab. Anim. Sct. —
24 888-894.

FEDERAL REGISTER, VOL. 41, NO. 176—-THURSDAY, SEPTEMBER 9, 1976
31, Barbeito, M. S., Mathews, C. T., and
Taylor, L. A. 1967. Microbiological laboratory
hazard of bearded men. Appl. Microbiol.
15:899-906.

32. U.S. Public Health Service, 1974, Guide
for the Care and Use of Laboratory Animals.
DHEW Publication No. (NIH) 74-23. U. 5.
Government Printing Office, Washington,
D.C. 20402. Price: 70¢. Stock No. 1749-0343.

33. Code of Federal Regulations, Title 9—-
Animals and Animal Products. For sale by
Supt. of Dochments, Government Printing
Office, Washington, D.C. 20402. (In the capi-
tal city of most states, a copy limited to 9
CFR Chapter 1, Subchapter A, Parts 1,2,8
can be obtained from the Federal Veterinar-

NOTICES
ian in Charge, Animal and Plant Health
Inspection Service.)

34. Seamer, J. (ed.). 1972. Safety in the
animal house. Laboratory Animal Handbook
5. Laboratory Animals, Ltd., London.

35, Gay, W. I. (ed.). 1965. Methods of ani-
mal experimentation, Vol. 1. Academic Press,
New York.

36. Perkins, F. T. and O'Donoghue, P. N.
31969. Hazards of handling simians, Labora-
tory Animal Handbook 4. Laboratory Ani-
mals, Ltd., London.

37. Melby, E. C., and Altman, N. H, (eds.}.
1974-1976. Vol. I, 11, IIT. Handbook of Labora-
tory Animal Science. CRC Press, Inc., Cleve-
land, OH.

38. Spaulding, E. H. 1972. Chemical dis-
infection and antisepsis in the hospital. J.
Hospital Res. 9:5-31.

39. Lawrence, C. A. and Biock, 5. 8. (eds i.
1976. Disinfection, sterilization, and pres-
ervation. Lea & Febiger, Philadelphia, PA.

40. Stecher, P. G, (ed.). 1968. The Merck
Index. Merck & Co., Inc., Rahway, NJ.

41, Taylor, L. A., Barbeito, M. S., aud
Gremillion, G. G. 1969. Paraformaldehyde for
surface sterilization and detoxification. Appi
Microbiol. 17:614-618.

42. Jemski, J. V. and Phillips, G. B. 1965
Aerosol challenge of animals. In W. I. Gay
(ed.). Methods of animal experimentation,
pp. 273-341, Academic Press, New York.

{FR Doc.76-95512 Filed 9-8-76:8:45 am]

FEDERAL REGISTER, VOL, 41, NO, 176—THURSDAY, SEPTEMBER 9, 1976