7,. kc
                  THURSDAY, SEPTEMBER 9, 1976









                                                                           I








`,






/I                            PART 111:


            DEPARTMENT OF
             HEALTH,




                National Institutes of Health



                                             O


                  RECOMBINA
                 RESEARCH GUIDELINES

                   Draft Environmental impact Statement


3s 126                             NOTICES

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, Education. and Welfare
and the Assistant Secretary for Health,
issued Guidelines that will govern the
conduct of NIH-supported research 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 potent.ial 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 Direct,or'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 potentia.lly 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 objectives were thor-
or~ghly explored, to maximize safety and
minimi7c potential risks. And an elab-
orate review st.ructure to ensure safet!,
has been created.

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
20_014.

Dated: August 26,1976.
      DONALD S. FREDRICKSON,
                 Director,
     National Institutes of Health.
DRAFT ENVIRONMENTAL IMPACT STATEMENT

GUIDELINES FOR RESEARCH INVOLVING RE-
     COMBINANT DNA MOLECULES

NATIONAL INSTITUTES OF HEALTH
    BETHESDA, MARYLAKD

August 19, 1976

GUIDELINES FOR RESEARCH INVOLVING
  RECOMBINANT DNA MOLECULES

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

(X) Draft    ( ) Final Environmental
Impact Statement.

Name of Action

(X) Administrative
Action.

Additioml Information

Additional information on the proposed
abtion, including technical documents psrti-
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
Involring Recombinant DNA Molecules" is
attached. (Appendix D)

"he Guidelines are premised on physi-
cal and bioiogical containment to pre-
vent the release or propagation of DNI\
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.

The Department. in issuing tlus 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 Qnality weekly notice in the FZDER.AL
REGISTER. Address comments to Dr. Donald
S Fredrickson.

           COrcTFEiTS
I. Foreword.
II. Authority.
111. Objective of the NIH Action
IV. Background.

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

A. Description of the recon?bi!xnt DNI
   experimental process.
B. Events leading to the development
   of guidelines.

C. Description of issl.les raised by re-
   combinant DNA research.

( ) Legi9ative

2. Expected benefits of DN.4 recom-

   binant rffiearcil.
3. Long-range implicatloxs.
4. Possible deliberate misuse

V. Description of the proposed action
VI. Description of alternatives.
   A. No action.
  B. NIH prohibition of fm~ding of al!
     experiments  u-it,h  reconll,lllaI~t
      DNA.

C. Development of different guideline;
D. No guidelines but NIH consideration
  of each proposed project on an in-
   diridual basis before fUnding
E. General Federal regulation of all
   such research

VII, Environmental impact of the guidelines.
  A. Impact of i%aance of NIH gnide-
       lines.

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

spread of possibly haznrdoas
agent- Y.

3. C&impact.
4. Secondary impacts.
B. Impact of experiments conduc:ed
   under the guidelines.
  1. Possible undesirable impacted
2. Beneficial impacts of DNA recom-
     binant research.

       *PPENDICES

A. Glwsarp.

B. Suggested references for add!tional
   reading.

C. Documents describing the impie-
   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 FF.DER.AL
   REGISTER, Part II, July 7. 1976.

FOREWORD

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 j
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 m@tipl:;. it will propagate
these newly acquired genes as part of its
own genetic complement. It appears
likely that any kind of gene from any
kind of or::anism could be introduced
it?to E. 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 cl;ange il:
the field of genetics. Non`, for the first
time. there is a methodology for cro~sin::
very large evolutionary boundaries. and
for maying genes betv;een organi,cms that
are believed to hai-e previously had litrle
genetic contact.

FEDERAL REGISTER, VOL. 41, NO. I76--THURSDAY, SEPTEMBER 9, 1976


NOTICES

38427

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 wssible hazards remain
spe&ative. 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 nublic. and the environment
should tde putative hazards prove real.

The issuance of Guidelines establish-
lng conditions and precautions with re-
sect to such exneriments is viewed bv
NIH as a Fed&al action that may
significantly affect the quality of the
&nan environment, and NIH Director
Dr. Donald S. 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.

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 significa.nt 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 auestion-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 issu,es involved,
in the Interest of gaining the under-
standing and views of the broadest DOS-
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 2421).

III. OBJECTIVE OF THE NLH 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 involvinK 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 pa.rt

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 exneriments will be
carried out with minimal risk to labora-
tory workers, the general public, and the'

environment.

IV. BXKGRO~ND

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 cbntain DNA. Figure IV-l il-
lustrates a typical cell.

DNA plays two roles: (1) Provides in-
formation for the reproduction, growth,
and functions of the cell. and (2) Dre-
serves and directs replication of thg 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 mnny viruses. The DNA cjf
cells is mainly found in organized strut-
tures called chromosomes.

Intracellular DNA also occu:`.s c::t;;ide
Of the Cll?O~lO.~Gi~l~S as separately rep-
licating molecu!cs. S:~ch DNA4 molecules
include the plasmids, found in bact.erin;
the DNA of chloroplasts, common to
green plants; nr.d the DNA of mito-
chondria, the e:iergy-producing units of
the cells of complex organisms. These
DNA% while not st,rictls part of the in-
herent genetic make-up of a cell, help
define the cell's functional capability.
Another type of DNA con:mo:?ly fou::d in
cells is the DNA of infecting vYusec.

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


3S428

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.

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
cells 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 illustrat.ed in Figure IV-3.

FIGURE 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 per-
tions of the t,wo 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 ma.y be
DNA from a virus or a plasmid, usually
derived from the same species as w-ill
serve as the host of the recombinant
DNA. From a growth cuhure of the host
cells, those containing the DNA frag-
ment of particular interest are selected

   wu
FIGURE IV-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 endonucleasea) to yield
linear 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 1s 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 o 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 member of this com-
mittee in voluntarily deferring * o o [cer-
tain] experiments * o *.

Second, plans to link fragments of ani-
mal DNAs to bacterial plasmid DNA or
bacteriophage DNA should be carefully
weighed + o o .
Third, the Director of the National In-
stitutes of Health is requested to give lm-
mediate consideration to establishing an ad-
visory committee charged with (i) 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
(111) devising guidelines to be followed by in-
vestigators working with potentially hazard-
ous recombinant DNA molecules.
Fourth, an international meeting of ln-
valved 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, 19'74, the NM Recom-
binant DNA Molecule Program Advisors
Committee (hereafter "Recombinant
Advisory Committee") was established to
advise the Secretary of HEW, the As-
sist.ant Secretary for Health, and the Di-
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


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, physica sciences, research,
education. and communications. In addi-
tion to &rent 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.

1 38.429

struction of recombinant DNA mole-
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 revort 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 biohazards. Until
these are established, we urge individual
scientists to use the proposa,ls in this
document as a guide."

The NIH Recombinant Advisors 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 snecific tidelines. and
that NIH establis`h a newsletter %r 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 IIde,
Massachusetts.

FolIowing this meeting, many Ietters
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 m-as held
on December 4-5, 1975, in La Jolla, Cali-
fornia. For t,his meeting a "variorilm edi-
tion" had been vrevared. comanrine line-
for-line the Hog&s, 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-

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 t0 the public with
scientific freedom to pursue new knowl-
edge.

Public responsibility weighs heavily in
this genetic research area. The scientific
com&mity 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 jnternational
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-
merits. Notification of all the meetings
was published in the FEDERAL REGISTER
and all the meetinps 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 broac?ly represented.

The guidelines n-ere reviewed in light
of the comments and suggestions made
by particivants at that meeting. as well
as the written comments received after-
ward. As part of that review the Recom-
binant Advisory Committ,ee was asked
to consider at its meeting of April l-2,
1976, a number of selected issues raised
by the commentators. Those issues and
the response of the Recombinant Ad-

visory Committee were tallen 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 FEDERAL REGISTER,~~~~
II, July 7, 1976 (See Appendix D).

C. DESCRIPTIONOFISSIJESRAISPD BP
  RECOMBINANTDNARESEARCH

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 cc) 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 foreian DNA on the
survival of-recipient sp&ies (host cells
or viruses). The effect of foreign DNA
on the surviva1 of recivient svecies is im-
portant to the discussion of possible haz-
ards of recombinant DNA experiments
because although a recipient species may
acauire a notential for harmful effects
as a result bf 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 cgent. depending on
the agent.

There are various i:ldir ?tie% that bar-
teria and Mruses containin~ir!iertedfc\r-
eian DNA are less liire!v to survive and
multiply than are the oricinnl organisms.
Natural evolution rc;;ilts in the survival
of well-balanced and eiiiOcient organisms.
Essential functions Rre carefullv co::-
trolled, and can be switched on &d off
as needed. It is unlikely that uncon-
trolled, nonessential properties such as
might be introduced by foreign genes
would result in ang advanbage to the
survival and multiplication of an cther-

FEDERAL REGISTER, VOL. 41, NO. I76-THURSDAY, SEPTEMBER 9, 1976


38430

NOTiCES

desirable elects. 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-
orga.nism, or it could funct,ion 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 Gela-
tive ability of the two organisms to grow
and infect an animal or plant species at
risk.

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-
centmtion of 90 percent to a concentration
of 0.0001 percent (1 part in l,OOO,OOO) 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 nresence 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 acquisiton and expres-
sion of a foreign gene conferring the
ability to metabolize particular nu-
trients. In an environmental niche con-
taming the metabolite, such a recombi-
nant might compete succesfillly against
organisms native to the niche. This
could result in destruction of an environ-
mental componenGthat 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 eflect of bacteria and viruses
containing recombined DNA m 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 life 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 pathe-
genicity or toxicity as a result of a reei-
dent recombinant :

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

The evidence available at present is in-
sufficient to predict whether or not for-
eign genes derived from a comnlex orga-
nism(animals, plants, yeasts, and fungi)
will be expressed in a bacterium in any
particular instance. It may be that spe-
ciflc manimrlations will be reouired to
permit bacteria to express information of
a foreign DNA efficiently. Faithful ex-
pression of a gene requires ac&rate func-
tioning of the complex bacterial machin-
ery involved in protein synthesis. At each
step, speciilc signals originating in the
fore@ 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 E. coli which c nnot manu-
facture the amino acid I! lstidine (5).
(Histidine is a comnonent of most oro-
teins and therefore is required for-the
growth of all organisms.) After insertion,
some cells no longer required histldine;
thus, the presence of the yeast DNA over-

came the requiremenl for histidine. This
is the first suggestion that a foreign gene
from an organism more comulex 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 protect.6
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 packagpd
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 mav
be expressed, resulting in <he>ormation
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 mau itself
cause pathogenic or toxic ebects. 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
comnetitive 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 oiga-
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-
tions in which DNA molecules themselves
may escape from the laboratory or from
the exoerimental 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 air, water, or any other medium, is
considered remote), they can be pro-
tected in nature in a variety of ways and
zTlea.sed either into, or close to, a living

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 eellula.r cem-
ponent, or it could recombine by natural

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


NOTICES

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-
mane), a.nd 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 hke-
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 pas-
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 oraanisms, 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
eukarvotes in nature? It is generally held
that &combination in nature is-more
likely if similar or identical sequences of
bases, (rungs in the DNA ladder) occur
in the two recombining DNA.?. The
greater the degree of similar sequences,
the more likely is recombination. In gen-
eral. the more closelv two snecies are re-
lated, the more likely it s that similar
sequences will be found in their DNA?,.
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
interspe&s 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 deadlntes-
tlnal 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-
es&-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-S).

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

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 ant.ibiotics 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 simale 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-
abilitv to isolate discrete segments of the
DNA"in a form that permits detailed
molecular analysis. Recombinant DNA
methodoloPlv remove this barrier. Pur-
thermore, &cillary techniques 6ave 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 endoaenous viral senomes are
Meg&ted into- the cell's chromosome.
This bears on the question of how the
expresdon of the integrated viral genes

::p.:::1

affects cellular ?egu!~?:ion. thus leading
to the abnormal grovvvth characteristics
of cancer cells. With the recombinant
DNA techniques for isolation and purifi-
cation of specif?c genes, this research
problem is reduced to manageable pro-
portions. It, is possible t.o isolate the de-
sired DNA segment in pure form. Large
quantities can be obtained for detailed
study by simply ext.ract.ing a culture of
the bacteria carrying the viral DN,4 seg-
ment in a plasmid.

b. Potential practical applicatiom for
medicine. aariculture and industru. Cer-
tain of ihe 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 efEcient translation of
eukaryote genes in bacterial (prokary-
ate) hosts has get to be proved, these po-
tential applications are speculative at
this time. ApplicaUons that depend on
the expression of foreign prokaryotic
genes in prokaryotic recipient cells are
presently more cerbain.

(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
uuantities of the desired material. A
nymber of costly and/or rare substances
would be prime candidates for such syn-

thesis :

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

Human growth hormone (preeenbly avaiI-
able only from human cadavers and in short
SUPPlY) i

Clothing factor VIII rfor xeatme:lt of

hemophilia).

Specific antibodies and antigens (for pre-
venting and treating infectiow, allergic, and
autoimmune dise-lse, ar.d per!:aps even ca!l-
cer) ;

Certain enzymes, such as Ebrinolysin and
urokinase (promismg agents In the trent-
ment of embolism) and lysosomal enzJ`mes.

(2) Endouiment 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 photosgntte:ic f%eEion 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 Qvallty or q2iantiry 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

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


::5&32

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
exueriments involving insertion of DNA
&to cells capable of d&eloping into com-
plex, multicellular organisms. The Guide-
iin& 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 succe&ull~
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 &`I- 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 con+
plex and diftlcult 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
migl& be considered fir 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
tdbiolotical warfare. a July 3. 19%let-
ter to cr. David Baitimore- frbm James
L. Malone. General Counsel of the United
States A&s 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

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

(2) Singer, M. F. and D. Soil (1973). Guide-
lines for DNA Hybrid Molecuies. Science
182:1114.

(3). Berg, P., D. Baltimore, H. W. Bayer,
S. N. Cohen. R. W. Davis. D. S. Hoaness, D.
Nathans, R. 6. Roblin, J. ti. Watson, 5. Weiss-
man, and N. D. Zinder (1974). PotentiaZ Bio-
hazards of Recombinant DNA Molecules.
Science 185:303.

(4) Berg, P.; D. Baltimore, S. Brenner, R.
0. Roblin, and M. F. Singer (19'75). Summary
Statement of the Asilomar Conference on
Recombinant   DNA   Molecules.   Science
188:991; Nature 225:442; Proc. Nat'l. Acad.
Sci. 72:1981.

(5) Struhl, K., J. R. Cameron, and R. W.
Davis (1976)  Functional Expression of
Eukaryotic DNA in Esoherichia Colt. 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: 23-726.

(9) Novick. R. P. and S. I. Morse (1967).
In Viva Transmisston 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. 6:461473.

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

(14) Brbckman, 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 543384-397.

(15) Gillespie, D.. W. C. Saxinger and R. C.
Gallo (1976). Information Transfer in CeZls
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. &d M. A. Baluda
(1973). Integrated State of Oncornauirus
DNA in Normal Chicken Cells and in CeZ&
Transformed by Atian Myeloblastosis Vfsw.
J. Viral. 12:721.

(17) Hill, M. and H. Hillova (1972). Virus
Recovery in Chicken CeZZs Tested with Rous
Sarcoma Cell DNA. Nature New Biology
237:35.

V. DESCRIPTION OF THE PROPOSED ACTION

The Director, Nat&iii&l 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 MH-
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 MH 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 MH
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 1eveIs approximate those rec-
ommended by the Center for Disease
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 u;Sual and spe-
cial microbiologicA safety practices,
priniary 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 mol&les 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 Gtide-
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 fudgti to be of Poten-
tially moderate or high risk, the propcr-
ties of the bacterial strains to be used

FEDERAL REGISTER, VOL. 41, NO. 176THURSDAY. 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
(CM: 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
classes 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 or 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 ta be free of harmful char-
6cteristics,

(3) The biohazard associated with the vec-
tor that 6erve6 to transmit the 6ource DNA
to a recipient host cell,

(4) The ability of the vector to survive in
natural environment6 or habltsts.

(6) The kinds and number of different
organism6 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 abilfty of the recipient cell to
6urvlve in natural environments or habitats,

(8) The ability of the recipient 6611 to
transmit the recombinant DNA molecule to
other cells capable of surviving in natural
environments or habitats.

  (9) The potential of the recipient cell to
obtain the source DNA by natural means, and
  (10) The evolutlonary relatedness of the

DRA source to humans.

- The Guidelines prohibit a number of
types of experiments, including those in
which an organism contributing DNA
i itself a biohazard of greater than low
risk 8s 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.
`I'bus, 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

host-vector system employing a variant
of 1. 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 requirement6
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
carry out the institutional responsibility,
and stipulate the qualmcations and ex-
pertise of the committee membership.
NIEI responsibilities are detailed in the
Guidelines and are divided among (1)
NIH Initial Review Groups, (2) the NIH
Recombinant DNA Molecule Program
Advisory Committee, and (3) the NIH
staff.

Physica 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-
mentP1, 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 physica.
containment at the Pl level is shown in
Figure V-l. Such a laboratory passesses
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-
eess is permitted. Control of biohazards

is provided by standard microbiological
practices.

P2 Level (LouJ). 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 Pl 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 experiment
requiring Pa-level physical containment.
arc being conducted.

FIC~RE V--l

RGURE v--2

PS Level (Moderate). As shown in Frg-
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 corridor6
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 safetg
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 i6
provided by waste sterilization tech-
niques. The surfaces of walls, floors,

FEDERAL REGISTER, VOL. 41, NO. ?76-THURSDAY, SEPTEMBER 9, 1976

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

P4 Level (High). As shown ln Figure
V-4, experiments involving recombinant
DNA molecules requiring physical con-
tainment at the P4 level shall be con-
fined to work areas ln a maximum-secu-
rity facility of the type designed to con-
tain mieroorganlsms that are extremely
hazardous to man or may cause serious
epidemic disease, The facility is either 8
separate bulldlng or a controlled interior
area completely isolated from all other
areas of a buildllg. Access to the facility
is under strict control. Class ID hiolog-
ical safety cabinets are available.

Fxcrrxx V4

A P4 facilit,y has engineering fea-
tures, shown in Figure V-5, designed to
prevent the escape of 7 icroorganlsms
to the environment (l-4 . The special
features in a P4 facility include:
Monolithic walls, floors, and ceilings in
which all penetrations such ax for air ducts.
electrical conduits, snd utility pipes are
sealed to ensure the physical isolation of
the work area and to facilitate houaekeep-
lug 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 autcclaves to sterilfie and
safely remove wastes and other materials
from the faculty. .
A biowesti treatment system to steriliee
liquid effluenta lf facility drains are In-
stalled.

k

             --iw-
   a
           .._ .
              w-m
    "*g$
         FIGUFS v-8
A separate ventilation system that maln-

tains negative air pressures and directional
airflow within tha facilitv.
A treatment system to decontaminate ex-
haust air before It Is dlsperaed to the at-
mosphere. A c+ral vacuum utility system
Ia not enmuraged:  lf one b lnstall~
each branch line leading to a laboratory
shall be protected by 6 high-efficiency par-
ticulate air filter.

%CFERENCES

1. Design Criteria for Viral Oncology Re-
oearch  Facuftfes.  UA Department OL
Health, Education and Welfam, Publie
Health Service, National Institutes of
Health: DHEW Publication No. (NIB) `76-
891, 1976.
2. Kuehne. R. W. (1973). B<~bgfcd Con-
tainment Factlfty @r Studying In~eCtiour
Df.%?u.ve. Appl. Mlcrobiol. 26.239-145.

8. Runkle. R. 8. snd G. B. Phillips (1060).
b?imobial Containment Control Facilities.
Van Nostrand Reinhold, New York.

4. Cbatigny, M. A. and D. L Clinger (1969).
Contamination control in Aerobiology. In.
R. L Dimmick and A. B. Akem (e&a.). An
Introduction to Experlmental Aerobiology.
John Wiley L Sons, New York, pp, 194-
263.

VI. DESCRIPTION OF ALTERNATIVES

The following general classes of action
have been considered ae 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 lm-
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 f&e 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-
duclng DNA segment of SV40) require
P3 or P4 according to the NIH Gulde-

lines. In addition, while the Asflomar
guidelines recommend that certain ex-
periments be deferred, the list of experi-
ments to be deferred is expanded h 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-l.

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 ani 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 signiilcant 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 beneflt 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

RDERAL REGISTER, VOL. 41, NO. 176THURSDAY, SEPTEMBER 9, 1976


NOTICkS

38435

tie United States in biological research
would be threatened. Further, historiqal
precedents indicate that measures which
interfere with free inquiry in one area
of interest, often inhibit the vitalit,y of
other aspects of society.

C. DEVELOPMENT OF DIFFERENT GUIDELINES
Each of the stipulations in the NIH
Guidelines was made after assessment of

the pcesible 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 speciflcatlons 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 investf-
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 regiona. cen-
t43-S:

a. It m-ould he le!;a expensive io construct
and staff s few such regional ce~?ters than
many 6uch facilities.

b. ~!rraining would be centralized.
E. PB facilities would be more unifornllv

accessible to qualified investigator6 from a
variety of institutions.
d. There would be greater assurance that
the facilities meet the specified require-
ments.
e. Banks of cells cont.aining recombinant
BNA could be maintained, with a view to
decreasing the number of times the actual
recombination process would be performed
(such banks can also be maint.ained in the
absence of centralized P3 facilities).
1. The sliks could be placed away from
populat,ion centers.

The disadvantages of establishing re-
gional centers include:
a. Long-range planning would be neces-
SarY.

b. Scheduling would be a problem.
e.. The Investigator's independence would
be diminished.

d. Competition for access might favor es-
tiblished investigators or established ideas.
e. The nature of the urocess, which might
require only brief acce& of p3 facilities & a
given day but over 8 lengthy period of time.
f. Acce.~ problems might unnecessarily dis-
fxxxrage valuable research.

2. AU permissible recombinant DNA
experiments be conducted in P4 facili-
ties. This alternative implies no distinc-
tion among experiments. It does not rec-
ognfze that certain recombinant DNA
experiments are widely agreed to pose
little, if any, possible hazard. It is equl-
valent t.e a total prohibition on much
recombinant DNA research because af
the limited number of P4 facilities that
a,re available a.nd the high cost of con-

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

4. Exaeriments vrohibited 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 uossible hazard. Alteina-
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
to 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 water.
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 aarticular strain
_-- -~ .~~~~.~
of E. coli called K12. ?I'he forms are
called EKl, EK2 and EK3 in the Guide-
lines where they are discussed in detail.)
Some of these forms already exist, others
need ta 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 E
coli partially fulfill many of the criteria
in the definition of the ideal strain. At
present, no other bacterial species is
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

properties of already known species may
be shown to approach the ideal more
closely than E. coli strain K12 and its de-
rivatives, 85 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 am-
mals, There is little evidence that SV40
results in disease in humans. However,
SV40 infects humans, and demonstrable
antibodies to SV40 indicate that h&c-
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 immunl-
zation aganist polio virus before SV40 was
identiiied as a contaminant in the VW-
tine. 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-
nifirantly affect their health (2). The
Guidelines restrict the use of SV40 DNA
to DNA from strains of the virus that. are

I--

defective in the infection process. In
addition, stringent physical containment

is required.

d. Sanction of experiments involv*iW
the transfer of uncharacterized mfxtures
of DNA segments deri,ved from wam-
blooded animals into bacteria. Such fiv-

periments are believed to present a
greater possible risk than others because
they involve a conglomeration of un-
defined genes that might include DNA
ca.pable of causing disease.

e. Sanction of the use of oncogenic
uirmes. It has been argued that tir*

. . ..-

int~roduction into E. cob of the whole
DNA or any purified segment of the DNA

_- .--

of any virus oncogenic ln any species
should not be permitted.

II. n;o guidelines but NIH consideration
oj eucil proposed project on an individual
oasis before funding. With this alterna-
tive, mclividual investigators requesting
NIH funds for projects involving recom-
binant DNA research would bring plans

for proposed experiments to an NIH
comrl~itt.ee t,hat would, without the us* of

_- -_

foirnal guidelines, recommend suitable
ronminnaent measures. Depending on the
criteria used by the committee, this might
result in lower or higher containment
levels than a.re currently imposed by the
Guidelines. The advantages of such a

procedure would include constant -reI
eraluation of potential hazards and con:
taimnent 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 opportunny
for arhil,rary decisions, and the bypasx-
uig of local input in asscisment. of
hazar&.

It should be pointed out that under the
present NIH Guidelines, loc:ll institu-
tional biohazards committees must con-
sider proposed research projects on an
IndivldunJs basis and may im5)ose more

FEDERAL REGISTER, VOL 41. NO. 176THURSDAY, SEPTEMBER 9, is'76


, 3&m

-:NQF##ES

stringent safeguards than those required
bv the Guidelines. The judgnients of the
i&estigator and hts lo&l c&mittee 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-
wsition of the Guidelines either bs indl-
iidual agency adoption or through an
Executive Order. Non-Federa. 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 fbr. Occupational Safety and
Health (NIOSH), or the Occupational
Safetv and Health Administration. De-
partment of Labor (OSHA) . For ex$mple
NIH could petition OSHA ti 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 85 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 aeneiallv reauired 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 l3e-

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 DNA
research.

REI`EREiW'ES

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

(2) Shah, K. and N. Nathanson (1975).
Human Eqosure to SV40: Review and Cm-
ment. A resource document for the meeting
on Recombinant DNA molecules. Asilomar
Conference Center, February 24-26, 19'75.

VII. ENVIRONMENTAL IMPACT 0F THE
        GUIDELINES

A. IMP.\CT OF ISSi'ANrE 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 laboratorg 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 to materials
containing recombinant DNA molecules,
in case they should prove hazardous. The
Guidelines govern only work supported
by the NIH, including NM 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
wrsonnel and m the swead of vossiblv
jurzatdous agents by injected iadorator$
perso?meZ. The ND3 Guidelines are di-
re&l. concerned with reducing and elim-
inating exposures of 1aborat.G~ 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 immble 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-
lng 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. PiotecUon of personnel from min-
lmrd risk materials is provided by or-
dinary microbiological techniques. SincS

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 ol organisms, e.g., due
to accidental ingestion by poor pipetting
techniques or self-inoculation by needIe
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-
ion-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 biolonical safety cabinets. Po-
tentlal 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 ca.binets 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 asrents (no or

minimal risk) and that animal virus iec-
tars 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 agent3
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-
auired to be surrounded by absolute Prl-
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.
Mate&& 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 reference 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 bc
standardized, and the Guidelines require
that research personnel be well trained
and proficient in the necessarv onera-
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. uotentialls 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
1s concerned, potentially harmful effects
from experiments judged to present the
possibility of very severe hazard should
bc prevented completely since those ex-
periments are prohibited.

2. Impact on the environme&al spread
of possibly hazardous agents. The NTH
guidelines a,re directly concerned with
preventing the release of cells and micro-
organisms containing recombinant DNA
m&cules, 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 liquid 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.

The potential for accidental release of
recombinant DNA materials into the
atmosphere, however, increases with de-
creasinsz   containment   reauirements
(moderate --) minimal). Harmful sec-
ondary effects from such a&dental 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
1925-19'75 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, liquid
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 tc 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 (Pl 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 II). 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 eabinet
will be required for every three persons

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

Directional air flow, single-pass ven-
tilation, and provisions for ensuring re-
stricted access are facility recmirementc
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-
ovat.ion 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 tP4) research involving recombinant
DNA materials be conducted only in class
III biological safety cabinets (glove
boxesi that are installed in maximum
security facilities. Fewer than 30 facil-
ities within the United States have the
potential for meeting the requirements
specifieti in the Guidelines for such facil-
ities. A smaller number may actually be
available for this research. It is esti-
mated that approximately $75G,OOO would
be required to construct and equip a
maximum-security facility having two
IO-font by 20-foot laboratory modules
with class III cabinetry. This great cost
is due to sophisticated mechanical sup-
port s:;stcms (e.g., negative pressure, ex-
haust sir filtration, air waste treatment
plant) and architectural barrier 1e.g..
clothes-change rooms, air locks. waste-
staging are&  and `monohthic walls
floors, and ceilings), The cost of class III
csbinetry  installed is' approximately
$3000 per linear foot. In addition, the
c.abinei;ry line and the facilny each re-
quire a double-door autoclave, costing a
minimum of $15;000 and $65,000 re.;pec-
tively.

4. Scc~~cia-ry 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 contaiti-
me& 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 emri-
mentti minimizes the probability of acci-
dental exposure of laboratory workers
and subsequent seconda.ry environmcmal
impa.ctr.

b. Safety uwarencss. The safe perform-
a.nce of biomedical research is dependent
on an awareness of the risks and the
safegutirds 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-
socia tw3 with biomedical research.

FEDERAL REGISTER, VOL. 41, NO. I76--THURSDAY, SEPTEMBER 9. )?76


c. Early recognition of potential iLo:-
ards. The Guidelines require that the
principal investigator notify NIH of any
serious or extended illness or accident
that may result in serious 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 emeriments can be required to
minimize the hazard to other lnvestiga-
tom. conducing similar studies. This up-
grading willalsoreduce anypotentin! for
environmental effects.

9. IMPACT OF EXPERIMENTS CONDUCTED
     UNDER THE GUIDELINES

1. Possible  undesirable  impact--a
aspersion of potentianr haaardous
agents. The hypothetical mechanisms by
which insertion of foreign genes into
cells or viruses might result ln the for-
mation of hazardous agents are de-
scribed in Scct.ion 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 specn-
latlon that such agents may be produced
and an equaliy speculative assessment of
the nature and extent of hazards that
may follow upon a particuh recombinant
DNA experiment. This is the underlying
reason that the thrust of the Guidelines
is to mlnimize 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
notentialls hazardous agents emnhasizes
the likelihood of sign&ant dispersion
rather than the nature of the hazard lt-
self. The analysis given does not appiy
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 mu&+

(a) contain foreign genes,
(b) Escape from the eXperlnle?>I;ii -.`~a-
uon,
(c) Survive after eecape.
-(d) Become established in an environment
permitting Its growth and multlpllcation,
(e) Contact other living organisme In a
signfI%ant manner, including contact by a
sufllcient number of organisms to ensure sur-
vival and growth and to cause infection.
(Note that the environment in (d) map be
a living organism itseli).       \

In those cases where the detrimental
effect results from the formation of a
harmful protein, the organism contain-
ing the recombinant DNA must-
(r) Contain a gene for a potentially harm-
rui protein,
(g) Be able to express the foreign gene-
that is. synthesize the foreign stein,
(h) Synthesize the protein in sufacteti
quantity to be deleterious to the infected
organfsm.

       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
inltlal host cell or virus, the inserted
DNA must-

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

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

-I& 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 reciplcni wit.hout
being destroyed,
(1) ~urvlve transfer to another cell,
(m) Become associated wlth the other cell
ln a stable manner. either aa an independ-
ent element or by natural recomblnatioa

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 ass&n-
ment of probabilities of : 1 for (a) ; of
lo4 (1 in 100) for fb) ; of l(r (1 in lo.-
000) for (19; and of lOa (1 ln a mfl-
lion) for (d). La& of any pertinent
knowledge concerning events (e) through
(h) would make assignment of probablll-
ties impossible. Even assuming a proba-
bility of one for each event (e) through
(h) , the overall probability of a deieterl-
ous effect on a member of a species at
risk in this hypothetical situation would
then be the product of ail probabihtiee
(a) through (h), namely lOmu (one in a
trillion). This probability then needs to
be compared with the number of orga-
nisms grown for the experiment. ml-
tally, bacteria are grown ln liquid mix-
tures to a concentration of between 1V
and 10" organisms per ml. The probabil-
ity will also need to be corrected for the
length of time over which the experiment
ls to be conducted In reality, it may fre-
quently be difficult to assess the relevant
probabilities.

It is currently impossible to assign
specific probabllltles for many exneri-
ments, although crude estimates ~821
often be made from current knowledge
of laboratory-acquired infections, from
prototype experiments set up to measure
bacterial or viral escape (4). and from
knowledge concerning the stability of or-
ganisms and DNA. NlH is currently SUP-
porting research designed to improve the
ability to evaluate certain of these prob-
abilities.

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

(1) Monitoring for r&XZ.W of Org&
nisms containing recombined DNA. Con-
trol of the spread of any agent outside of
an experimental situation to laboratory
workers or the outside environment ti
greatly assisted by adequate means for

monitoring the agent in question. A per-
tinent example is the monitoring for spli-
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-
mentilly different in the case of orga-
nisms or viruses containing recombined
DNA. No simple general procedure exlsts
for identifying an organism released
from the <ab&atory a&ins% the large
background level of related and un-
related organisms occurring naturally.

It is possible, however, to devise special
pertinent procedures for detection of
some of the agents used ln recombinant
DNA experiments. For example, develop-
ment of bacterial strains, phases, or
plasmlds carrying readily detectable
genetic traits would enable the monitor-
ing of labcratory personnel, people work-
ing in the am. and their families for the
presence of those agents. This would be
analogous to the examlnatlon 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-T (1 part ln 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-eg., production
of toxic foreign proteins. We might
therefore be aware of the potential prob-
lem soon after disnersal of the organism.
and reasonable means for mln~%n.izlng
further dlsnersal could be undertaken.
In other instances--e.a.. a cancer-urO-
duclng DNA fragmt%+evldence~ of
harmful effects might not be apparent
for many years. The connection between
the causative organisms and the ob-
served harmful effects could be difilcult
to establish Further, dispersal of the
hazardous agent might then be so wlde-
spread as to make control difficult or
impossible.

(2) Natural occurrence of DNA re-
combination between unrelated orga-
nfsm. Concern over the ootential for
hazard in organisms contalnlng 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 recomblnatlons 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 ebeen selected against
in evolutlon. However tests for shared
base sequences are of hmited seIUltlJ%`.

FEDERAL RKGISTER, VOL. 41, NO. I76-THURSDAY, SEPTEMBER 9, 1976


NOTICES

(2) Wednm, A. G. ( 1976). The De!i%:l; EZ-
perfence As a Guide to t7ze Probable Efiicacy
of P4 Mfcrobfologfcal Containment Facflftier
for Studfes on MicsobfaI Recombinant DNA
Facilitfes. 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 01 the AsiIomar
Conference on Recombinant DNA Moleculer
(P. Berg, D. Baltimore, S Brenner, R. 0. Rob-
lin and M. Singer, eds.). Submitted to the
National Academy of Sciences.
(4) Din&k, R L., W. Vogl and Chatigny,
&I. k (1973). Potential for dccfdental Mfcro-
bfal Aerosol Transmission in the Biological
Laboratory (in) Bfonarards in Biologfcal Re-
search, Helman, A., M. N. Oxman and R. Pol-
lack, eds Cold Spring Harbor Labora'c!~.
N.Y.
           &PENDIX A

GLOSSARP

I. Aerosol; A colloid of liquid or soiid par-
ticles suspended in a gas, usually air.
2. Antibody: A protein which is 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 !a
fitted with a gauge that automatically regu-
late the pressure, and therefore the degree
of heat to which the contents are subjected.

5. Bacteriophage:`A virus that infects Onig
bacteria.

8. 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 die.-
ruption of the environment.

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

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

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

11. Biohazardous Mat.erlal: Any substance
which contains or potentially contains bio-
hazardous agents.

12 Biowsste: Liquid wastes from biological
research procedure&

13. CDC:  Center for Di.9ea.90 Control.
United States public Health Service, Atlanta,
Georgia

35-130

13) Relative irreaersibility 01 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, tid 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
mea31s will not ,be available, or that they
will be available too late to Prevent or
stop untoward events.

As described earlier, the' likelihood is
that newly constructed organisms will be
less At than those occurring naturally
and therefore will disappear over time.

2. Beneficial impacts of tecmbimnt
DNA research. Section Iv-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-l-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 h-n-
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 mmnmization 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 realization 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 F3 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
Ml3 Recombinant Advisory Committee.
Time will also be required for the various
review Processes that are require&

R.EFXRENCE4

il) Chattgny, LYL A., W. E Barkley and W.
Vogl (19'74). Aerosol Efohazard fn Microbfo-
logfed Laboratorfea and How It Is AffGCted
bar Aff Conditfonfng Systems. Am. Sot. Rest.
Ref. Alrcond. Engr. 8O:Wl-t 1.

14. CDG ClassFBcation of etiologic agenta
on the b&S Of hazard: A sv&em for evaluab
lng the harm& assochk with various
etiologlc agents, and definition of minimal
safety conditions for their management in
microbiological investigations. The b&s for
Aeent Clsssiflcation in &R follows:
-Clam 1: Agents or no or minimal hazard
wer ordtnary conditions of handling.

Class 2:  Agents of ordinary ' potent.ial
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
    red++ ram out&de the United
States which   uh a federal permit for
lmportat.ion unless they 851) specified for
hiphe+ elaw~flcatlon. Tbfs class includes

p&hogr IIS which require special conditi~:ns
lar 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 cl&% includes Class 3
agear*; from outside the United States when
they are employed in entomological experl-
ments or when other entomological experi-
~F!J:$ are conducted in the same laboratory
L-IX.:;.

6~aiz 5 Foreign animal pathogens that are
r?i-luded from the United States by law or
wlio,?e entry is restricted by USDA admini+
rrailve policy.

Norm: Federally licensed vaccines contain-
ing iive bacteria or viruses are not subject
10 i.lxse classifications. These clssshlcatlons
:lre applicable, however, to cultures of the
rirains used for vaccine production, or fur-
`her pa.s<ages of the vaccine strains.

15. Class I biological safety cabinet: A
ventilated cabinet for personnel protection
uniy, having an open front with inward now
of air away from the operator. The cabinet
esl:a%t air ia filtered through a high ef&
c%irncy particulate air (HEPA) filter before
being discharged to the outside atmosphere.
l'bls cabinet can be used for work with low-
`0 moderate-hazard risk agents where no
pr(Yicct protection is required.

15. Class II biological safety cabinet: An
! pen-front cablnet for personnel and product
prot-ection with ma.% recirculated airflow
A it,h HEPA filtered exhaust and BEPA filtered
recirru!ated air. This cabinet can be used for
v orb with low- to moderate-hazard risk
sagen?c lt is not suitable for use with cx-
plOsiVF  ;md flammable substances, tonic
iyenls, or radioactive materials.

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

18. Clone: A nouulatlon of cells derived. bv
a*xual reproduXon, from a single cell. Ever+
cell in the population is presumed to be
genetically identical. In recombinant DNA re-
bearch, every cell in a clone contains the
fame recombinant DNA species.

IQ. Coding sequence: The orderly arra)- of

codons which are subunits of a gene.
30. Chromosome: One or more small nxl-

shaped body{s) in the nucleus of a cell that
contains genetic information for that cell A
t-ollection of genes.

21. Deoxyribonucleic acid, or DNA: A com-
plex substance of which genes are composed.
22. Eilluent: A liquid or gs8 flowing from
a process.

23. Endogenous: Developing or originating
withh the organism, or arising from causes
within the organism
24. Escherichia ~011: A bacterium com-
monly found in the intestinal tract of
enimals.

26. Etiologie agent: A viable microorga-
nism or lte toxin which causes, or may cause+
human disease.

26. Eukaryotic cell: A-cell that contains 8
nucleus with a nuclear membrane surround-
ing multiple chromosomes: also contains ex-
tranuclear organelles.

27. Gene: The smallest portlon of a chrom-
osome that contalns the hereditary informa-
tion for the production of a protein.
28. Genetic engineering: Directed inter-
venlion with the content and/or organization
of an organism% genetic complement.
2Q, Genome: The complete set of hereditary
lnformatfon In a cell as the chromosomes l?S

FEDERAL REGISTER, VOL. 41. NO. )76-THURSDAY, SEPTEMBER F \5;7C


33440

NOTICES

R eukaryote or the slnglc chromosome in a
prokaryote.

30. HEPA fllt.er: (High EfiiCienCy Particu-
late Air Filter) A disposable, extended medl-
urn, 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, repllcatlng, and causing an al-
tered host reaction commonly referred to as
a disease.

32. Lamlnar 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 blohazard-
0218 materials in a laboratory environment.
Exposure may be the result of * specific acci-
dent or inadequate biohazard control proce-
dure or equipment.

34. Messenger rlbonucleic acid (mRNA):
A complex molecule that transmlt.9 the ln-
formation from the gene to a template OIL
which a protein ls formed.

35. Mltochondrlon: A DNA-containing
structure present in all aerobic eukaryotic
cells. The mltochrondia produce energy for
the cell and divide by fission after cell divi-

sion has occurred.

36. Nucleotide: A basic unit of the noly-
merit structure of DNA. Each unit contiiti 8
sugar (deoxyrlbose) , phosphoric acid, and one
of the followlng organic substances: adenlne,
guanine, thymine or cytceine.
37. Oncogenesis: The process of tumor for-
mation.

38. Organelle: An independent structural
body existing within cells, generally related
to a particular uellular function, and contain-
ing a special group of genes wlthln an extra-
chromosomal DNA molecule (e.g., mltochron-
&la 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 oukide of

.   the cromosome that is capable of replicating
  independently of the chromosome.
   42. Polymer: A large molecule composed
  of simpler repeating uniti. DNA is a polymer
  composed of nucleotldes, while star& and
  cellulose are polymers composed of sugars.
   43. Prokaryotic organisq or Prokaryote:
  Cells of bacteria or blue-green algae which
  are charaoterized 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. Shot.gun experiment: An experiment in
which ail the DNA fragments cleaved by a
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 veo+&r
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-
riophrrge.

50. Viable: Literally, "capable of life."
Cienerally 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 oenisma may
be viable under.one set of culture conditions
and not under another set, making it ex-
tremely important to deflrie precisely the
conditions used for determining viability.
           APPENDIX B

SUGGESTED REFERENCES FOR ADDITIONAL RE.tDING

Alderson, T. (198'7). Induction of Genefi-
tally Recombinant Chromosomea fn the Ab-
se7tce of Induced Mutation. Nature 215:X28&
3.

Basu, S. K. et al. (1975). Factor Which Al-
tecfs the iWode uf Genetia Recombination
in E. Coli. Nature 253;138-40.
Berg, P. et al. (1974). Potential Hazards ol
Recombinant DNA Afolecules (&f&r.). Scl-
ence 185:303.

Berg, P. et al. (1975). Asilomar Conference
on Recombinant DNA Molecules. Science
188:9914.

Berg, P. et al. (1975). Summary Statement
o/ the AsiZomar Conference on Recombfnunt
DNA MolecuTes. Proo. N&l. Aca. Scl. USA
72:19814.
Berg, P.  (1976). Genetic Engineering:
Challenge and Responsibitfty ASM News 42:
273-277.

Capaldo, F. N. (1974). AnaZyysfr oj the
Growth of Recombfnafion-Deficient Strains
of E. CON K-12. J. Bact. f18.%%2-Q.
Clark, A. J. (1987). 2% Begfnnfng ol a
Genetic ,Arurlysis 01 Recombination Prop&
ency. J. Cell. Physlol. 70rSuppl.:188-#XL
Clark, A. J. (1971). Toward a %fetaboZfc In-
terpretation oj Genetic Recombfnution of 8.
Colt and Its Phage.?. Ann. Rev. Mlcroblol. 25:
437-64.

Clark, A. J. (1974). Progress Toward a
Metabolic Interpretation of Genetic Recom-
bination of B. Coff and Bacterfophage Zamb-
da. Genetics 78:26Q-71.

Cohen, S. N. (1975). Manipulation of Genes.
Scl. Amer. 233.24-33.

Curtis% R. III (1968). UZtTatiotef-induced
Genetic Recombination in a Partially DfpZoid
Strain oi E. Colf. Genetics 58.9-84.
Felsensteln, J. (1974). The Evolutionary
Advantage oj Recombination. Genetics 78:
737-56.

Fowler. J. V. (1974). Molecular Biologists
Cult for Temporary Ban on DNA Experimenta.
Bio. Science 24:533.

Green. M. M. (1968). Some Genetic Pfop-
etfies of Intrachromosomul Recombfnatbn.
Moleo. Oen. &met. 103.309-17.
Harvey, C. L. (1973). Lambda Phage DNA:
Joining of a Chemically Synthesfzed Cohesive
End. Science 179.2915.
Helling, R. B. (1969). Recognition of AZ-
tered DNA in Rcccmbination. J. Bact. 100:
224-30.

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

Hotchkiss, R. D. (1974). Models 01 Genetic
Recombfnatfon. Ann. Rev. of Microbial. 28
(0) :445-68.
Hotchkiss. R. D. (1974). Molecular Basis for
Genetic Recombination. Qenetics 78:247-57.
Hubacek, J. et al. (1967). Formation o/
Recombfnants in E. CoZi and a Gene Ccmtrol-
ling the E:cpression of a Donor Marker. Folla
Microbial. (Praha) 12:422-31.
Kilbourne. E. D. (1969). Future Influenza
Vaccines and the Use of Genetic Recombi-
nunts. Bull. W.H.O. 41:643-5.
Kushner, S, R, et al. (1971). Genetic RC-
combinatfon in E., Coli: The Role oj Ezonu-
&ease. Proc. Nat1 Acad. Scl. USA 68:824-7.
Lederberg, J. (1975) a DNA SpZfCfng: Will
Fear Rob Us of Its Benefits? Prism, Nov. 1975,
p, 331                     m

Marx, J. L. (1973). Restriction Enaynw:
New Tools for Studying DNA. Science 180:
482-5. _ - -
McCahon, D. et al. (1973). Use of Recom-
Mnatiun fn the Production oj Influenza VIZ-
cfne Strains. Posterad. Med. J. 49:195-Q.

Meselson. M. 8: et al. (1975). A General
Model for Genetic Recombination. hoc. Nat?.
Aca. Sci. USA 72~358-61.
Nash, H. A. (1975). Integrative Recombina-
tion fn Bacteriophage Lamb&: Analysis ol
Recombinant DNA. J. Mol. Blol. 91:501-14.
Norkln, L. C. (1970). Marker Specffc Effects
fn Genetic Recombfnutia. J. Mol. Biol. 52'
491-O.

Putrament, A. (1971). Recombination and
Chromosome Structure in Eukaryotes. C&net.
Rea 18:8SD5.

Potential Hazard8 01 Recombtnant DNA
&fOZecUZeS. Proc. Nat?. Acad. Sci. USA 71:
25934.

Russo. V. E. (1973). On the PhysfcaZ
Structure of Lambda Recombinant DNA.
MoL Gen. C3enet. 222.355-86.
Signer. E. et aL (1988). The General Re-
combination  System of  Bacterfophage
Lambda Sympos. Quant. Blot. 33~114.
Slmchen, 0. et eL (lQ69). Pine ctnd Coarse
Confro& oj Genetic Recombination. Nature
(London) 222.32%32.
Sinsheimer. R. (1975). Troubled Dawn
for Genetio Enggtneeting. New Scientist.
Oct. 16, 1976.

SmItL J. M. (1974). Recombination and
the Rate of Evolution. C3enet.l~ 78.aQQ-304.
Smithie6, 0. (1974). Zettef: RecomMnant
NDA ~Okcuks. Genetics 77:81&20.
Strauss, B. a (lQ68). DNA Rep& Mesh-
tt?ltSnta and Thefr Relation to Mutation and
Recombfnatfon. CWT. Top. Mlcroblol. Im-
munol. 4X:1-85.

Tomlzaws. J. L (1967). RloZecuZar iUecha-
nfzms of Genetb Recombfrbatfon {?a Bac-
teriophage: Jofrrt Moluxles and Their Con-
~ersfon to Recombinant Molecules. J. Cell.
PhyeioL 70:Suppl. 201-13.
Wade, I?. (1976). Recombinant DNA: The
Last Look Before the Leap. Science 192.230-
8.

Walmsley, R. IX. (1969). The General The-
ory 01 Yapping Functiona @r Random Ge-
netic Recombinutfon. Blophye. J. 9.421-31.
Whitehouse, H L. (1970). fhe dfechmrbm
ol Gene%? Recombination. Bid. Rev. 45.665-
315.

Wood, T. H. (1967). Genetic Recomblna-
tfon in 8. CoZf: Cbne Hetrogenity and the
Kfnetfcs of Segfegaffon. Science 157:3X+-21.
         Appendix C

  DEPARTMENT OF HEALTH, EDVCATION.
    AND . WELFARE,  PVBLIC HE.UXE
    SERVICE,   NATIONAL   INSmS
    OF HEALTH
                 June 18,1976.
To:  Director. NIH. Through:  Director,
  NIGhfS. NIH.
From: Executive Secretary, Recombinant
  DNA   Molecule  Program   Advisory
  Committee.
Subject: Operation of the Ofilce of Recum-
  binant DNA Activities (ORDA) .
The proposed structure and responsibill-
tles al 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 oresent our views as to how such
__.--. .-
sn office C'Bn function effectively at the
NIH. Consequently, the following relntion-
ships and activities are discussed:

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

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


NOTICES

38441

particularly the Deputy Director for Scl-
ence informed concerning the activities of
ORDA. the status of BID extramural nro-
grams and intramural research involving re-
combinant DNA molecules, and on sclen-
tmc 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
M chairman and vice-chairman, respectively,
of the Recombinant DNA Molecule Proeram
Advisory Committee (Recombinant Ad&Fry
Committee).

OD. NIH, ln turn, should involve ORDA
in all major planning activities leading to
formation of NIH policies and procedures.
As is current practice, the Executive Secre-
tariat, NIH will provide ORDA with lnfor-
m&ion copies of ail 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
various matters relating to this technology.

An Executive Recombinant DNA Commit-
tee (Executive Committee) should be estab-
llshed in the Oflice 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-
nine and Evaluation: Director. OWce of Re-
search Safety, NCI; &d Assoda'ti Director for
Environmental Health and Safety, DRS. The
Special Assistant for Intramural AfIairs will
serve as Executive Secretary of the Executive
Committee. There wlll 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 BIDa, 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, mall or presentation at the
next meeting. The Deputy Director for
Science wlll have the ultimate responsibility
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
lnd.lvlduaJ BIDS various projects developed
by the Executive Committee or Recombinant
Advisory Committee or solicited by ORDA.

The Ofllce 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 ao-
tivities and/or need for accountabillty~ and
may make appropriate recommendations in
these circumstances.

II. Dissemination of information. The Of&e
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 ln-
stltutlons and research laboratories through
its mailing keys and use of the appropriate
instrument. such as the "ND3 Guide for
Grants and Contracts" (NIH Guide), NIH
Manual Issuance (NIB Manual), etc. There-
after, ORDA wlI1 be responsible for dlstrlbue
lng Guidelines and responding to requests by

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

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

III. Division of research grants. ORDA will
work with the Associate Director for Scien-
tlfic 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.

rV. Institutes and divisions. Institutes and
Divisions will be required to report to ORDA
on all activities involving recombinant DNA
technology, including: intramural research
prefects. 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-
lng 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 ln-
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 facllltata interchange of lnfor-
matlon. Because of the procedures proposed,
NIH awarding components may wish to con-
sider naming speciiic stafI for liaison with
ORDA.

V. Special NIH relationships. ORDA wlli
maintain close working relationships with
the Director, Ofllce of Research Safety, NCI,
the Associate Director for Environmental
Health and Safety, DRS and the NIII Bio-
hazards Committee. and will coordinate their
activities on matters relating to recombinant
DNA technology. The OflIce of Research
Safety, NC1 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 lnspec-
tlons 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 coordl-
nated through ORDA, and copies of site visit
reports will be flled 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 Foundatlon
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-

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

VII. Non-FederaZ and international rela-
tiowhips. ORDA will attempt to develop ln-
terrelstlonships with private foundations,
professional societies, scientific journals 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 Biology Orga-
nization.

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

As stipulated in the ffuldelines, 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 is 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 commlttess. 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, lnvestlgators 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 Quldellnes
also require that investigators report to
ORDA and their biohazards committee anv
problems pertaining to operation and lmple-
mentatlon of biological and physical con-
tainment safety practices and procedures, or
equipment or facility failure.

ORDA will receive from investlgstors ln-
formation on nurnorted El22 and EIU svs-
terns, and 1nfo;mation bearing on the Gulde-
lines, such as technical information relating
to hazards and new safety procedures or in-
novations.

ORDA will receive and Ale these reports
and, as appropriate bring them to the at-
tention of the Deputy Director for Science,
NIH, the Omce of Research Safety. NCI, and
the Recombinant Advisory Committee or
subcommittees thereof. ORDA may, after re-
view, recommend approplrate 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 gsther-
lng. analysis and dissemination of lnforma-
tion, the presentation of issues, etc.

XI. Transition and imolementetion. 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 ln 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. 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976


38442

NdTlCES

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

WILLWM J. GAARTL~D, Jr., ph. D,

PROCESSING, REVIRW,  AND MANAGEMENT OF
MTRAEJL60RAL PROJEC-TB INVOLVING RECOM-
BINANT DNA TDzfiNOLoGY

I. General. The purpose of this appendix ls
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., 8s
defined in MH 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.

II. Capture of information. One of the
primary functions of the Offlce of Recombi-
nant DNA Activities (ORDA) is to maintain
8 central register of NIR 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 MH to modify application forms to in-
dicate on the face sheet whether or not re-
comblnant 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-
matton would be captured, as is the case for
human experimentation, and permit sorting
by different parameters such as awarding
component, geographic location of projects,
etc.

III. Receipt of applications. Through the
use Of  appropriate  instrument (NIH
Guide, NIH Manual, etc.), the NIH will ln-
form applicailts of the necessity for aaeess-
ing the physical and biological containment
required for the proposed experiments as
stipulated in the NIH guidelines. This asBesB-
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 (MDA) and a Certiflca-
tion Statement (Certification) from the in-
stitutionsl biohazards committee. The MH
must and will require that a properly exe-
cuted MUA and Certification accomnanv 811
spplications proposing to use reco&biGant
DNA technology. This will eliminate the need
for tracking these documents after the appli-
oation is accepted for review. The originals of
these documents will be DlaCed in the ofacial
fflee of the MH awarding components with
copies on ffle in ORDA
IV. Review ot appZ<cation.% The executive
secretaries of initial review groups am re-
sponsible for identlfylng all applications in-
volving recombinant DNA technology, and
for placing on the first page of every sum-
mary statement the following special note:

REc!OMBIN*NT 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 8s to whether the containment levels
proposed by the investigator meet the levels

stipulated in the NIH guidelines. If the pro-
posed containment levels are inadequate, the
initial review group should discuss, in as
much detail 84 possible, the inadequacies of
the proposed oontainment 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 8 copy
of the Guidelines to permit them to make
these judgments. Problems relating to as-
sessment of biolosical and nhvsical contain-

- I

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 8re exoected to carefullv
scrutinize proposals, involving recombinant
DNA technology,  and make appropriate
recommendations.

V. Award of neza 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
olearance 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 nrenare a memo-
randum outlining the reasons ior disapprov-
al but emphasizing that the action is inde-
pendent of scientific merit or other reaBous.
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 Advisorv Committee. for
review. The fln8l de&don `will rest witd the
Deputy Director for Science, MH.

BIDs will forward to ORDA 8 oopy of 8lI
award Statements 1nVOlving these applica-
tions.

ORDA will retain in its files the dmumente
cited above. In the event that the volume of
filed documents b ecomes excessive, ORDA
will retain a copy of the face sheet of 8
funded application rather than the entire
application. In those cakes in which the
proposed project involving recombinant DNA
technology is but one component of a mul-
tiproject application, ORDA wlll retain in
its flies the face sheet of the application and
the section(s) describing the project(s) in-
volving recombinant DNA technology.

It must be emnhasixed that the nrimarv
responsibility forknsuring that appl&ti&
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 st8iI for advice and con-
sultation, but it can not be expected to make
decisions for review unite and awarding
components.

VI. Award of non-competing renewals and
WcrementuZZy-funded contracts. Bach non-
competing renewal of a grant and subsequent
budget. period of 8n incrementally-funded
contract utilizing recombinant DNA tech-
noloav must be Bccomoanied bv 8n UDdated
Certiftcation Statement from -the i&titu-
tional biohazards committee. prior to any
award of this type the program oiBcial 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
th6n w&v reouired in the anolication as re-
viewed by the initial revieG'group, and for
ensuring that the required documents are
properly executed. The program ofacial will
then forward to ORDA one copy of the ap-
plication and the certification statement,
8long 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 nronosed contaln-
ment 1evei.s are adequat& &hereafter, 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 DrocedureB described in VII
must be follow&. It is the responsibility of
the program official to ensure that all the in-
formation an(l properly executed documents
required in VII are present in the application
prior to forwarding the request to ORDA.

VII. Chanaes in awarded moiects. Since in
many cases"the NIH suppbr& projects -for
project periods longer than one year, 8 num-
ber of situations will arise in funded projects.
One situation arises when an investigator
makes 8 decision to utilize recombinant
DNA technology after the project has been
reviewed and awarded. Another situation
8rises when an investigator decides to close
DNA segments other than thOBe 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 slgniiic8ntly alter an approved pro-
tocol, the investigator must first apply to
the MH awarding component for permission
before proceeding. This requirement should
be stated in the annrooriate MH instrument
(MH Manual, MKGAde. etc.) The investi-
gator will be required to submit to the award-
ing component 8 proposed protocol, an
assessment of the levels of physical and bio-
logical containment required by the Quide-
lines, an MBA, and a Certification Statement
from the institutional biohazards commit-
tee. The program oilicial in &he awarding
component has the responsibility for review-
ing the request in light of the Quidelines, for
ensuring that the required documents are
properly executed, and for forwarding to
ORDA 8 CODY of all ;the documents 8lone
with a recommendation. The latter should in:
elude the program 05clal's independent
assessment of the levels of physical and
biological containment required by the MH
GUidelineB, and 8 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 consultante.

VIII. Reouests for Zowerina of containment
levels. Under "cliaracterized ciones of DNA
recombinante derived from shotgun experl-
merits," the Guidelines state:

o o o before containment conditions lower
than the ones used to clone the DNA efur be
adopted, the Investigator must obtain sp-
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
levels of  containment for  characterized
cloiles derived from shotgun experiments
must state, in writing, the justification for
the request; to the program official of the NIH
awardmg component. Such justification will
provide data- on (a), (b) and (c) as
stated above. The DroPram official will
retain the original r&u&t in the award-
ing component's file, and forward a copy
to ORDA which will submit the request
to the Recombinant Advisory Committee or
to a subcommittee thereof 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 Deoutv Director for Science. NIH.

IX. .f.ar.&-scale experiments. The Guide-
lines state that:

o * * 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 orovided that these ex-
periments are kxpressly approved by the Re-
combinant DNA Molecule Program Advisory
Committee.

An investigator a-ho wishes to conduct such
experi1nent.s 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 &ttention 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-
dependently.

APPENDIX B TO APPENDIX C

NIH INTRAXIURAL 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 prese:lt, the Chief of the Laboratory
in n;hich 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, IiIH with copies to the Associate
Director for  Environmental Health and
Safety, DRS. The request for approval is in
the form @f a draft Memorandum of Ucder-
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-

NQWES

tar 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
Sssociate 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
Reccmbinant Advisory Committee.

APPENDIX C TO APPENDIX C

TRANSITION AND IMPLmIENTATION

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 0: 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 Dire<qtor
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
Dlrector for Science, NIH.

II. Extramural programs. ORDA u-ill brief
the Executive Committee for Extramural Af-
fairs on NIH policies and procedures.

BIDS will be required to report to ORD.~
all presents or planned workshops, training
courses, con!erences, etc., relating to recom-
binant DN.A technology. BIDS must also re-
port all present or planned EPFs and RF&
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 Execut.ive Committee.
With regard to active grants and contracts,
BIDS will be rec&ired to submit to ORDA a
copy of the application, summary state-

w

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 ouerv will be retained in BID of-
ficial file.& 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 oppor&unlty to
petition the Recombinant Advisory Commit-
tee to permit co ,tinued use cf rharacterired
clones already in existence and constructed
under Asilomar guidelines. Presumably, the
use of thcqe 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-
vestigat.ors are already at least in compli-
ance with Asilomar guidelines. If projects
are identified which appeaf: not to be in com-
pliance with Asilomar guidelines, they will be
brought to the immediate attention of the
Deputy Director for Science, NrH and the Re-
combinant Advisory Committee.

APPENDIX D

RECO?.IEINANT DNA RESEhF.CH

     Guidelines
  as published in the
FEDERAL REGISTER, Part II,
    July 7, 19'76

On Wednesday, June 23, 19'76, 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 N:H 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
"Summarv Statement of the Asilomar Con-

ference on Recombinant DNA hfolecules."
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 Piilds of experiment; until an intcr-
national meeting could be con~eneci to con-
sider the potential hazards of recombinant
DNA mclcrulcs. T!ley hl'o cal!ed upon the
NIH to estnblisb a committee to provide ad-
vice 0-1 recomhi:lnrit DNA techno:o~y.

The internationnl meering XT;S beid eit the
A~ilornar Conference Center, Pacific Grace,
California, in Februiirr 1975. Ti:e consensus
3f this meeting was that certain experiments
should not be done at ?!:e present time, but
that most of the work on construcrion of re-
:ombinant 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

NOTICES

made interim assignme?.& 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-
tirlc 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 tc the NIH
Director. The Recombinant Advisory Com-
mittee extensively debated three different
versions o the Guidelines during this period.
The Ad 4 isory Committee to the NIH Direc-
tar, augmented with consultants representing
law, ethics, consumer affairs and (the envlron-
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-
ilnes will be sent to all of the approximately
26,000 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 beneilts 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
REGISTER. 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 tc 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 Rcckville Pike,
Bethesda, Maryland 20014.

   DONALLI S. FREDRICKSON, M.D.,
                  Director,
     Nctionul Institutes of HeaWl.
JUNE 25, 1976.

DECISION OF THE DIRECTOR. 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 Withln the NIH

II.

III.

IV.



V.



VI.



VII.

C. Implementation Beyond the Purview
   of NIH

D. Environmental Policy
Methods of Containment (See Guide-
lines II)

Prohibited Experiments (See Guide-
lines III, A)

Permissible Experiments: E. Coli K-12
Host-Vector Systems (See Guidelines
III, 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

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-
nact 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.
Thev will renlace the current Asilomar guide-
lines, discuf;sed 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 a
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 tc 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 tc Dr. Philip Handler, President
of the National Academy of Sciences, and t0
Dr. John R. Hogness, President of the Insri-
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 iOr
preventing- their spread, scientists through-
out the world loin with the members of this
committee in-voluntarily deferring * o o
[certain] experiments * * *.

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

Third, the D'rector 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 bioloaical and ecoloeical 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 Pronram Advisorv Committee
(hereafter "Recomibinant Advisbry 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
recombinant&"

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 pokntlal
biohazards of the work. Participants felt that
experiments on construction of recombinant
DNA molecules should proceed, provided that
appropriate biological and physical contafn-
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 Commit.tee on May 20, 1975. A
summary statement of the report was pub-
lished in "Science 188," 991 (1975), "Nature
225," 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


NOTICES

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 Mny 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
draft 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 maioritv of critics felt that thev were
too lax, Uoth& that they were too st&t. All
letters were reviewed by the committee, and
a 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 Tnvolrinrr Recom-
binant DNA Rlolecules," which wei; referred
to the Director, NTH, for a final decision in
December 1973.

As Director of i-he National In~tit?rtes 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 NH-I, 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 NJH
policy, program development, resource allo-
cation, and administration. T%e members of
the committee are knowledgeable in the fields
of basic and clinical biomedical sciemes, the
social sciences, physical sciences, research,
educat,ion, and communications. In addition
to crwrent 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 srssion.

The purpose of the meeting was to seek
the committee's advice on the guidelines
propo,ed by the Recombinant Sdvisory 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.

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 ralues of our society. A key
element in achieving and maintaining this
public trust is for the scientific community to
ensure an ope:mess and csodor in its pro-
ceedings. The meetings of the Director's Ad?-
sory Committee, the Asilomar group, and the
Recombinant Advisorv Committee have rt-
fleeted the intent of "science to be an open
community in considering the conduct of
recombinant DNA experiments. At the Direc-
tor's Advisory Committee meeting, there n-as
ample opportunity for comment and an air-
ing of the issues, not only by the committee
members but by public witnesses as v~ell. ,411
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 comment.s received afterward. As
nart of that review I asked the Recombinant
Advisory Committee to consider at their
mrrting of April l-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 CO~`SIDER.+TIONS

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 ccl1 particles (plns-
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 geneticsly homogeneous cul-
t;ure. In general, there are two potential uses
for the c!ones 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 t,he treatment of
various diseases. There is a:so a potential
risk-that microorganisms with transplanted
genes may prove hazardous to man or other
forms of life. Thus special procisicns arc
necessary for their coutaimnent.

All commentators ackpowledged the es-
emnlarv resoonsibilitv of %he 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 t.he
process leading to the formulation of the
proposed guidelines was a most responsible
nud responsive cne. Suggestions by the com-

mentators on broad po!icy considerations are
presented below. Thev relate to the science

policy aspects of the"guidelines. the imple-
mentation of the guidelines for XIH qrantees
and contractors, and the scope and impact
of the guidelines nationally and in:er!in-
tionally.

Commentators were divided on how SC-it to
steer a course between stifling research
through excessiT-e regulation and alloy ing

It to continue with su6icient controls. Sev-
eral emphasized that the public must have
assurance that the controls afford adequate
protection against potential hazards. In the
views of these commeniators, the burden is
on the scientific community to :how that the
danger is mi:iimal and that the beneflt.3 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 exafgegrate safety
procedures that inquiry would be unnece+
sarilp retarded, while others found the guide-
lines weighted toward promoting research.
The issue was how to strike a reasonable bnl-
ante-in fact, a proper policy "bins"-be-
tween concerns to "go claw" 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 cnuid
initiate an frreversible 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 c'iirlelines is to ens:ue
that experimental DP:*4 recombination wi!l
have no ill edects on the.%? engaged in the
work, on the general public, or on the en-
vironment. The esseixe of their construction
is subdivision of potential experiments by
class. decision as to which exuernnents shoirlrl
be permitted at present, and assignment to
these of certain procedures for containmen
of recombinant organisms.

Containment is defined as phy-iienl and
biological. Physical coli?ainment irivolres the
isolatioil of the resenrcli by procedures which
have erolied o!er many years of esperience
in la'xwatories st;ldxi:-,y infectiozrs micro-
organiwx 1'1 coritaiclncl:t-tile first physi-
en1 containment lercl--is that used in most


3546

NOTICES

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-
errbly less certain than are the benefits now
c!enrly 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
eil'ect in many areas of biology. No other pro-
cedure, not even chemical 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 signlf-
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 fore;@ 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
a 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. EXamDleS 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 oil
spills in the sea. Potential benefits in agrl-
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 it8 survival in some niche within the
ecosystem. Other commentators believe that
the endless experiments in recombination
of DNA which nature has conducted since
the beeinnine of life on the earth. and which
have accou&d in part for the &olution 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 eukaryotlc hast and that tGe failure of
the altered prokaryotes to be detected at-
tests to a sharply limited capacity of such
recombinant8 to survive. Thus nature, this

argument runs, has already tested the proha-
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 preparn-
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 ia?ued a press release in July 1975;
and the European Molecular Biology Organi-
zation Standing Committee on Recombinant
DNA, meeting in February 19'76.

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 contajnment practices
in other work that is not technicallv 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 research 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 exnlore the CaDabilitv 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 guideline% 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. IMPLFMENTATION CONSIDERATIONS WITHIN

THE NIH

All the commentators had suggestions con-
cerning the structure and function of deci-
sion making as it relates to the principal ln-
vestigator, the local biohazards 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 pr+
sented below.

Of considerable coccern 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 pubhc commentators recommended
the regulatory process.

Many who opposed changing the propo-ed
guidelines into Federal regulations esprenzed
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 experimenti
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 eauivalent to that Drovided if Steps
towards re'gulatlons were to proceed immecii-
ately. The guidelines will be published in the
FEDERAL REGISTER forthwith to allow for fnr-
ther public comment.

Special concern has been expressed by
many commentators regarding the applica-
tion of the euidelines to research outside
NIH by invescgators 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 schoo!s
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 undert.ake to present the
recommendation to all appropriate journals.
We are also prepared to take steps to dis-
seminate the guidelines widefy, and to ar-
range for a continual flow of information
outw-ard concerning the activities of the Re-

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


NOTICES

combinant Advisory Committee and the .4d-
visory Committee to the Director, NIH. in
the evolution of the guidelines and theh
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 MH 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
bv all Federal agencies. Encouraged bv these
eiforts, we held-a meeting on .?une 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 industrv commit-
tee-under the auspices of the Pharmaceutical
Manufacturers Association will be formed
to review the guidelines for potential ap-
allcation to the drue industrv. Further meet-
ings will be schedul&l with oiher 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 guldelinee will obtain throughout the
rest of the world. MH 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 Scientlflc Unions. with a
review to achieving a consensus on safety
standards in this most important research
area.

There has been considerable internationd
cwneration and activitv in the nast. and I
expkct it to continue -in the iuture. The
aforementioned Ashby Report, presented to
Parliament in January 1976. describes the
advances in knowledge and possible bene-
fits to societv of the exneriments fnVolvinZ
recombinant *DNA molecules, and attemp&
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 guldellnes for recombinant DNA
research. They have closely followed the ac-
tlvities of NIH. and will thus be encouraeed.
I believe, to monitor their research with r&g-
mented cooperation and coordination. For
example, EMBO recently announced plans
for a voluntary registry of recombinant DNA
research in Europe. Following this EMBO
lnitlatlve, MH shall similarly maintain a
voluntary registry of investigators and insti-
tutlons engaged in such research in the
United States. Plans for establishing this reg-
istry are under way.

D.  ENVmONMENT*L POLlCY  CONSmEsATIONS
A number of commentators urged MH 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.

I am in full agreement that the pot.entlsIly
harmful effects of thfs research on the en-
vironment should be assessed. As discussed

throughout this paper, the guidelines are
premised on physical and biological contain-
ment. to prevent the release or propagation
Of DNA recombinants outside the Isboracory.
Deliberate release of organisms into the en-
vironment is prohiblted. 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 cocducted 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 Oi
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 min1mlze potential risk. And
an elaborate review structure has been cre-
ated to achieve these safety objectives. From
a public policy viewpoint, however, the envl-
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.

II. METHODS OF CONTAINIMENT

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-
ness 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 effect.lve-
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
Oillce 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 l-2, 1976, reviewed the recommenda-
tions of the Barkley group. These are lncor-
norated. 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 oncogenlc viruses. Fbr
each of the proposed levels, optional items
have been excluded, and only those items

deemed absolutely necessary for safety are
presented. Necessary facilities, practices, and
equipment are specified. To give further
guidance to investigators and their inst.itu-
tions, a supp:ement to the @delines explains
more fully safety practices appropriate to re-
combinant DNA4 research. And a new section
has been added to ensure that shipment of
recombinant DNti materials conforms, where
appropriate, to the standards, prescribed by
the U.S. Public Health Service, the Depart-
ment of Transportation, and the Clvll 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 reasonable 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 MH
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-
lems of P4   installations.  Several com-
mentators advocated that MH arrange for
sharing of P4 facilities, both in the NIH
intramural program and in institutions sup-
Dorted throueh NIH awards. In resnonse to
these sugges~ons, we are currentld 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 lnvestlga-
tars. 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 comnlex ex-
perlments in recombinant DNA research Ls
cons!derably reduced.

1. Practically all commentators supported
the aresent urohibition of certain exoeri-

menis. There-were suggestions for * 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 r&stance to any
antibiotic, irrespective of its use in medicine
or agriculture. Conslderat.ion of such a sue-
gestion must take into account that anti-
biotic resistance occurs naturallv amone bac-
teria, and that resistance is a vaiuable 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 prohihl-
tion relating to drug resistance was intended
to ban those experiments that could com-
promise drug usa in controlling dlseacs
agents in veterinary as well aa human medl-
clne and this is now clearly stated.

In the draft guidelines t&m were two
statements concerning rfdstemca to dmgm
which related to experimentd with B. co&
The statements appeared to allow expM-

FEDERAL REGISTER, V61. 41,dNO. 176-THURSDAY, SEPTEMBER 9, 1976


384418

NOTICES

ments that would extend the range of resist-
ance of this bacterium to therapeutically
useful drugs and dlslnfectants, and thus
seemed to be ln conflict with the general
prohibition on euch research. There are
numerous reports ln the sclentlilc literature
indicating that E. co11 can acquire resistance
to all antibiotics known to act against it.
Since E. co11 acquires resistance naturally,
the prohibition directed against increasing
resistance does not apply. The ambiguous
statements have been deleted from the pres-
ent guidelines. Cm the other hand, new
language has been inserted ln the section
dealing with other prokaryote species to set
containment levels for permitted experi-
mental

2. The Recombinant Advisory Committee
was also asked to clsrlfy whether the pm-
hlbltion of use of DNA derived from patho-
genic organisms (those classified as 3, 4, and
6 by the Center for Disease Control, USPHS)
also included the DNA from any host ln-
fected with these organisms. The committee
explained that this prohibition did extend
to experiments wlth'cells known to be SO
infected. `IJo avoid misunderstanding, the
prohibition as now worded includes such
cells. In addition. the prOhibitions have been
extended to include moderate-risk oncogenic
viruses. as dellned bv the National Cancer
Institute. and cells known to be infected
with them.

3. Two other issues relating to the section
on prohibited experiments were raised by
Roy Curl&% III, Ph.D., professor, Depart-
ment of Microbiology, University of Alabama
School of Medicine, Birmingham, who ls a
member of the Recombinant Advisory Com-
mittee. Dr. Curtlss noted that for the class
of experiments prohibited on the basis of
production of highly toxic substance& only
substances from microorganisms were cited
as examples. He suggested that other exam-
ples be included. such as venoms from inSeCti
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. Curtlss felt that the guidelines should
provide greater speclflcity 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
contalnlng 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-
comblnant organism that ls universally ac-
cepted as belg bene6cial to introduce into
the environment. When the sclentlflc evl-
dence becomes available that the potential
benefits of recombinant organisms. partlcu-
larly for agriculture, are about to be realized,
then the guidelines can be altered to meet
the need for release. It ls most important
that the potential environmental impact of
the release be considered.

IV. PERMISSIBLE EXPERIMENTS: E. COLI K-12

HOST-VECTOR SYSTEMS

The continued use Of E. co11 as a host has
drawn considerable comment, including
some suggestions that its use be prohibited
presently or within a specified tlme limit.
It should be stressed that the use of E. co11
as detailed in the guldellnes ls limited to E.

*Speclflcally, experiments that would ex-
tend resistance to therapeutically useful
drugs must use p3 physical containment plus
a host-vector comparable b Egl, or P2 con-
tainment plus a host-vector comparable to
EK?2.

coli K-12, a strain that has been carried in
the laboratory for decades. and does not ln-
volve the use of any strain of E. co11 that is
freshlv isolated from a natural source. E. co11
K-12 hoes not usually colonize the normal
bowel, even when given in large doses, and
exhibits little lf any mullplication 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 it-s genetic markup and
recombinant behavior exceeds greatly that
pertaining to any other organism. I believe
that because of this exnerience. E. co11 K-12
will provide a host-vector system that ls
safer than other candidate microorganisms.

NIH recognizes the importance of support-
ing the development of alternative host-vec-
tor svstems (such as B. subtills. which has
no e&dog& niche in man) and will en-
courage such development. It should be
noted, however. that for each new hoat-vec-
tar system, the same questions of rlsk from
altered properties attendant upon the pres-
ence of recombinant genes will apply as ap-
ply to E. toll. NIH does not believe it wise
to eet a time limit on replacement of E. toll
systems by other organlama.

There were specific suggestions concerning
the three levels of biological containment
prescribed for use of E. co11 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 @Xl).* Sections of the
guidelines dealing with physical containment
and roles and responsibllitles now specify the
need for safety practices and accident plans.

For the second level of containment (EK2),
it ls required that a cloned DNA fragment be
contained ln a host-vector system that has
no greater than a lo-8 probability of eur-
viva1 in a nonpermissive or natural envlron-
ment. It was suggested that the selection of
this level of biological containment and the
appropriate tests for vertlflcatlon be more
fully explained ln the guidelines. The com-
mittee, ln responding to a request for fur-
ther examlnatton of this point, reviewed at
considerable length the testing for an EK2
system and recommended certain modlflca-
tlons. 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 blo-
logical. containment afforded by these al-
tered host-vector systems are outlined in this
section. Because this ls such a new area of
sclentiflc research and development, however,
it ls inappropriate to standardize such test-
ing at the present time. Standards will grad-
ually be set as more experience with EK2
host-vector systems ls acquired. The commit-
tee, for example, during its April 1976 meet-
ings gave its flrst approval to an EK2 host-
vector system. What is necessary ls that new

`The EKl system presently conslsls of a
battery of different vectors and of E. co11
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 restrlc-
tlon 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 ls
supporting the development of more EK2
host-vector systems, it la not expected that a
battery equivalent to that available for the
EKl system will be certified by the Recom-
binant Advisory Committee in the near fu-
ture.

and more effective tests be devised by investl-
gators, and this effort ls very likely to occur
under the present guidelines. For example,
one task recognized by the committee ls 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-
mltteefor EK2 biological containment jeop-
ardize considerably the capacity of such crlp-
pled organisms to survive and replicate even
under permissive laboratory conditions. More
experience will-be required to determine
whether EK2 containment will permlt 6ome
lines of important research to be followed.

  Several  commentators  suggested  that
methods and orocedures to confirm an EK
system at the third level of containment
(EK3) be more fully explained. The Recom-
binant Advlsory Committee was asked to con-
sider this suggestion. After considerable dls-
cusslon the committee declined to define the
procedures more fully at this time, because
development of an EK3 system ls still far
enough in the future not to warrant specific
testing procedures. Further, it ls not clear
what tests are best suited The language,
therefore, remains general. The committee,
however, ls aware of the concerns for a more
completely deflned system of testing. and has
considered 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 svstem. In thls reaard
`the NIH ls prepared through the Nat&al
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 Advlsory Committee
has certified it. I share the concern of the
commentators that new host-vector systems
require the highest quality of scientlflc re-
view and scrutinv. At this earlv staee of
development, it g most important that the
committee provide that scrutiny. Further, I
believe that until more experience hes been
gained, the committee should encourage and
the NIH support research that will lndepend-
ently confirm and augment the data on
which certlllcatlon of EK2 host-vector sys-
tems are baaed.

v. CLASSIFK!ATION OF KPERIMENTS USING THE

E. COLI K-12 CONTAINMENT SYSTEMS

The guidelines assign different levels of
containment for experiments in which DNA
from different sources ls to be introduced
into an E. coli K-12 host-vector system. The
variation ls based on both facts and as-
sumptions. There are some prokaryotee (bac-
teria) which constantlv exchange DNA with
E. c&i. Here it is assumed that experlmen-
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 everylnstance 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
poesiblllty that normally repressed genes of
the host may be expressed and thus change,
undesirably. the characteristics of the cell.
It ls assumed that the more similar the
DNAs of donor and hOst, the greater the
probability of expression of foreign DNA. or of
possible derepreesion 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


NOTICES

38449

bin&ion experiments will create new genetic
combinations. When prokaryole donors not
known to exchange DNA with B. calf ln
mature are used, however, there ls a greater
potential for new genetic combinations to
be formed and be expressed. Therefore, it ls
required that experiments involving prokary-
otic DNA from a donor that ls not known
to exchange DNA wlth E. COU ln nature be
carried out at a higher level of containment.
Recombination using prokaryotic DNA from
an organism known to be highly pathogenic
ls prohibited.

There are only limited data available con-
cemlng the expression of DNA from higher
forms of We (eukaryoles) ln E. calf (or any
other prokaryote). Therefore, the contaln-
ment prescriptions for experiments lnsertlng
eukaryotlc DNA into prokaryotes are based
on risks having quite uncertain probabilities.
On the assumption that a prokaryote host
might translate eukaryotic DNA, it ls fur-
ther presumed that the product of that for-
eign gene would be most harmful to man
lf it were an enzyme, hormone, or other
proteln that was similar (homologous) to
proteins already produced by or active in
man. An examnle is a bacterium that could
produce 11~~11~. Such a "rogue" bacterium
could be of benellt ii contained, a nuisance or
possibly dangerous lf capable of survlvlng
in nature. This is one reason that the higher
the phylogenetlc order of the eukaryote, the
higher the recommended containment, at
least until the elllclency of expression of DNA
from hlgher 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. Thls is the
concern that viruses capable of propagating
in human tissue, and possibly causing dls-
eases, can contaminate DNA, replicate in
prokaryote hosts and infect the experlmen-
tallst. Such risks are gre&test when total
DNA from donor tissue is used in "shot-
gun" recombinant experiments; it diminishes
to much lower levels when pure cloned DNA
la used.

The commentators were clearly divided on
the classlflcatlon of containment criteria for
different kinds of recombinant DNA.% Many
wmmentatcrs considered the guidelines too
stringent and rigid. Others viewed the gulde-
lines ln certain instances as too permlsslve.
And still others endorsed the guidelines e,s
sensible and reasonable, affording the public
an enormous degree of protection from the
speculative risks. Several suggestions were
made for the speclfm 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 bv the wmmentators
and the response of the commtttse 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 blo-
logical containment levels are very strict. For
example, biological containment levels are at
EK2 or BK3, and will effectively preclude ex-
perimentation until useful EK2 and RK3 sys-
tems are available. EK2 systems are still in
the initial stages of development. and the
llrst system was only certlfled at the most re-
cent meeting of the Recombinant Advisory
Committee. An BK8 host-vector system has
yet to be tested, and ita certification ls far
enough In the future to place a moratorium

on those experiments requiring biologlcal
containment at an EK3 level. The physical
containment levels of P3 or P4 themselves
afford a very high degree of protection. I am
satlsiled 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 derlved
from embryonlc tissue or germ-line cells. This
ls based on evidence that embryonic material
ls less llkely to wntain viruses than is tlssue
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 wld-
blooded animals, because several commenta-
tors questioned the b&s of lower physical
and biological containment levels for DNA
from these species. The Rewmblnant Ad-
visory Committee has debated this exten-
sively, and they were asked to do so once
again in April.* The committee hes now rec-
ommended high containment levels (P3+
EK2) when the DNA ls from a cold-blooded
vertebrate known to produce a potent toxin.
That recommendation ls included ln the
present guidelines. Where no toxin ls ln-
volved the commlttes supported lower wn-
tainment levels. The guldellnes specify P2+
EK2 levels for such work. There was wnslder-
able discussion concerning the advisability of
recommending lower containment (Pa+ RKl)
when the DNA ls isolated from embryonic
tissue or germ-line cells from cold-blooded
vertebrates. Those supporting lower wn-
talnment levels argued that the justlfmation
for P2fEK2 was the posslbllity that wld-
blooded vertebrates may carry viruses and
that the dlstlnctlon between adult and germ
cell tissue ls 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 dls-
tlnction should be made on the basis of tis-
sue origin. Flnally, the committee recom-
mended, on a divided vote (8 to 4). to adopt
PS+EKl when the cold-blooded vertebrate
DNA ls isolated from embryonic tlssue or
germ-line cells. Upon reviewing these wn-
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 moue&. the classlilcation of exnerl-
ments where'DNA ls derived from other kld-
blooded anlmals or lower eukaryotes. Sev-
eral commentators, for example, had been

`A eommlttee member, David 8. Hcgness,
Ph. D., Professor, Department of Biochemls-
try, Stanford University. California, submit-
ted a statement in support of lower contaln-
ment levels based on current scientific evl-
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 mlght be
encountered wlth ditrerent levels of shotgun
experiment ls the degree of sequence homol-
ogy between the DNA of the given species and
that of humans. This criterion is used to
estimate the llkellhood that segmenta of
DNA from the given species might be lnte-
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
ls a considerable degree of homology be-
tween human DNA and DNA from other prl-
mates, much less homology between pri-
mates and other mammals, and even lower
but detectable homology between birds and
primates. By contrast, no slgnlflcant homolo-
gies between cold-blooded vertebrates and
primates have been detected.

concerned about the fact that insects are
known to carry agents pathogenic to man.
In the committee revlew. It was noted that
viruses carried by insects and known to
transmit dlsesse 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 thls class. New
language ls 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 ln
the guidelines the requirement tnat any in-
sect must be grown under laboratory condi-
tions for at least 10 generations prior to its
use as a DNA source.

2. As alluded to above, certain commenta-
tors expressed concern that when B. cdi be-
wmes the host of rewmblnant DNA from
prokaryotee with wblch DNA ls not usually
exchanged, there ls hazard of altered host
chamcterlstlca resulting from translation of
the DNA into functioning protelns. The
wmmittee was asked to review the gulde-
lines and take lnt.o account this potentisl
haxard. They agreed that the containment
levels should be increased for thls cateeorv
of experiment, from P2fBKl to either b$
KK2 or PS+EKl. That recommendation Is
included ln the present guldellnes.

Comments were made concerning that class
of experiments in which the r&omblnant
DNA. re~ardleee of source. has been cloned
A clone L a population of'cells derived from
a single cell and therefore all the cells m
presumed to be genetically identical. As out,-
lined ln the proposed guidelines. clones wuld
be used at lower containment levels lf they
had been rigorously characterized and shown
to be free of harmful genes. Several wm-
mentatom lnqulred how the characterization
wss to be performed and the freedom from
harmful genes demonstrated. Although the
Wmmittee acknowledees that these terms are
unavoidably vague, they do cite appropriate
scientltic methods to make relevaPf deter-
mlnations. Again, this is a rapidly chang-
lng area and more clarity and precision can
be expected with experience. Reduced eon-
talnment reaulrements for this class of ex-
perlment are*warranted because of the purl-
fled 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.
1. Another comment was related to the use
of DNA from organelks (intracellular ele-
ments that contain special groups of genes
for particular cell functions). Concern was
expressed about the potential contamination
of purlfled organelle DNA with DNA from
viruses because of the similarity of thelr
Structures. The wmmlttee aarees. and the
guidelines now specify a req&ement. that
the organelled be isolated prior to extracting
DNA, as a further means of reducing the
haxard of viral wntamlnatlon.
6. Some commentators were troubled about
the lowering of containment for that class of
experiments involving recombinations with
cell DNA segments purifled by chemical or
physical methods. They asked that proce-
dures for determining the state of purliica-
tlon be more fullv detailed and that the Re-
combinant Advl&y Committee certify the
purlty. 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 resulte
beyond repeating the experiments themselves.
These techniques are well documented and
described ln the literature. I do not believe
1% b necessary or feaslble for tie committee
to review each procedure for purl8cation of
DNA.

6. COlDlllPlitS were made concerning the
use of DN.4 d~ired from animal viruses. It

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


I 38450

NOTICES

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 hax-
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 recomblnants have
been shown to be free of oosslblv harmful
genes by suitable biochemical and-biological
tests. This also pertains to DNA that ls
copied from RNA viruses. In no instance are
the guidelines more lenient, and in most ln-
stances they are more stringent than condl-
tions obtaining in manv laboratories where
such viruses ar'e studied-in non-DNA-recom-
binant experiments.

VI. CLASSIFICATION OF EXPERIMENTS Uswe
CONTAINMENT SYSTEMS OTHER THAN E. COLI
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.
SV40 is not known to produce any disease in
man. although it can be grown ln human
and on very rare occasions has been is01

from humans. Many humans have received
SV40 virus inadvertently in vaccines prepared
from virus crown ln monkev kldnev-cell cul-
tures. An ln;ensive search h-s been-made and
ls continuing for evidence that SV40 might
cause cancer or be otherwise pathogenic for
man. At present. it ls my view that the ex-
tensive knowledge we have of SV40 virus nro-
vldes us with s&lcient 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 Commlttec to
review this section of the guidelines. The
committee reconsidered the containment
condltlons for this class of experiments and
judged them appropriate to meet the poten-
tial hazards.4

This class of experiments will proceed un-
der the most careful and stringent condl-
tlons. Work with SV4.0 virus wili be done at
the maximum level of physical containment
(P4). The extraordinary precautions required
in a P4 facility lessen the llkellhood of a
potential hazard from this work. Only defec-
tive SV40 virus will be used as vector: that la,
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 pro&ion. It should be noted
that SV40 virus and its DNA can be efilclently
disinfected by Clorox and autoclavlng. These
are customary procedures for disinfecting
glassware and other items used in SV40 anl-
mal-cell work.

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

4 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 lte inserted non-SV40 DNA seg-
ment, does not replicate in human cells with
elgnllIcantly more efilciency than does SV40.

2. Several commentators found the guide-
lines inadequate regarding experiments with
plant host-vector systems. Because NIH
shared these concerns, a group of extensive
experience with plants was appolnted to re-
view this section. The group met comxr-
rently with the Recombinant Advisory Com-
mittee in April i976 and made several modl-
ficatlons. The suggested revisions were ac-
ceptable lo the full committee, and we have
,lncluded 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
modlfied to take into account operations wlth
whole slants. On the whole the resnec-
tive po-;tions of the guldellnes relating to
plants are more fully explained and the ln-
tent is clarified.

I have also accepted the recommendation
of the subcommittee to lower the biological
containment level from EK2 to BKl for ex-
periments in which the DNA from plants la
used in conjunction with the E. COB K-12
host-vector system. thereby setting contaln-
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
NH-I. Commentators generally urged open-
ness, candor, and pubiic partlclpation in the
process, emphasizing shared responsibility
and accountability from the locai to the na-
tional level. We reviewed that section of the
guidelines in llght 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 he ln the wisdom with which
they are implemented. Because of the im-
portance of this section, especially ln terms
of safety programs and plans, we have care-
fully weighed the comments and suggestions
made in this r&zard. NIH has a sueclal re-
aponslbility to t&e a leading role hi ensuring
that safety programs are part of ail recombl-
nant DNA research. Dr. Barkley and a spe-
claily convened committee were asked to pro-
vide greater detali for safety, accident, and
training plans for this section of the guide
lines. Bssed on their recommendations. the
section has been extensively rewritten tc
clarify the respective responslbliltles of the
principal lnvestlgator, the institution (ln-
eluding the institutional biohazards com-
mlttee) , the IiIH initial review group (study
section), the NIR Recombinant DNA Mole-
cule Program Advisory Committee, and NIH
staff.

This section has a deflnltive admlnistrs-
tive framework for assuring that safety is an
essential and inteerated comwnent of re-
search involving re&nbinant DNA moleculea
The guidelines require Investigators to ln-
stltute, monitor. and evaluate containment
and safety practices and procedures. Before
research is done, the investigator must have
safetv and accident ~1an.s ln mace and train-
ing exercises for the-staff weh under way.

Some commentators suggested that the ln-
vestlgator be required to obtain lnformed
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 hls program and support staff as
to the nature and assessment of the reai and
potential biohazards. He must explain and
provide for any advised or requested precau-
tionary medical pollcles, vaccinations, or
serum collections. Further, an appendix to
the guidelines includes detailed explanations
for dealing with accidents, as well as lnstruc-

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

In response to suggestions for epidemlolog-
lcai monitoring, the guIdelines now require
the principal investigator to report certain
categories of accidents, in writing. to appro-
priate officials. NIH ls investigating procp
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 blohax-
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 MH 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-
neerlng.

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 flndlngs ln terms of applicable laws.
regulations, standards of practice, communl-
ty attitudes, and health and environmental
considerations. The names of and relevant
background information on the committee
members will 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 blo-
hazards committee to serve as a source of
advice and reference to the investigator on
sclentl3c and safety questions. It was fur-
ther suggested that the committee's respon-
sibility be broadened in ,&he development,
monitoring, and evaluation of safety stand-
ards and procedures. In response to these
sueeestlons. the euldelines now indicate that
thz-lnstltutlonai- biohazards committee has
the responsiblllty 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-
sarv for Pl facilities. but we believe that all
fac~ltles 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 responslbil-
lty). The Recombinant Advisory Committee
took exception to this suggestion. They urged
NIH not to include these condltlons as local
requirements, arguing among other things
that review by the NM study sections would
provide the necessary scrutiny at the national
level and assure uniformity of standards ln
application of the guidelines. I do not believe
that NIB 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 lnstltutfon
from having Its biohazards committee per-
form this function. Accordingly. I have de-
leted the prohibitlon that appeared in the
proposed guidellnes.

Another suggestion was that the local com-
mittee ensure that research le carried out in
accordance wlth standards and procedures
under the Occupatlonal Safety and Health

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


NOTICES                            38451

Act (OSHA) . This is an area of importance t0
rhe local lnstltutlons under Federal and
state law, but need not be included as a re-
quirement in the guidelines. NIR will maln-
lain liaison with the Occupational Safety and
Health Admlnlstratlon (Department of La-
bor) to ensure maximum Federal cooperation
Yn this venture.

I would also encourage all institutions. as
suggested by several commentators, to review
their insurance compensation programs to
determine whether thelr 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 clsssl-
flcatlon of the proposed research under the
guidelines, along with the customary judg-
ment of the sclentlilc merit of each grant
application. This additional element of review
will ensure careful attention to potential
hazards in the research actlvltv. The studv
sections will also scrutlnlze the proposed
safeguards. Blologlcal safety expertise shall
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 resoonsibilltv of the NIH Recombinant
DNA Molecule Program Advisory Committee.

Comments on possible structural mechan-
isms for decision maklng included sugges-
tions that there be a scientific and technical
committee and a general advisory public
policy committee. It was also suggested that
the scientlfm 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-
tlflc 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-
Lessor of Government and Public Affairs at
the Lyadon B. Johnson School of Public Af-
fairs, University of Texas at Austin, serves in
lhat capacity. An ethlcist 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, 19'76, 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 RK2 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 recomblnants are to be used,
and on questions concerning potential blo-
hazard nnd adequacy of containment provl-
sions.

Ckmmentators also asked that the com-
mlttee review ongoing research Initiated
nrlor to the lmnlementatlon of the eulde-
&e. Now that -tie guidelines are bei-& re-
leased, NIH-funded investigators ln this field
will be asked to give assurance, within a
given period, that they will comply. Any ln-
vestlgators who constructed clones under the
Asllomar guldellnee will be asked to petition
NIB for special consideration of their case,
if the new guidelines require higher contaln-
ment than did the Asllomar guldellnes. Here
the advice of the Recombinant Advisory
Committee will be sought.

There were also suggestions that the com-
mittee certify chemical purlflcation of recom-
binant DNA, but 11s I indicated earlier, these
procedures are too well known to require
NIH monitoring.

6. In light of comments received, NIH will
provide review, through appropriate NIB
offices, of data from institutional biohazards
committees (including accident reports) and
will ensure dlssemlnation of these findings as
appropriate. Dr. William Gartland will head
the newly created NIH Ofiloe of Becombl-
nant DNA Activities for these purposes. In
addition, NIR will provide for rapid dls-
semination of lnf'orrnatlon tluwugh~lta Nu-
cleic Acid Recombinant Sclentl6c Memo-
randa (NARSM) , distributed by the National
Institute for Allergy and Infectious Dlsesses.
NIH will also provide an appropriate mech-
anism for approving and certifying clones
before  containment  conditions  can be
lowered.

With these extended modlfloations, the
section of the guidelines dealing with roles
and responslbllltles now sets forth a more
fully developed review structure involving
the principal lnvestlgator, local biohazards
committees, and the Recombinant Advisory
Committee, as well as peer review commlt-
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 scientlflc merit and conditions
for safety.

The Recombinant Advisory Committee ln
conjunction with the Director's Advisory
Committee shall continue to serve ae an
ongoing forum for examlnlng 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. i!l a sense, is just beginning.

JXINALD S. WEDRICKSON, M.D.,
              Director,
  h'ational Institutes of Health.

NATIONAL INSTITUTES OF HEALTH

CU'IDELINES FOR RESEARCH INVOLVING
  RECOMBINANT DNA MOLECULES

JCNE 1976

              II.
Containment.
A. Standard practices and training.
B. Physical containment levels,
  Pl Level (Minimal).
P2 Level (Low). '
P3 Level (Moderate).
P4 Level (High).
C. Shipment.
D. Biological conf.:~inment levels.

III.

Experimental Guldellnes.
A. Experimenta that are not to be per-
   formed.

B. Contalnnient guidelines for permlsslble
   experiments.

1. Biological containment crlterla using E.
   Coli K-19 host-vectors.

IX1 host-vectors.
EK2 host-vectors.
EFX3 host-vectors.

9. Classlilcatlon of experiments using the
   E. Coli K-12 containment systems.
<a> Shotgun experiments.
(1) Eukaryotlc DNA recomblnants.
(ii) Prokaryotlc DNA rewmblnants.
(iii) Characterized clones of DNA recom-
blnants derived from shotgun experiments.
<b> Purified cellular DNAs other than
-plasmids, bacteriophages, and other vlrusea
<c> Plasmlds, bacteriophages, and other
viruses.
(1) Animal viruses.
(ii) Plant viruses.
(iii) Bukaryotlc organelle DNAs.
(iv) Prokaryotic plasmld and phage
DNA%
3. Experiments wit.h other prokaryotic host-
   vectors.
4. Experiments   with  eaukaryotlc   host-
   vectors.

<a> Animal host-vector systems.
<b> Plant host-vector systems.
<c> Fungal or similar lower eukeryotic

systems.

Roles and Responsibilities.
A. Principal investigator.
B. Institution.
C. NIR Initial Review Group (Study Sec-
   tlons) .
D. NM Recombinant DNA Molecule Pre
   gram Advisory Committee.
E. NIH Staff.
              V.
Footnotes.
              VI.

References.

VII.

Members of the Recombinant DNA Molcc-1:e
Program Advisory Committee.

APPENDXCES

A. Statement on the use of Bacillus subtdti
   in recombinant molecule technology.
B. Polyoma and SV40 Virus.
C. Summary of Workshop on the Design &
  Testing of Safer Prokaryotic Vehicles
   & Bacterial Hosts for Research on Re-
   combinant DNA Molecules.
D. Supplementary Information on Phrhiinl
   Containment   (Including   De? ,! ilcd
   Colltents).

I. INTRoDnCTIoN

lhc p:irpo.se of these guidelines ih tn rei-
omniend 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
t.hat consist of different segments of DNA
which have been joined together in cell-free
systems, and which have the capacity to in-
Sect and replicate in some host cell, either
autonomously or as an integrated part of
the host's genome.

This is the first attenint to Drovide a de-
tailed set of guidelines for use by study sec-
tions as well as practicing scientists for ev:ii-
uating research on recombinant DNA moie-
culen. We cannot hope to anticipate a11 PO+
sible lines of imaginative research that are
possible with this powerful new method-.
ology. Nevertheless, a considerable volume of
written aud verbal contributions from sci-
entifits in a variety of disciplines has been
received. In many instances the views pre-
sented to us were contradictory. At pre>er?t,
the hazards may be guessed at, speculated
abo;:t.. or vo:ed upon, but they cannot be

FEDERAL REGISTER, VOL. 41. NO. 176--THURSDAY, SEiTEMBER 9, 1976


:, i&M52

`NOTEES

known absolutely in the absence of firm ex-
perimental dst-nd. unfortunately, the
needed data w&e, more often than not, un-
available. Our DrOblem then has been to con-
struct guidelldes that allow the promise o!
the methodology to be realized while advocat-
ing the considerable caution that is de-
mand@ 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 1075 (3) : (1) There are certain ex-
periments for which the assessed potential
hazard ls so serious that they are not to be
attemnted to the oresent time. (iii The re-
mainder can be undertaken at thk present
time provided that the experiment is justlfl-
able on the basis that new knowledge or
beneflls 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 addl-
tlon to an insistence on the practice of good
mlcroblologlcal techniques. these safeguards
consist of providing both physical and blolo-
glcal barriers to the dissemination of the po-
tsntlallv hazardous aeents. (ill) The level of
conU&ment 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 modlfled 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 modifled to reflect improvement% 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 reaog-
nlze that these definitions do not take into
account existing and anticipated special pro-
cedures and information that will allow psr-
tlcular 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 dontainment levels recommended here.

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

II. CONTAINiVENT

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 facilltles and the selection of
laboratory procedures applicable to orgs-
nisms carrying recombinant DNAs (4-17).
The existing programs rely upon mechanisms
that, for convenience. can be divided into
two categories: (i) A set of standard prac-
tices that are generally used in mlcrohiolog-
ical laboratories. and (ii) sUecia1 orocedures.
equipment, and `labor&&y install~tlons that
provide physical barriers which are applied
in varying degrees according to the estimated
biohazard.

Experiments on recombinant DNAs by
their verv natUre lend themselves to a third
contalnnient mechanism-namely, the ap-
pllcatlon of highly speclftc biological barriera
In fact, natural barriers do exist which either
limit the infectivtty of a vector or vehicle

(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 ln which they
replicate can be genetically designed to de-
crease by many orders of magnitude the
probability of dissemination of recombinant
DNAs outside the laboratorv.

As these three means ofVcontainment 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 uractices. 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 mlcroblological 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 traln-
ing in aseptic tecbnlques 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
DmCedUres to be followed lf an accident con-
taminates personnel or environment. The
principal investigator must ensure that
everyone in the laboratory is famlllar with
both the potential hazards of the work and
the emergency plan. If a research group is
working with a known Dathoeen for which
an effective vaccine ls a&iabie. all workers
should be immunized. Serological monitor-
ing. where appropriate, should be provided.

B. Physical contatnment 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 assocl-
ated with the "Classification of Etlologlc
Agents on the Basis of Hazard" (5). four
levels were recommended in the "Summary
Statement of the Asllomar Conference on
Recombinant DNA Molecules" (3); and the
National Cancer Institute Uses three levels
for research on oncogenlc viruses (6). We
emphasize that these are an ald to. and not a
substitute for, good technique. Personnel
must be competent in the effective Use of all
equipment needed for the required contaln-
ment level as described below. We deflne 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-
rant a more detailed classlflcatfon, and be-
cause additionai flexibility can be obtained
by combination of the physlcai wlth the bio-
logical barrlers. Though different in detail,
these four levels (Pl<P3<P3 < P4) approxl-
mate those given for human etiologlc agents
by the Center for Disease Control (i.e., classes
1 through 4; ref. 6). in the Asllomar sum-
mary statement (le., mlnlmal, low moderate,
and high: ref. 3). and by the National Can-
cer Institute for oncogenic vlnises (I.e.. low,
moderate, and high: ref. 6). as ls lndlcated
by the P-number or adjective in the fol-
lowing headings. It should be emphasized
that the descriptions and assignment% of
physical containment detailed 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 facll-
ltv installations lndlcated for es&h level of
physical containment are requlred for the
safety of laboratory workers. other persons.
and for the protection of the environment.
Optional items have been excluded; only
those items deemed absolutely necessary fpr
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 ls
provided in a supplement to the guidelines
(Appendix D) .
Pl Level (Minimel). A laboratory suitable
iOr experiments involving recombinant DNA
molecules requiring physical containment at
the Pl 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 in this
laboratory ls generally conducted on open
bench tops. Special containment equipment
ls neither required nor generally available in
this laboratory. The laboratory ls not sepa-
rated from the general traffic patterns of the
building. Public access is permitted.

The control of biohazards at the PI level
is provided by standard microbiological prac-
tices of which the following are examples: (i)
Laboratory doors should be kept closed while
experiments are in progress. (11) Work sur-
faces should be decontaminated daily and
following spills of recombinant DNA mat%-
rlals. (iii) Liquid wastes containing recom-
binant DNA materials should be decon-
tamlnated before disposal. (iv) Solid wastes
contaminated with recombinant DNA mate-
rials should be decontaminated or packaged
in a durable leak-proof container before IR-
moval from the laboratory. (v) Although
pipetting by mouth is permitted, it is prefer-
able that mechanical plpettlng 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.
(vii) Facilities to wash hands should. be
available. (viii) An insect and rodent con-
trol program should be provided. (lx) The
use of laboratory gowns, coats. or uniforms
is discretionary with the laboratory super-
visor.

P2 heue2 (Lou:). A laboratory suitable for
experimenti involving recombinant DNA
molecules requiring physical containment at
the P3 level is slmllar in construction and
design lo the Pl laboratory. The P2 laboratory
must have access to an autoclave within the
building; it may have a Biological Safety
Cabmet. Work which does not produce a
considerable aerosol is conducted on the open
bench. Although thls laboratory is not sepa-
rat%d from the general traffic pattern% of the
building. a&es% to the laboratory 1s limited
when experiments requiring P3 level physical
containment am belng conducted. Experl-
ments of lesser biohasard potential can b%
carried out concurrently in carefully demar-
cated areas of the same laboratory.

The P3 laboratory h commonly used for
experiments involving mlcroorganlsma of low
biohazard such ae those which have been
classhled by the Center for Disease caaroI
as Class 2 agents (6).

See footnotes on p. 33459.

FEDERAL REGISTEk, VOL. 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976


NOTICES

38553

me f~~l~~in~ practices shall applp to all
experiments requiring P2 level physical con-
tainment: (1) Laboratory doors shall be kept
closed while experiment8 are in ProgTW.
(ii) Only persons who have been advised
of the potential biohazard shall eater the
laboratory. (iii) Children under 12 years of
age shall not enter the laboratory. (iv) Work
surfaces shall be decontaminated dally and
immediately following spills of recombinant
DNA materials. (v) Liquid wastes of recom-
binant DNA materials shall be decontarni-
nated before disposal. (vi) 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 materials shall
be disposed of by incineration or sterilized
before disposal by other methods. Contami-
nated materials that are to be processed
and reused (i.e., glassware) shall be decon-
taminated before removal from the labora-
tory. (vii) Pipetting by mouth is prohibited;
mechanical pipetting devices shall be used.
(viii) Eating, drinking, smoking. and stor-
age of food are not permitted in the working
area. (ix) Facilities to wash hands shall be
available wi%hjn 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,
coat.s, or uniforms is required. Such cloth-
ing shall not be worn to the lunch room
or out.side the building. (xii) Animals not
related to the expe~lment shall not be per-
mitted in the laboratory. (xiii) Biological
Safety Cabinets 1 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
needle and syringe shall be avoided when
alternate methods are available.
P3 Leoel (jModerale) A laboratory sKitable
for experiments involving rwombinnnt DIV.4
molecules requiring physical con~t~ainment 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
acce& corridors, air locks, locker rooms or
other doable-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 wlthln 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 ts 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
1s conducted on the open bench. All such
procedures are confined to Biological Safety
Cabinets.
The following practices shall apply to all
experiment8 requiring P3 level physical con-
tainment: (I) The universal biohazard sign
is required on all l&oratory access doors.
Only persons whose entry into the laboratory

See footnotes on p. 38459.

Is required on the basis of program or sup-
nort needs shall be authorized to enter. Such
A---- -~~
persons shall be advised of the potential bio-
hazards before entry and they shall Comply
with posted entry and exit procedures. C'hil-
dren under 12 years of age shall not enter the
laboratory. (11) Laboratory doors shall be kept
closed while experiments are in progress. (ut)
Biological Safety Cabinets1 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, sonlcating, shaking,
etc.). (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 m&erials shall be decont.am-
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 (i.e., 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 he used, (viii) Bating, drinking, smok-
ing, end storage of food are not permitted
in the laboratory, (lx) Facilities to wash
hands shall be available within the labora-
tory. Persons shall wash hand9 after experi-
ments involving recombinant DNA materials
and before leaving the laboratory. (x) An ln-
sect and rodent control program shall be pro-
vided. (xi) Laboratory clothing that protects
street clot.hlng (i.e., long sleeve solid-front
or wrap-around gowns, no-button or slipover
jackets, etc.) 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,
ha&, caps, and such street outerwear shall
not be kept in the laboratory. (xiii) 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 wlJen
alternate methods are available. (xvi) If ex-
periments of lesser biohazard potential are to
be conducted in the same Iaboratory 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 Cabinet *
with attached glove ports and gloves. All ma-
terials before removal from the Biological
Safety Cabinet1 shall be sterilized or trans-
ferred to a non-breakab!e, 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.
Pd Lerrel High. Experiments involving re-
combinant DNA molecules requiring physical
containment at the P4 level shall be con-
flned to work areas in a facility of the type
designed to contain microorganisms that are

extremely hazardous to man or may en-
serious epidemic disease. The facility Is either
a separate bullding 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 avall-
able. Class III Biological Safety Cabinets *
are avaiiable 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; (11) air locks through
which supplies and materials can be brought
safely into the facility: (iii) 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 blowaste
treatment system to sterilize liquid efauents
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
vacuum utility Sy.%em is not encouraged: if
one is installed, each branch line leading to a
laboratory shall be protected by a high efX-
ciency particulate air filter.
The following practices shall appIy io all
experiments requiring P4 level physical con-
Ininment: (i) The universal biohazard sign
is required on all facility access doors and
all lntprlor 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 shall
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-
SOILS shall comply with the instructions and
all older posted entry and exit procedures.
Under *!o condition shall children under 15
years of age be allowed entry. (11) Personnel
shall enter into and exit from the facilitv
only through the clothing change and show-
er rooms. Personnel shall shower at eac!~
exit from the facility. The air locks shall not
he used for personnel entry or exit except
for emergencies. (ill) Street clothing shall be
removed in t.he outer facility side of the
clothing change area and kept there. Com-
plete laboratory clothing including under-
garments, pants and shirts or jumpsuit.%
shoes, head cover, and gloves shall be pro-
vided and used by all persons who enter into
the facflltv shall be olaced in an entrv &jr
be stored `in lockers provided for this `pur-
pose or discarded into collection hampers
before personnel enter into the shower area.
(iv) Supplies and materials to be taken into
the facility shall be placed in an entry a.ir
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 lock door. This door shall be
secured after supplies and materials are
brought into the facility. (v) Doors to labors-
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 IlI Biological Safety Cabinets.' All ma-
ter'bls. before removal from these cabinets,
shall be sterilized or transferred to a non-
breakable sealed container, which is then re-
moved from the system through a chemical
decon:amniated tank, autoclave, or after the

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


NOTICES

entire system has been decontaminated.
  (vii) No materials shall be removed from the
facility unless they have been sterilized or
decontaminated ln a manner to prevent. the
release of agents requiring P4 physical con-
  t.aimnent. 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 or a cham-
ber designed for gas sterilization. Othey m,tc
terials which may be damaged by tempera-
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. (lx) 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 provld-
ed. (xl) Animals and plants not related to
the experiment shall not be permitted in the
facility. (xii) If a central vacuum system is
provided, each vacuum outlet shall be pro-
tected by a filter and liquid trap in addition
to, the branch line HEPA filter mentioned
above. (xiii) 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 1
and other equipment. shall be decontaml-
nated following the completion of the ex-
perimental activig contained within them.
Mechanical pipetting devices shall be used.
All other practices listed above w1t.h the cx-
ceptlon of (vi) shall apply.
  C. Shipment. To protect product, per-
sonnel, and the environment, e,ll 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 Tmnsportation (Sec-
tion 173.38'7 (b) of Part 173, Title 49. Code of
Federal Regulations) and the ClvIl Aero-
nautlcs Board (CA.B. No. 82. OfRcial Air
Transport Restricted Articles Tarlff No. 8-D)
for shipment of etlologic agents. Labeling re-
quirements speclfled in these Federal regu-
lations and tarifls will apply to all viable re-
combinant DNA materials in which any nor-
tlon of the material is derived from ati dtio-
logic agent &ted in paragraph (c) of 42 CFR
`72.25. Additional information on nacklne and
shipping Is given in a supple&lent to' the
guidelines (Appendix D, part X) .
  D. Biological containment levels. Biologl-
cal barriers are speclAc to each host-vector
system. Hence the criteria for this mecha-
nism of containment cannot be generalized
to the same extent 8s for physical contain-
ment. Thls ls particularly true at the present
time when our experience with existing host-
vector systems and our predictive knowledge
about projected $yst.ems are sparse. The clas-
slflcatlon of experiments with recombinant
DNAs that 1s necessary for the construc-
tion of tbe exnerimental guidelines (Sec-
tion III) can beaccompllshe;i 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
classiflcatlon. It ls therefore convenient to
suecifv the nature of the bioloelcal contaln-
Aent -under host-vector headings such as
those given below for Escherf&ta coli K-12.

A general rule that, though obvious, de-
serves statement is that the level of contaln-
ment required for any experiment on DNA
recombinanti shall never be lees than that
required for the most hazardous component
used to construct and clone the recombinant
DNA (i.e. 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 l&elf effectively pre-
cludes certain experiments-namely, those in
which one of the components is in Class 5
of the "Classification of Etlologlc Agents on
the Basis of Hazard" (6). 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. Exmriments that are not to be wr-
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, OUI estimates of
the possible dangers that may ensue if that
containment fatls are of such magnitude
that, we consider it the wisest policy to at
least defer experiments on these recombl-
nant DNAs until there is more information to
accurately z%?.sess that danger and to allow
th& construction of more effective biological
barriers. In this respect, these guidelines are
more stringent than those initially recom-
mended (I)-.

The following experiments are not to be
initiated at the present time: (I) Cloning of
recombinant DNAs derived from the patho-
genic organisms In Classes 3, 4, and 5 of
"Classification of Etiologic Agents on the
Basis of Hazard" (6). or oncogenlc VirUSeS
classified by NC1 as moderate risk (6). or cells
known to be infected with such agents, re-
gardless of the host-vector system used. (ii)
Deliberate formation of recombinant DNAa
containing genes for the biosynthesis of po-
tent toxins (e.g., botulinurn or diphtheria
toxins; venoms from insects, snakes, etc.).
(iii) 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 contalnlng a recombinant DNA
molecule. (v) Transfer of a drug resistance
trait to mlcroorganlsms that. are not known
to acquire it naturally lf 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) width recombinant DNAs known to
make harmful products are not to be carried
out. We differentiate between small- and
large-scale experlemnts with such DNAs be-
cause the probability of escape from contain-
ment barriers normally increases with ln-
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 permfsdble
experiment'l. It is anticipated that most re-

combinal!t DIiA experiments initiated be:ora
these guidelines are next reviewed (Le., with-
in the year) will employ E. co2i K-12 host-
vector ssytems. These are also the systems fur
which we have the most experience and
knowledge regarding the effectiveness of the
containn!ent provided by exfsting hosts and
vectors necessary for the constru&on of
more effective biological barriers.
For these reasons, E. 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 hy uqing an organism as intimately
connected with a man as is E. coli. Thus.
while proceeding cautiously with E. ooli,
Serious efforts should be made toward devei-
aping alternate host-vector systems; this sub-
jeCt is discuysrd in considerable detail in Ap-
pendix A.

We therefore consider DNA recombinant3
fn E. c&i K-12 before pioceeding to other
host-vector systems.

1. BioZogicaZ colltalnrrlent miterin using E
coli K-12 host-vectors EKl host-1wtor.q.
These are host-vector systems that can be
estimated to already provide a moderate level
Of COntainme!lt. and include most of the
presently available systems. The host is al-
wavs E. COli K-12, and the vectors include
nonconjugative plasmids [e.g.. pSC101, ColE1
or derivatives thereof (19-26) 1 and variants
of bacteriophage 7 (27-29)

The E. coli K-12 nonconjugative plasmid
system is taken as an example to illustrate
the approximate level of containment refer-
red to here. The availaee data from experi-
ments involving the feeding of bacteria to
humans and calves (30-32) indicat,e that E.
coli 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 lOI 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
lnoculum. Moreover, since viable E. coli K-12
can be found in the feces after humans are
fed 10' bacteria in broth (30) or 3x101 bac-
teria protected by suspension in milk (31).
transductional and conjugational transfer of
the plasmld vectors from E. coli K-12 to resl-
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 plssmid for
mobilization and transfer to other bacteria.
When present in the same cell with dere-
pressed conjugative plasmida such as F or
Rldrdl9, the nonconjugative C&El, ColEl-
trp and pSClO1 plasmlda are tramferred to
suitable recipient strains under ideal labora-
tory conditions at frequencies of about 0.5.
10-t to 10-6, and 10-s per donor cell, respec-
tively. These frequencies are reduced by
another factor of 109 to l(r if the conjugative
plasmid 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
flrst acquire a conjugative plasmid from
another cell before it can transfer the non-
conjugative plasmid to a secondary recipient.
With colEl. the frequencies or transfer are
103 and lo-' to 103 when using conjugative
plasmld donors possessing derepressed and
repressed plaemids, respectively. Mobilization
of ColEl-trp and pSClO1 under rdmilar 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, 1976


NOTICES

x-155

sion of donor fertility, the frequency at which
nonconjugative plasmids are mobilized and
transferred by this sequence of events i?z
o&o is difficult to estimate. However, in C%lVeS
fed on an antibiotic-supplemented diet, it
has been estimated that such triparental
nonconjugative R plasmid transfer occurs at
frequencies of no more than 1O-`o to lo-`:
ner 24 hours oer calf (32). In terms of con-
;liderlng othe; means ior' plasmid transmis-
sion in nature, it should be noted that trans-
duction does operate in oiuo for Staphylococ-
CUJ aure~s (34) and probably for E. coli &s
well. However, no data are available to indi-
cate the frequencies of plasmid transfer in
oiuo by either tranirluction or tmnsforma-
tlon.

These observations indicate the low prob-
abilities for possible dlsseminatlon of such
plasmid vectors by accidental Ingestion,
which would probably involve only a few
hundred or thousand bacteria orovided that
at least the standard practices *(Section II-A
above) are followed, particularly the avold-
ante of mouth pipetttig. The possibility of
colonization and hence of tmnsfer are in-
creased, however, if the normal flora in the
bowel is dk3rUDted bv. for examale. anti-
biotic therapy i35). F& this reasoh, tiersons
receiving such therapy must not work with
DNA recombinants formed with any E. coZi
K-12 host-vector system during the therapy
period and for seven davs thereafter: simi-
iarly, persons who have Lhlorhydrla br 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 A variants.
Bacteriophage can escape from the labora-
tory eltber aa mature infectious phage par-
ticles or in bacterial hc& cells in which the
phage genome is carried as a plasmid or
prophage. The fate of E. co.% K-12 host cells
carrying the phage genome as a plasmid or
prophage ls similar to that for plasmid-con-
&Lining host cells as discussed above. The
survival of the X 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 X-sensitive E. coli strains.
Although the probability of survival of x
and its infection of resident intestinal E. calf
In animals and humans has not been mea.s-
wed, it Is estimated to be small given the
high sensitivity of x to the low pH of the
stomach, the lnsusceptlbillty to h infection
of smooth E. coli cells (the type that nor-
mally resides ln the gut), the infrequency of
naturally occurring ~-sensitive E. coed (36)
and the failure to detect infective x particles
In human feces a.fter ingestion of up to lOI*
h particles (37).
Moreover, A particles are very ieusitive to
desiccation.
EstablLhment of x as a stable lysogen ls a
frequent event (100 to I@`) for the att* int+
cI+ phage so that this mode of escape would
be the preponderant laboratory hazard: how-
ever, most EKl x vectors currently in use
lack the att and int functions (27-29) thus
reducing the probability of lysogenizatlon to
about 104 to l@* (33-40). The frequency for
the conversion of x to a plasmid state for
persistence and replication is also only about
Ied (41). Eforeover, the routine treatment of
phage lysates with chloroform (42) should
eliminate all surviving bacteria including
lysogens and x plasmld carriers. Lysogeniza-
tlon could also occur when an infectious x
contaln%ng cloned DNA infects a h-sensitive
cell In nature, and recombines with a resident

lambdold prophage. Although i-sensitive E.
coli strains 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 anorcied 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 x vector systems provide a moderate level
of biological cont,ainment. Other noncon-
jugative plasmids and bacteriophages that,
in association with E. coli K-12 can be esti-
mated to provide the same approximate level
of moderate containment are included ln the
EKl class.

EK2 host-rcciors. These are host-vector
systems that have been genetically con-
structed aud 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. coZi 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-S. This meas-
ure of biological containment has been se-
lected because it is a measurable entity. In-
deed, by testing the condributlons of pre-
existing and newly introduced genetic prop-
erties of vectors and hosts, individually or in
various combination& 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 svstem 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 108
host cells should be able to oernetuate the
vector and/or a cloned DNA fiagbent 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 fraement bv transformation. transduc-
tion or ckjugatfon to a host witd propeTt1e.s
common to those in the natural environment.

In terms of potential EK2 plasmld-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 CoIEl-trp and
pSlO1 in an E. coli. K-12 strain possessing
a mutation eliminating host-controlled re-
strfctlon and modification fhsdSI result6 in
about Ip-fold reduction in modilizatlon to
restriction-proficient recipients. The com-
bination of the dapD8, AbloH-asd, Agal-chlr
and rib mutations in E. coli K-12 results ln
no detectable survivors in feces of rats follow-
ln~! feedine bv stomach tube of 1010 cells in
milk and $mi"larly leads to complete lysls of
cells suspended in broth medium lacking
dlaminopimelic acid. E. coli K-12 strains with
MhyA and deoC(dra) mutations undergo
thymineless death in growth medium lack-
ln$ thvmine and alve a 10s-fold reduced sur-
&al &ring passige through the rat lntes-
tine compared to wild-type thy' E. coli K-12,
(However, the AthyA mutation alone or in
combination with a deoB(drm) mutation
only reduces in viva survival by a factor of
lo'.) Other host mutations, as yet untested,
that might further reduce survival of the
plasmid-host sys%em or reduce plasmid trans-
mkisfon are:  the combination polA( TS)
recA(TS) AthyA which might interfere wlth
ColEl replication and lead to DNA degrads-

tlon at body temperatures; Con mutations
that reduce the ability of conjugative
plasmids to enter the plasmld-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
a specific host, to make its replication
thermosensitlve 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 t.he construction of ERB plasmid-hont
systems it is important to use the moht
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 p&mid-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 po6-
sible in nature and that are also m&t ad?aI:-
tageous for its perpetuation. For example, 01-e
might conduct a triparental mating with il
primary donor possessing a redeprehsed F-
type or I-type conjugative plasmid, the safer
host with AbloH-asd, dapD8, Agal-chl', rfb,
AthyA, deoC, trp and hsdS mutations and R
plasrnid 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 thymlne and survival
of the Apr or trp marker in any of the three
strains followed as a function of time. Sur-
viva.1 of the vector and/or a cloned marker h?
t,ransduction could also be evaluated by in-
t,rociucing a known generalized transducing
phage into the system. Similar experimenL>
should also be done using a secondary recipi-
ent that is restrictive for the plasmld vector
as well as with primary donors possessing re-
pressed conjugative plasmlds with incom-
patibility group properties like those com-
monly found in enterlc mlcrocrganlsms.
Since a common route of escape of plasmid-
hoat systems in the laboratory might be by
accidental ingestion, it is suggested that the
same types of experiments be conducted in
suitable animal-model systems. In addition
to these tests on survival of the vector audj
or a cloned DNA framnent. it would be useful
to determine the s&lval' of the host strain
under nongrowth conditions such as in water
and as a function of drying time after a
culture has been spilled oti a-lab bench.

For EK2 host-vector svstems in which ihe
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 t,he natural environment either (aj as
a DroDhage or Dlasmid in the laboratorv ho&t
u&d ior phage-propagation or (b) by &vlv-
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 ~a-
tural environment.

In terms of potential EK2 X-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
X lysogeny in the normal laboratory host
should be reduced by removal of the phage
att site, the Int function, the reprea5or
gene(s)  and adding virulence-enhancing
mutations. The trequency of plssmld forma-
tlon, although normally already less than
10 *, could be further reduced by defecte ir;

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


38156

NOTICES

the PR-Q region, including mutations such
as vir-s. cro(TS), ~17, fi=, 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 Xaft site and inclusion
of one or more of the mutations described
above for nlasmid-host svstems to further
reduce the ^chanoe of form&ion 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 EKl sys-
tems. The infectivity of the phage particles
could be further reduced by introducing mu-
tations (e.g.,  suppressed ambers)  which
would make the phage particles extremely
unstable except under special laboratory con-
ditions (e.g., high concentrations of salts or
putrescine j .  Another meana would be to
make the nhaee itself a two-COmDOnent SYS-
tern, by eiin&ating the tall genes and ie-
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 safetv factor in this reaimen
is the extreme Instability of the heads, &less
they are stored in 1OmM 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
bv addine 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- (hsdS) laboratory host, which
produces phage with unmodified DNA which
should be restricted in r+ m+ bacteria that
are probably prevalent in nature. The likeli-
hood of recombination between the ,J vector
and lambdold prophages which are present
in some E. culi strains might be reduced by
elimination of the Hed function and the pres-
ence of the recombination-reducing Gam
function together with mutations contribut-
ing to the high lethality of the X phage.
However,  these second-order precautions
might not be relevant if the stability and
infectivity of the escaping h 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 (10'0 -10" partl-
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 A-host systems indicate that
cohtainment well in excess of the required
lo* or lower survival freauencv for the h
&or with or without a Eloneci DNA frag-
ment should be attained. Obviously the pres-
ence of all mutations contributing to this
high degree of biological containment mUSt
be veri5ed 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. SfmIlarly. the potential for
perpetuation of a cloned DNA fragment car-
ried by lnfectlous phage particles can be
tested by challenging typical wild-type E. coli
strains or a X-sensitive nonpermissive labora-
tory K-12 strain, especially one lysogenic `for
a lambdold phage.

In view of the fact that accurate assess-
ment d the probabilltiea for escape of in-

fectious A grown on r- m- Su. hosGs is de-
pendent upon the frequencies of r-, Su-, and
X-sensitive strains in nature, investigators
need to screen E. coli strains for these prop-
erties. These data wlll 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 safetv
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
1lOSt.s.

EK3 host-vectors. These are EK2 systems
for which the specified containment shown
by laboratory tests has been independently
conArmed 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 EKP 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 con5rm
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 svstem will be considered to
be a bona tide Ei(3 host-vector system, until
it is so certified by the NIH Recombinant
DNA Molecule Program Advisory Committee.

2. Classfflcatfon of experiments using the
E. coEi K-2 containment systems. In the fol-
lowing classl5cation 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 (EKS>EK2>EKl) are
to be used if they are available and are
equally appropriate for the purposes of the
experiment.

<a > Shotgun experiments. These experi-
ment.? 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.

(i) Eukaryotic DNA recombinants-Prf-
mates. P3 physical contalnment+an EK3
host-vector. or P4 physical containmentfan
EK2 host-vector, except for DNA from un-
contaminated embryonic tissue or primary
tissue cultures therefrom, and germ-line cells
for which P3 physical contalnment+an EK2
host-vector can be used. The basis for the
lower estimated hazard in the case of DNA
from the latter t&sues (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 J an EKP
host-vector.

Cold-blooded vertebrates. P2 physical con-
tainmentfan EX2 host-vector except for
embryonic or germ-line DNA which require
P2 physical containment+an FXl host-
vector. If the eukaryote is known to produce
a potent toxin, the containment shall be
increased to P3+EK2.

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

(I) Species that are known to produce a
potent toxin or are known pathogens (i.e.,
an agent listed in Class 2 of ref. 6 or a plant
pathogen) or are known to carry such patho-
genic agents must us-e P3 physical contain-
ment+an EK2 bc&-vector. Any species that
has a demonstrated capacity for carrying par-
ticular pathogenic agents is 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 FZtEKl. However, any insect
In this group should have been grown under
labomtory conditions for at least 10 genera-
tions prior to its use as a source of DNA.
Plants. P2 physical containment-&an EKl
host-vector. If the plant carries a known
pathogenic Rgent or makes a product known
to be dangerous to any species, the contain-
ment must be raised to P3 physical contain-
ment L- an EK2 host-vector.

(ii) Prokaryotes DNA recombinants-
Prokaryotes tlLat exclrange genetic infor-
matio with E. coli I.

The level of physical containment IS di-
rectly determined by the rule of the most
dangerous component (see introduction to
Section III). Thus Pl conditiona can be used
for DNAs from those baoteria in Class 1 of
ref. 6. ("Agents of no or minimal haz-
ard o o *") which naturally exchange genes
with E. co% and P2 conditions should be
Used for such bacteria if they fall in Class
2 Of ref. 6 ("Agents of ordinary potential
hazard o o *"). or plant pathogens or sym-
blonts. EKl host-vectors can be used for
all experiments requiring only Pl physical
containment; in fact, experiments in this
category can be performed with E. calf K-12
vectors exhibiting a lesser contaiment (e.g..
conjugative plasmids) than EKl vectors. Ex-
periments with DNA from species requiring
pd physical containment which are of low
pathogenicity (for example, enteropathogenic
Escherfchfa colt, Salmonella typhfmurfum,
and Klebsfella pneumonfae) can use EKl
host-vectors, but those of moderate path-
ogeniclty (for example, Salmonella typhf,
Shigella dysenteriae type I, and Vfbrfo
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 gelle of Agrobacteriztnr
tumefacfens.
Prokaryotes that do not exchange genetic
information with E. coli.The minimum con-
tainment conditions for this class consist
of P2 physical containmenttan EK2 host-
vector or P3 physical containment+an EKl
host-vector. and apply when the risk that the
recombinant DNAs will increase the path-
ogenicity or ecological potential of the host
is judged to be minimal. Experiments with
DNAS from pathogenic species (Class 2 ref.
6 plus plant pathogens) must use P3 f EK2.

(iii) Characterized clones of DNA tecombi-
nants derfved Jrom shotgun experfments.
When a cloned DNA recombinant has been
rigorously characterized' and there is suffi-
cient evidence that it is free of harmful
genes,' then experiments involving this re-
combinant DNA can be carried out under
Pl+EKl conditions if the inserted DNA is
from a species that exchanges genes with
8. co& and under P2+EXl conditions if not.

<b> Purified cellular DNAs other than
plasmids, bacteriophages; and other r:iruses.
The formation of DNA recombinants from
cellular DNAs that have been enrichedc by
physical and chemical techniques (l.e., not
by cloning) and which are free of harmful
genes can be carried out under lower con-
tainment conditions than used for the COP-
responding shotgun experiment. In general,
the containment can be decreased one step
in physical containment (P4+P3+P2+Pl)
while maintaining the biological contain-
ment specified for the shotgun experiment.
or one step in biological containment (EK3-r
EK24EKl) whlle maintaining the speci5ed
physical containment-provided that t.he

See foot,notes on p. 38459.

FEDERAL REGISTER, VOL. 41, NO. 17dTHURSDAY. SEFTEMBER 9, 1976


NOTICES

38437

new condition is not less than that specified
above for characterized clones from shotgun
experiment8 (Section <a> -ill).

cc> PZasmfds, bacteriophages, and other
utfuses. Ftecom~binanta 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
condltlons given below a.pply only to propa-
gation of the DNA recomblnants in E. coZi K-
12 hosts. They do not apply to other hosts
where they may be able to replicate 83 a re-
sult of functions provided by the DNA ln-
serted into the EK vectors. These are con-
sidered under other host-vector Systems.

(1) dnfmal uftuses. P4+EK2 or P3+EK3
shall be used to isolate DNA recombinanti
that include alI 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 psrtlally
characterized subgenomic segments of viral
DNAs (for examole. the eenome of defective
vlruaes, DNA %g&eniX i$olated after treat-
ment of viral genomes with restriction
enzymes, et&). When cloned recomblnants
have been shown by Suitable biochemical and
biological t&a tc be free of harmful regions.
thev can be handled in .P3+EX2 conditions.
In iae case of DNA vlru~e& `harmlesS regions
include the late region of the genome: ln
the csse of DNA cupies of RNA viruses, they
might include the genes coding for capsld
proteins or envelope proteins.

(U) PZant vfruses. F?+EKl or PZ+EK2
cond1t.ion.s shall be used to form DNA re-
combinanta t)rcrt include all or part of the
genome of a plant virus.

(ffl) Eukaryotic ofganelle DNA.% The con-
tainment conditions given below apply only
when the oreanelle DNA has been ~urlfled*
from &olate$ organelles. Mitochon&al DNA
from primates: P3qEKl or P2+EK2. Mlto-
chondrial or chloroplast DNA from other
eukaryotes: P2+EKl. Otherwise, the condi-
tions given  under shotgun experiments
apply.

(iv) Prokaryotfc p&mid and phage DNAa.
PZasmkls and phage from hosts that ex-
change genetfc-fnformatfon wfth E. calf.
Experiments with DNA recombinanti formed
from plasmlds or phage genomes that have
not been characterized with regard to
presence of harmful genes or are known tc
contribute slgniflcantly to the pathogerdclty
of their normal boats must use the contaln-
ment conditions specitled for shotgun experi-
ment6 with DNAs from the respective host.
If the DNA recombinanti are formed from
pla~mids or phage that are known not to con-
tain harmful genes, or from purified a and
characterized plafcnld or phage DNA segments
known not to contain harmful genes, tqe
experiments can be performed with PI physl-
cal containment fan EKl host-vector.
PZasmfdp and phage from hosts that do
not ezchange genetfc fnformation with
E. calf. The rules for shotgun experiments
with DNA from the host. apply to their plas-
mlds or phages. The minimum contaln&en~
conditions for this cateeorv IP2 CEKP. or
P3+EKl) can be used-f"; &s&i a&i
phage, or for purified* and characterized
segments of plasmld and phage DNAS, when
tie risk that the recombinant DNAs will
Increase the pathogenlclty or ecological po-
tential of the host is iudeed to be minilnal.

NOTE :  Where appilcaile, cDNAY (i.e.,
complementary DNAs) synthesized in vitro
from cellular or viral RNAs are included
within each of the above classlflcat~ions.
For example, cDNAs formed from cellular
RNAS that are not purified and character-
ized are included under <a>. shotgun ex-
periments; cDNAa formed Urom purified and

See footnotes on p. 33459.

characterized RNAs are included  under
<b>; cDNAs formed from viral RPv'Pls are
included under <c>; etc.

3. Experiments with other prckaryotic
host-vectors! Other prokaryotic ho&-vector
systems are at the speculative, planning.
or developmental stage, and consequently
do not warrant detailed treatment here at
this time. However, the containment crl-
teria for different types of DNA recombi-
nants formed with E. CoZf 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-for instance. ther-
mophilic organism or other host-iectors
WhQSe major habitats do not include humans
and/or economically important animals and
plants. In general, the straln of any pro-
karyotic species used 86 the host fs to con-
form to the definition of Class 1 etiologlc
agents given in ref. 5 (Le., "Agents for no
or minimal hazard o * o "), and the plasmid
or phage vector should not make the host
more hazardous. Appendix A gives a de-
tailed discussion of the B. aubtflfs system,
the most, promising alternative to d&e.

At the initial stage, the host-Ve&Qr mu&
exhibit at least a moderate level of biological
containment comparable to EKl Systems,
and should be capable of modification to ob-
tain high levels of containment comparable
t0 EK2 and EK3. The tvne of confirmation
test(s) required to movka host-vector from
an EK2-type classification to an EKS-type
Will clearly depend upon t e
habitat of the host-vector. +Fu preponderant
                  r example, ii
the  unmodified  host-vector  propagates
mostly in, on, or arQURd higher plants, but,
not appreciably in warm-blooded animals,
modidcatlon should be designed to reduce
the probability that the host-vector can e.s-
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 ln
using the containment criteria given for ex-
periments with E. calf K-12 host-vectors ss a
guide for other prokaryotlc systems. Experl-
ments wit+ 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 cla.%ses with E. calf
K-12 host-vectors applied. Experiments with
recombinanti between plasmld or phage vec-
tors and DNA that extends the range of re-
sistance of the recipient species to thera-
peutically useful drugs muse use P3 physical
containment + a hose-vector comparable to
EKl or P2 Dhv&Cal containment + a host-
vector compa&ble to EKZ. Transier of re-
combinant DNA to plant pathogens can be
made safer by using nonreverting, doubly
auxotrophic, non-pathogenic variants. Ex;
Derimentz3 usine: a DlaDt, Dat.hok%n that af-
?ects an element oi the focal gora will re-
qullp more stringent containment than if
carried out In areas where the host plant is
not common.
Experiments with DNAs from cukargotes
(and- their plssmlds or viruses) can also fol-
low the criteria for the corresponding experi-
ments with E. colt K-12 vectors if the major
habitats of the given host-vector overlap
those of E. 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 iOr some eUkaryQtiC
recombinant DNAs ned to be increased (for
instance. higher p1ant.S and their viruses in

the preceding example) while others can be
reduced.

4. Experiments with eukaryotfc host-vec-
tors<a> AnfmaZ 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. Olven good microblologlcal
practices, the most, likely mode of escape of
recombinant DNAs from a physically con-
talnecl laboratory is carriage by humans;
thus rectors should be chosen that have llt-
tie or no ability to replicate in human cells.
To be used as a vector in a eukarvotic host. a
DNA molecule needs to display ail of the f&-
lowing properties:

(1) It shall not consist of the whole ge-
nome of any agent that is infectious for hu-
mans or that replicates ta a significant ex-
tent, in human ce124 in ttgsue 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 studled genetically.
It is desirable that mutants be available in
adequate number and variety. and that
Quantitative studies of recombination have
&en 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 lnutant 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,

Currentlv onlv two viral DNAs can be con-
sidered as &e&g 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 viva and in vitro, and
tQ infect laboratory personnel, as evidenced
by the frequency of their conversion to pro-
ducing SV40 antlbodles. 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 complementatlon. with Yhe
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 uftro. How-
ever, this system still needs to be studled
more extensively. Appendix B gives further
details and documentation.

Taking account of all these factors:
1. Polyoma tiirus. a Recombinant DNA
molecules con&ting of defective polyoma
virus genomes plus DNA sequences of any
nonpathogenic organism, including Class 1
viruses (5), can be propagated in or used to
transform cultured cells. P3 conditions are
required. Appropriate helper virus can be
used if needed. Whenever there is a choice, it
is urged that mQuSe cells, derived preferably
from embryos, be used 83 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, recomblnants
form6d i?~ ?ilro between polpoma virus DN.4

FEDERAL REGISTER, VOl, 41. NO. 176--THURSDAY, SEPTEMBER 9, 1976


33458

NOTICES

and mouse DNA are presumably co: novel
irom an evolutionary point of view.

b. S!$h experiments are to be done under
P4 conditions if the recombinant DNA con-
tains segments of the genomes of Class 2
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 eenome isee
Section III%2-c-l) and that thi host ra&ge
of the polyoma virus vector has not been a.l-
iered, experiments can be conti!?ued under
P3 condit,ions,

2. SV40 Virus. a. Defective SV40 genomes.
with appropriate helper, can be used as a
vector for recombinant DNA molecules con-
z,aining sequences of any non-pathogenic
organism or Class I vicsus (5). (I.e., a shot-
gun type experiment). P4 conditions are re-
quired. Establjshed lines of culturrd cells
should be used.

b. Such enperi&nl.s are to be carried
out in P3 (Or P4) conditions if Tile non-
SV40 DNA segment is (a) a purified 0 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 eP-
ficiently in human cells in tissue culture
Wan does SV40, following infection at a mul-
t.iplicity of infection of one or more helper
SV40 viruses per cell.
c. A recombinant DNA molecule conslst-
ing of defective SV40 DNA lacking sub-
stantial segments of t,he late region, plus
DNA from non-pathogenic organisms or Class
I virllses (6), can be propagated as an alaton-
omous cellular element in established lines
of cells under P3 conditions provided that
there 1s no exogenous or endogenous helper,
and that it Is demonstrated that RO infec-
tious virus particles are being produced. Un-
til this has been demonstrated, t.he 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 (6) can be used
to transform established lines of non-permls-
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 Wansfection techniques must be car-
ried out in P4 conditions.

3. Efforts are to be made to ensure that all
cell lines are free of virus pa.rUcles and myco-
plasma.

Since SV40 and polyoma are limited in
their scope to act as vectors, chiefly because
the amount of foreign DNA that the normal
virlons can carry probably cannot exceed
2 X 10' daltons, the development of systems
in which recombinants can be cloned and
propagated purely in the form of DNA, rather
than in the coati of infectious agents fs
necessary. Plasmid forms of viral genomes or
organelle DNA need to be explored as possible
cloning vehicles in eukaryotic cells.
<b> Plant host-vectof s@ems. For cells
in tissue cultures, seedlings, or plant parts
(e.g., tubers, stems, fruits, and detached
leaves) or whole mature plant.9 of small
species (e.g., Arabidopais) the Pl-P4 contain-
ment conditions that we have specified prevl-
ously are relevant concepts. However, work
with most plante poses additional problems.

See footnotes on p. 38469.

The greenhouse facilities accompanyfug p2
laboratory physical containment condition3
can be provided by: (1) Insect-proof green-
houses, (ii) appropriate sterilization of con-
t.aminated plants, pots, soil, and runoff water,
and (iii) adoption of the other standard
practices for microbiological work. P3 phyai-
cal containment can be suRicient.ly approxi-
mated by coniining the operations with
whole plant5 to growth chambers like those
nsed for work with radioactive tsotopes: Pro-
aided, That (i) such chambers are modified
to produce a negative pressure environment
Y&h the exhaust air appropriately filtered,
(ii) that other onerations with Infections
materials are carried out undqr the specifiih
P3 conditions, and (iii) to guard against in-
advertent insect transmission of recombinant
DNA, growth chambers are to be routinely
fumigated and onIy used in insect proof
roomy. The P2 and P3 conditions specified
earlier are t,herefore extended to include
these cases for work on higher plants.
The host cells for experiments on re-
combinant DN.4.s may be cells in culture, in
seedling or plant parts. Whole plant.5 or plant.
parts that cannot be adequately contained
shall not be used as hosts for shotgun ex-
periment,s at this time, and attempts to in-
fect whole plants with recombicant 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
`a9 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 recombinanti 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
itself 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 hoat 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 puriflede 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 a 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 blologi-
Cal containment permit 8 decrease of one
step in the physician containment speclfled
above (P4+P&P2+Pl).

<G> FvngaZ or similar lower euharyotk
host-uector systems. The containment cri-
teria for experiments on recombinant DNA8
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 experiment& with B. c02i K-12 and
other prokaryotic host-vectors (sections
IIIB-1 and -2, respectively) provide adequate
direction for experiments with tiese lower
eukaryotic host-vectors. This is particularly
true at this time since the development oi
these host-vectors is presently in the specu-
:ative stage.

N. ROLLS AND R~s~o~sx~~~rrres

Safety in research involving recombinant
DNA moleculea 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-
hure protection of personnel, the public, and
rile environment.

The guidelines given here are to help &he
principal investigator determine the nature
of the safeguards t.hat should be fmple-
mented. These guidelines will be incomplete
1x1 acne respects because all conceivable ex-
oerimenls ivit,h recombinant DNAs cannct
now be anticipated. Therefore, they cannot
sl;bst:tut~e fc.r the investigator's own knou-l-
edgenble  and  di%riminating  evaluation
WIlenever !his evaluation calls for an In-
crease in cont,ainment over that indicated
in t.he culdelines. tlx InvestiEator has a

rcspon~~G`~lity to institute such an increw.
111 ,~o:`.trast.  :he containment conditions
called for in the guidelines should not be
decreased without review and approval at
the instiiutional and NIH levels.

The following roles and resp%ibilities de-
:ilit? an administrative framework in which
hafety is an essent,isl and integrated func-
tion of research involving recombinant DNA
molecules.

A. Prinecipal im:esliyulor. The principal i:l-
vestigator has the primary responsibility for :
(i) Determining the real and potential bic-
hazards of the proposed research, (ii) dc-
terniiniug the appropriate level of biological
and physical containment, (iii) selecting the
microbiological practices  and laboratory
techniques for handling recombinant DNA
materials, (iv) preparing procedures for deal-
ing with accidenta spills and overt personnel
contamination, (v) determining the appll-
cability of various precautionary medical
practises,  serological monitoring, and lm-
munization, when available, (vi) securing
approval of the proposed research prior tc
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
st.rains available to others, (viii) reporting
to the institutional biohazards committee,
and the NIH Offlce of Recombinant. DNA Ac-
tivit,ies new information bearing on the
guidelines, such as technical information re-
lating to hazards and new safety procedures
or innovations, (ix) applying for approval
from the NIH Recombinant DNA Molecule
Program Advisory Committee for large scale
experiments with recombinant DNAs known
ta make harmful products (Le., 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 rfgor-
ouslv characterized and tbere is sufecieni
evi8nce that tt is free of harmful genes.

Before work is begun, the principal in-
vestigator is responsible for: (i) Making
available to program and support staff copies
of those portions of the approved grant ap-
plicatlon that describe the biohazards and
the precautions to be taken, (11) advIsIng tba
program and support staff of the nature and
assessment of the real and potential bio-
hazards, (ill) instructing and training this
staf7 in the practfces and techniques required
to ensure safety, and In the pfoceduti for
dealing with accidentallv created bloh-ds.
and (ii) informing the-staff of the-reasons
and provisions for any advised or requesfed
precautionary medical practlses, vaccina-
tiona, or serum collection.

During the conduct of the research, tbs
principal investigator is responsible for: (rj

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


NOTICES

Supervising the safety performance of the
staff to ensure that the required safety prac-
t&es and techniques are employed, (ii) ln-
vestlgatlng and reporting in writing to the
NIH Offfce of Recombinant DNA Activities
and the institutional biohazards committee
anv serious or extended illness of a worker
or any accident that results in (a) inocula-
tion of recombinant DNA materials through
butaneous 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-
sonnel or danger of environmental contaml-
nation, (111) lnveslgatlng 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 blo-
logical and physfcal 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 physloal contaln-
ment (e.g-. biological safety cabinets) and
the biological containment (e.g., genotyplc
and phenotyplc characteristics, purity, etc.).

B. Institution. Since in almost all cases,
NIH grants are made to institutions rather
than to individuals. all the reeponslblllties
of bhe principal lnvestlgator listed above are
the responslbllltles of the institution under
the grant, fulfilled on its behalf by the prin-
cipa) investigator. In addition, the institu-
t:on ls responslble for establishing an lnsti-
tutional biohazards committee' to: (1) Ad-
vise the institution on policies. (ii) create
and maintain a central reference file and
library of catalogs, books, articles, newslet-
ters. and other communications as a source
of advice and reference regarding, for exam-
ple, the avallabillty and quality of the safety
equipment, the avalIabfl1t.y and level of blo-
logical containment for various host-vector
systems, sult.able training of personnel and
data on the potential biohazards associated
with certain recombinant DNA& (111) de-
velop a safety and operations manual for any
P4 faclllty maintained by the institution
and used in support of recombinant DNA
research, (iv) certify to the NIH on appllca-
tlons 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 inst~itutional biohazards
committee.

The biohazards commtttee must be sufff-
clently qualified through the experience and
exoertise of its membershin 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-
site of dlsclollnes relevant to recombinant
DNA technology. biological safety, and engi-
neering. In addition to possessing the profes-
sional competence necessary to assess and
review speclflc activities and facilities, the
commlt.tee should possess or have available
to it, the competence to determine the ac-
ceptabllity of lte flndlngs in terms of ap-
pllcnble laws, regulations, standards of prac-
tires, community attitudes, and health and
enrironmental conslderatlons. Minutes of the
meetings should be kept and made available
for public inspection The institution is re-
sponsible for reporting names of and relevant
background information on the members of
ita biohazards committee to the NIH.

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

to reviewing the scientific merit of each
grant application involving recombinant
DNA molecules,  are responsible for:  (1)
Making an independent evaluation of the
real and potential biohazards of the pro-
posed research on the basis of these guide-
lines, (11) determining whether the proposed
physical cont,ainment safeguards certified by
the institutional biohazards commit,tee are
appropriate for cont,rol of these biohazards.
(iii) determining whether the proposed blo-
logical containment safeguards are appro-
priate, (iv) referring to the NIH Recombl-
nant DNA Molecule Program Advisory Com-
mittee or the NIH Oi_ce of Recombinant
DNA Act,lvltles 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 Recombinmzt DNA Molecule' PTO-
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 potent&l 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 t.he capac-
ity to infect and replicate ln 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 responsiblllty
for: I II Revlslne and undatlne euldelines
to be `followed by~lnvestlg&ors Go&ring with
DNA recomblnante, (ii) for the time being,
receiving information on purported EKZ and
EK3 systems and evaluating and certifying
that host-vector systems meet EK2 or EK3
criteria. (ill) resolving questions ooncemlng
potential biohazard and adequacy of con-
talnment capability if NIH staff or NM In-
lt.ial Review Group so request. and (Iv) re-
viewing and approving large scale experiments
with recombinant DNAs known to make
harmful products (e.g., more thsn 10 liters
of culture).
E. NIH Staff. NH-I Staff has respolitlbillty
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) revlew-
lng and responding to questions or problems
or reports submitted by institutional blo-
hazards committees or principal lnvestiga-
tars. and disseminating findings, as appro-
priate, (iii) receiving and reviewing appllca-
tlons for approval to lower containment
levels when a cloned DNA recombinant de-
rived from a shotgun experiment has been
rigorously charact.e&ed and there is sum-
clent evidence that it is free of harmful
genes, (iv) referring items covered under (il)
and (111) above to the NIH Recombinant DNA
Molecule Program Advisory CommIttee, as
deemed necessary, and (v) performing site
inspections of all P4 physical containment
facllltles, engaged In DNA recombinant re-
search, and of ot.her facilities a.? deemed
necessary

APPENDIX D

V. Foororxs

1 Blological Safety Cabinets referred to in
this section are class~ed as Class I. Chsa II
or Class III cabinets. A Ckrss I cablnet ls a
ventilated cabinet for personnel protection
having an inward flow of air away from the
operator. The exhaust air from thls cablnet is
filtered through a high efflclency or high ef-
ficiency particulate air (HEPA) filter before
discharged to the outside atmosphere. This
cabinet is used in three operational modes:
(1) with an 8 inch high full width open
front, (2) w1t.h an installed front closure
panel (having four eight inch diameter open-
ings) without gloves, and (3) with an ln-
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 C&s II cabinet is a ventilated
cabinet for personnel and product protection
having an open front with inward air flow
for personnel protection. and HEPA filtered
mass recirculated alr flow for product protec-
tion. The cabinet exhaust air is filtered
through a HEPA filter. The face velocity of
the inward flow of air through the full width
open front 1s 75 feet per minute or greater.
Design and performance speciflcatlons 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 sas tieht construc-
tion which provides the highest-level of per-
sonnel protect.ion of 811 Biohazard Safet.y
Cabinets. The interior of the cabinet is pro-
tected from contaminants exterior to the
cabinet. The cablnet is Atted 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 Altered through
HEPA fllters or incinerated before belng dip-
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, plssmid and `or chro-
mosomal genetic information.
`The bacteria which constitute Class 2 of
ref. 5 ("Aeents of ordlnarv notentlal hazard
. _ .") rep&sent a broad sp&`um of etlologir
agents which possess different 1eveIs of vir-
ulence and degrees of communicablllty. We
think it appropriate for our specific purpose
to" further subciivlde the agents of Class 2
into those which we believe to be of rela-
tively low pathogeniclty and those which are
moderat.ely pathogenic. The several specific
examples given map surnc-e to illustrate t.he
principle.

4 The term "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 lnvestlgator must
obtain approval from the National Institutes
of Health. Such approval would be contln-
gent upon data concerning: (a) The absence
of potentially harmful genes (e.g.. sequen-
ces contained in indigenous tumor vlru.ses
or which code for toxic substances), (b)
the relat.ion 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.

6 A DNA preparation 1s defined as enriched
lf the desired DNA represents at least 99%
(w/w) of the tot.al DNA ins 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 ia

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


38460

NOTICES

markedly reduced. Thus, the probability of
cloning a harmful gene could, for example, be
reduced by more that IWfold 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 preparat,lon Is deflnsd as purl-
fled if the desired DNA reuresenta at least
99 percent (w/w) of the t&Z DNA in the
preparation, provided that it was verified by
more than one procedure.
`In special circumstances, in consultation
with the NIH OfBee of Recombinant DNA Ac-
tivities, an area biohazards committee may
be formed, composed of members irom the
institution and/or other organizations be-
yond its own staff, as an alternative when
additional expertise outside the instit.ution
is needed for Bhe indicated reviews.

V. REFERENCES

1. Berg, P., D. Baltimore, H. W. Boyer, S. N.
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2. Advisory Board for the Research Coun-
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3. Berg, P., D. Baltimore, 5. Brenner, R. 0.
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4. Laboratory Safety at the Center for Dis-
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6. Classification of Etiologic Agents on the
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6. National Cancer Institute S$fety Stand-
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7. National lnstitutcs of Health Biohazarda
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8. Biohazards fn BioZogfcaZ Research ( 1973).
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10. Bodily, H. L. (1970). General Adminis-
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11. Darlow, H. M. (1969). Safety fn th-e
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12. The Prevention of Laboratory Acquired
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13. Chatigny. M. A (1961). Protection
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Umbrelt (ed.). Advances in Applied Mlcro-
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14. Design Crfteria for Viral Oncology Re-
search Facflities, U.S. Department of Health,
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Publication No. (NIB) 76-891, 1975.

16. Kuehne, R. W. (1973). BioZogicuZ Con-
tainment Facilfty lor Studying Infectious
Disease. Appl. Mlcroblol. 26: 239-243.

16. Runkle, R. 8. and Q. B. Phillips. (1960).
Microbial Containment Control Facilities.
Van Nostrand Reinhold, New York.

17. Chatigny, M. A. and D. I. Clinger (1969).
Contfbmi?z.ation Control in Aerobiology. In
R. L. Dimmick and A. B. Akers (eds.) An In-
troduction to Experimental Aerobiology.
John Wiley & Sons, New York, pp. 194-263.

18. Grunsteln, M. and D. S. Hognsss (1975).
Colony Hybrtdieation: A Method for the Iso-
lation of Cloned DNtis That Contain a Spe-
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19. Morrow, J. F.. 6. N. Cohen, A. C. Y.
Chang, H. W. Bayer, H. M. Goodman and
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20. Hershfield, V., H. W. Boyer, C. Yanofsky,
M. A. Lovett and D. R. Helinskl (1974). Plas-
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Acad. Sci. USA 71,345&3459.

21. Wensink, P. C., D. J. Finnegan, J. E.
Donelson, and D. S. Hogness (1974). A Sys-
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22. Timmis, K., F. Cabello and S. N. Cohen
(1974). Utilization of Ttoo Distinct Modes ot
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in Vitro lrom Separate ReQliconS. proc. N&t.
Acad. Scl. USA 71,4&X%4560.
23. Glover, D. M., R. L. White, D. J. Fiune-
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lion Of Six Cloned DNAs from DrosoQhfZa
melanogaster. Including one that Contains
the Geaes lor rRNA. Cell 5, 149-155.
24. Kedes, L. H., A. C. Y. Chang, D. House-
man aud S. N. Cohen (1975). Isolation ol
Histone Genes from Unfraclionated Sea
Urchin DNA by Subculture Cloning in E.
colt Nature 255, 533.

25. Tanaka, T. and B. Weisblum (1975).
Construction of a Colicin EZ-R Factor Com-
posite Plasmid in Vitro: Means for Ampli-
floation of Deowibonucleic Acid. J. Bacte-
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26. Tanaka, T, B. Welsblum, M. Schnoss
and R. Inman  (1976). Construction and
Characterization of a Ohfmerfc Plasmid Com-
posed ot DNA from Eschertchfa col and
DrosophiZa meZanogastt?r. Biochemistry 14.
2064-2072.

27. Thomas. M., J. R. Cameron and R. W.
Davis (1974). ViabZe Molecular Hybrids o/
Bacteriophage Lambda and Eukaryotic DNA.
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28. Murray, N. E. and K. Murray (1974),
Manfpulation of Restttction Targets tn Phage
A to jorm Re&?ptor Chromosomes for DNA
Fraoments. Nature 251. 476-481.

25. Rambach, A. a& P. Tiollals (1074).
Bacteriophage X Having EcoRZ EndonucZease
Sites only in the Non-essential Region oj the
Genome. Proc. Nat. Acad. Scl. USA 71, 3927-
3930.

30. Smith, H. W. (1975) StuufuaZ of Orally-
Administered Escherichia eoZf Kit tn the
Alimentary Tract 01 Man. Nature 255, 600-
502.

31. Anderson, E. 8. (1975). ViabiZttg of,
and Transjer of a Plasmid from Escherichia
coli Ki2 in the human intestine. Nature
255, 602-604.

32. Falkow, S. (1076). Unpublished experl-
ments quoted in Appendix D of the Report
oj the Organizing Committee of the AsUo-

mar Conference on Recombfnunr DNA Mole-
cules (P. Berg, D. Baltimore, S. Brenner, R
0. Roblin and M. Singer. eds.) submitted to
the National Academy of Sciences.

33. R. Curtiss III. oersonal communtca-

              I *
tlon.

34. Novick, R. P. and S. I. Morse (1967).
In Viva Transmission of Drug Resistance
Factors between Strains ol Staphylococcus
aweus. J. Exp. Med. 125,46-59.
36. Anderson, J. D., W. A. Gillespie and
Ed. H. Richmond. 1974. Chemotherapy and
Antibiotic Resistance Transfer between En-
terobacterta in the Human Gaatrointestinol
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36. Ronald Davis, personal communication
97. K. Murray, personal communication:
W. Szybalskl, personal communication.
88. Manly. K. R., E. R Signer and C. M
Raddlng (1969). Nonessential Functfolu of
Bactetiophage A. Virology 37 177.
30. Gottesman, M. E. and R. A. WeLsberg
(1971). Prophage Insertion and Ezcisfon. In
The Bacteriophage Lambda (A. D. Hershey.
ed.) . Cold Spring Harbor Laboratory pp. 113-
138.

40. Shimada, K., R. A. Weisberg and M. E
Gottesman (1972). Prophage Lambda at Un-
usual Chromosomal Locatfons: I. Location
oj the Secondary Attachment Sites and the
Properties of the Lysogens. J. Mol. Biol. 63.
483-503.

41. SlMnner, E. (1969). PZasmid Formcrfio~.

A New *ode of L'ysogeny by Phage A. Nature
223. 15f&-180.

4& Adams, M. H. (1959). Bacteriophages.
Intersciences Pub&hers, Inc.. New York.
43. Jacob, F. and E. L. Wollman (1956).
Sut I-es Processus de Conjuqaison et de Rf-
combinasfon chez Escherichia coli. I. L'tndilc-
tfon par Conjugafson ou Induction Zygoti-
que. Ann. Inst. Pasteur 91, 486-510.
44. J. 5. Parkinson as cited (p. 8) by Her-
shey, k D. and W. Dove (1971). Introdug:-
tfon to Lambda. In: The Bacteriophage 1.
A. D. Hershey, ed. Cold Spring Harbor ~nl-,,-
ratory. New York.

CYAIRMAN

STETTEN. Hewitt, Jr., M.D., Ph.D., Dcp~.l~y
Director for Science, National Il;sti: .:tes
of Health

VICE CEAIRMAN

J,4COBS, Leon, Ph.D., Associate Dlrectclr i;~r
Collaborative Research, National Instltu:<+
of Health.

ADELBERG, Edward A., Ph.D.. Professor. De-

partment of Human Gene&s, Scho& of
Medlclne, Yale University.
CHU, Ernest. K Y, Ph.D., Professor, De-
partment of Human Genetics, Medica
School, University of Michigan
CURTIS& Roy, III, Ph.D., Professor. Dr-
partment or Microblolo,T, School or hwdi-
clne, University of Alabama.
DARRELL, James E., Jr.. M.D.. Pr0fe.ssc.r.
DepartmerIt of Molecular Cell Biology.
Rockefeller University.

HEXJNSKI. Donald R.. Ph.D., Professor DF-
partment of Blolocv. Universitv of Calm-
fornia, San Diego. --.
HOGNESS. David 5.. Ph.D., Professor. Gr-
partment of Biochemistry, Stanford UT-,!-
vend tv.

KUTTER, Elizabeth M., Ph.D., Member of
the Faculty in Biophysics, The Evergrttn
State College.

LI'ITLEFIELD, John W., M.D., Professor &
Chairman, Department of Pediatrics, Chll-
dren's Medical & Surgical Center, Johns
Hopkins Hospital. -
REDFORD, Emmett& S., Ph.D.. LL.D., Ash&I
Smith Professor of Government and Pub-

110 Aftairs, Lyndon B. John&ma School OL
Publ!c Affalq Eniversity of Texas at hxa-
1m

FEDERAL REGISTER, VOL. 41, NO. I76-THURSDAY, SEPTEMBER 9: 1976


ROWE, Wallace P, MD, Chief, Laboratory
of Viral Diseases, Natlonal Institute of Al-
lergy & Infectloua 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.
i3ZYBALSK.I. Waclaw, D.Sc.. Professor o!
Oncology. McArdle Laboratory, Unlver-
slty Of wiiconsin.

THOMAS, Charles A, Jr., Ph.D., ProfesSo?.
Department of Biologlcd Chemistry, Har-
vard Medical School.

GARTLAND, Willlam J, Jr., Ph.D., Health
Scientist Administrator, National Institute
of General Medical Sciences, National In-
stitutes of Health.

LIAISON REPRESENTATNES

HEDRICH'. Richard. Ph.D., Coordination Pro-
gram of Science Technology S Human
Value. National Endowment for the Hu-
-itieS.

LEWIS, Herman W.. Ph.D., Division of Bio-
logical and Medical Sciences, National Sci-
ence Foundation.

NIGHTINGALE, Elena 0.. Ph.D., Assembly of
Life  Sciences, National Academy of
Sciences.

BHEPHERD. George R.. Ph.D., Division of Bio-
medical and Environmental Research, En-
ergy Research and Development Adminis-
tratlon.

APPRNDIX A TO APPENDIX D

IN RECOMBINANT MOLECULE TECIINOI.O(iY

Unquestionably. Escherichfu calf 1s the
most well characterized unicellular organism
Years of basic research have enabled lnvestl-
gators to develop a well characterized genetic
map. to obtain detailed knowledge of v&u-
lent and temperate bacteriophages, and to
explore the physiology, genetics, and regula-
tion of plasmlds. 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 unite. The dis-
coverv of transformation of Bacillus subtiZfs
by S~iazen (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-
WY.

A. Current knozD&dge of the chromosomal
archftecture and mechanisms ot genetic ax-
ehanae in B. subtilis. Two mechanisms o!
geneilc exchange have been utIlLzed to estab-
lish the linkage map of B. subtilis, DNA-
mediated transformation (capable of trams-
ferrlng approximately 1 percent of the gen-
ome) and transduction with bacteriophage
PBS1 (capable of transferring b-8 percent of
the chromosome). Recent detailed genetic
atudles with PBS1 by Lepesant-Kejzlorova
et aI. (2) have resulted in the development
of a circular genetic map for this organism
The current edition of the map (3) contains
196 loci. Blophysical analyses have estab-
lished that the chromosome is circular (4)
and replicates bidlrectionally (6).

Transformation with purlfled fragments of
DNA is a highly efllcient process in B. subtflis
with frequencies of 1 to 4 percent usually
attained for any auxotrophlc or antibiotic
resistance markers. Frequencies of approxi-
mately X0 percent transformation can be
achieved with DNA prepared from gently
lysed L-forms or protoplasm (8). These large
fragments of DNA are readily incorporated
by the recipient cell. Qeneralised transduc-

tion occurs with bacteriophages SPlO (7).
PBS1 (8), and SPPl (9). while a low fre-
ouencv of soeciallzed transduction has been
-.
report& wib bacteriophage 0195 (10).

Although transformation is 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
oi the B. subtilis genospecies include 8.
Zichenijormis, B. pumilus, B. amylolique-
faciens, and B. globigif (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 trsns-
formation 1s low In the heterologous cross
[e.g.. B. amyloliquefaciens (donor) jB sub-
tilia (recipient) 1, the newly acquired DNA
from B. wnyloliquefaciens in the B. subtilis
background 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. subtilti ATCC
7993 appears to be the most useful since it
carries one to two Copies of a plasmid wit,h
a molecular welght of 48 x 1CP. This strain
is aho closely related to B. subtilis 168. An-
other strain of B. subtflis (ATCC 15841) con-
tains 16 copies of a plasmid with a molecular
wetoht of 4.6 X 108. Currentlv it is not known
.---w- ~~
whether genetic markers can be readily ln-
traduced into these plasmids. To date it has
not been possible to readily stabilize plas-
mids derived from E. pumilus in B. subtilis
even with heavy selective pressure I P. Lovett,
personal communication),
Two temper&e bacteriophages are under
development. as veotors in B. subtilis. +3T
and SPOZ. Lyeogeny of thymine auxotrophs
(strdns caxrying thyd thyB) by beoterio-
phage Q3T results In "conversion" to a Thy+
phenotype. The attachment sita for this
baateriophage and the bacteriophage gene
for thymldylate 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
oleaved by the aite-specific endonuclease,
Barn 1 (20). to produce 6 fragments (one
of which carriee the thyP gene). The thyP
carryiq gene can be integrated into the
bacterial genome in the absence of the intact
viral genome. Because deletions are available
that include the thyP region, it is theoreti-
eallv nossible to introduce thvP at manv
W&i on the chromosome. The thyP ge&
can be readily purlfmd for insertion ix&
plesmide or utilized BS a scafiold to integrate
other heterologous DNA lnt.o the ohromo-
some of B. subtflia. Alternatively, ie la pas-
eible to purify fragments of the chromcczome
bv ael electroohoresis (21. 22). for insertion
h& be&eriophage 48T or S&2. At present,
unfortunately, only the former carries a
selective marker, i.e., the gene for thymidyl-
de syntietase, thyP.
C. Development ol vehicles. B. subtilis h
a Gram-positive sporu.lat.ing rod that usually
inhabits solI. Although tt oan exist on
cutaneous surfaces of man (23) and experi-
mental animals, it r&rely produces disease.
To develop a suitable vebiole it is imperative
to have a hoot that Ss aeporogenic. The most
appropriate deletion mutation is deletion !IQ
(c.it D). In addition to a deficiency in
epmlatin thb mutant rapidly lyres when
# has reached the end of its growth cycle.
Presumably thle is due to tie failure fo
inwtlvate 0~9 of the fbut~lytlc enzymes (24)

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 (25.28). The
further introduction of thymlne anxotrophy
(defects ti the thyA thyB loci) wiU enable
the strain to survive only with a plasmid
vector carrying the purified thyP gene from
bactWiODhi%?e +3T or a defective bacterio-
phage &3T -&rying 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. Ii we ran
isolate a temperature-dependent lysogen
that will grow only at 48oC lt should be
possible to make an unusual vehicle.
D. Site-specific endonucleases. Recently
two restriction modification systems hare
been observed between B. subtilis 168 and
other bacilli. Trautner et al. have isolated
an effective system that Mibits infection of
the R strain of I?. subttlti by bacteriophage
SPPI propagated on 8. subtilis 168 (27). The
site-specific nnrlease recognizes the sequence
GGCC.
CCGG                    o
Young, Radnay,  and Wilson ObSeNSd II
restriction  modificaiton  system between
B. amyloliquefaciens and B. subtilis 168 (28).
The endonuclease from B. amyLoZiquefaciens
(20) recognizes the sequence GGTAC (29).
                    CCTAGG

bfore recently, two additional enzymes have
been isolated from B. gZobigii (30). The recog-
nition sequence is not known.
E. Adrantcrgm and liabilities of the B. sub-
tilis system-a. Advantages. 1. B. subtilis b
nonpathogenic. Asporogenic deletion mu-
tants are available to preclude the problem of
persistence through sporulation.
2. The circular chromosomal map is well
defined. At leir?t 196 10~1 have been posl-
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 endotexin in the
cell wall. Therefore the cells can be used as
a single cell protein source.
6. The frequency of transformation is very
high, facilitating the detection of rare events.
7. A unique bacteriophage, d3T. exists that
carries a gene that can be readily puritled for
"scaRoldlng" experiments.
b. Disadvantages. 1. The knowledge of
genetics and physiology of plasmlds and
viruses is prlmitlve compared with B. wolf.
2. High-frequency, spec&lized 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.

REPERENCES

1. Spieizen. J. 1958. Transformation of bio-
chemically deficient strains of Bacillus sub-
tilis by deoxyribonucleate. Proc. Nat. Acad.
Sci. U.S.A. 44: 1072-1978.

2. Lepesant-Kejzlarova, J., J. A. Lepesant,
J. Walle. A. Billault, and R. Dedonder. 1978.
Revision of the linkage map of Bacillus sub-
tilis 168: indications for circularity of the
chromosome. J. Bacterial. 121:828-884.
3. Young, F. E. and 0. A. Wilson. 1978.
Chromosomal map of Bacillus subtilfs p.
696-814. In P. Gerhardt, R. N. Costllow, and
H. L. Sadoff (ed.), Spores VI. American S+
eiety for Microbiology, Washington, D.C.
4. Wake, R. G. 197% Termination of Bb-
tillus sxbtilis chromosome repliratlon (L~I

FEDERAL REGISTER, VOL. 41, NO. I76-THURSDAY, SEPTEMBER 9, 1976


NOTICES

vlsusllzed by autoradiography. J. hIo1. Blol.
86:223-231.

5. Harford, N. 1976. Bidirectional chromo-
some replication in Bacillus subtills. J. Bao-
i,eriol. 121:835-847.

6. Bettinger, G. E. and F. E. Young. 1976.
`Transformation of Bacillus subtflis: Trans-
forming ability of deoxgrlbormcleic acid in
lysates of L-forms or protoplasts. J. Bacter-
iol. 222:987-993.

7. Thorne, C. B. 1962, Transduction in
Rmillus subtilis. J. Bacterlol. 83: 106-111.
8. Takahashi, I. 1961. Genetic transduc-
tion in Bacillus s-ubtilis. Blochem. Biophys.
Res. Commun. 5: 171-176.
9. Yasbin, R. E. and F. E. Young. 1974.
Transduction in Bacillus subtilfs by bac-
teriophage SPPI. J. Viral. 14:X%3-1348.
10. Shapiro, J. A., D. H. Dean and H. 0.
Halvorson. 1974. Low-frequency specialized
transduction with BmiZZus subtizis bacterio-
phage @IO& Virology 62:393-403.
11. Marmur, J., E. Seaman, and J. Levine.
1963. Interspecific transformation in B&Z-
Zus. J. Bacterial. 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 I-L 0. 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 F. E. Yonng. 1972.
Intergenotic transformation of the Bacillus
:;ubtiLis genospecies. J. Bacterial. 112:705-
716.
15. Yamaguchi, K.,  Y. Nagata, and B.
hIaruo. 1974. GeneOic control of the rate
of an amylase synthesis in Bacillus subtt?is.
J. Bactsriol. 119:410416.

16. Lovett, P. S., and F. E. Young. 1969.
Identification of Bacillus subtilis NRRL B-
3276 as a strain of BacilEus plc?nilus. J. Bac-
teriol. 100:65%661.

17. Lovett. P. S., and M. G. Bramuccl.
1975.  Plasmid deoxyritonuclelc acid in
BaciZlw subtilfs and Bacillus pur~flus. J.
Bacterial. 124:484490.

18. Lovet.t, P. S. 1973. Plasmid in B. pumnt-
Zus and the enhanced sporulatlon of plasmid
neeative variants. J. Bscteriol. 115:291-298.

19. Young, F. E., M. T. Williams. and G. A.
Wilson. Genebics ot Bacillus subtilfs. I?L D.
Schlessinger (ed.) Mlcroblology 1976, in

_ press.

20. Wilson, G. A. and F. E. Young. 1975.
Isolation of a seauence-wecific endonu-
clease (Barn 1) frdm BacfiZw amyiolique-
faciens H. J. Mol. Biol. 97:123-125.

21. Brown. L. Recombination analysis
with purified endonuclease fragments in the
RNA polymer- region of Bacillus subtilfs.
In D. Schlessinger ted.), Microbiology 1976,
in press.

22. Harris-Warrlck. R. M., Y. Elkana, S. D.
Ehrlich. and J. Lederberg. 1975. Klectro-
phoretic separation of BaciZZw subtfli,
genes. hoc. Nat. Acad. Sci. TJS.A. 72:2207-
2211.

23. Kloos, W. F., and M. S. Musselwhlte.
1975. Dlstributlon and pergistenca of Sta-
phylococcus and Micfococcw species and
other aerobic bacteria on human skin. Ap-
plied Micro. 30:381-395.

24. Brown, W. C., and F. E. Young. 1970.
Dynamic interactions between cell wall
polymers, extracellular proteasea and auto-
lytic enzymea B&hem. Biophpa Res,
Commun. 38:664-i%%

25. Clarb. V. L. and I'. E Young. 1974.
Active transport of D-alanine and related
amino adds by whole cells of BacUZus sub-
tilfa. J. Bacterlol. 120 : 108%109%
as. c&r& v. L. and F. E. young. Actvs
transport fn cells of B. subtflis 188: Lose of

endogenously energized transport in auxo-
trophs deprived of D-alanine or glycerol.
Submitted to J. Bacterial.

27. Trautner, T. A., B. Pawlek. S. Bran,
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 G. A.
Wilson. Manuscript in preparation.

29. Wilson, G. A. and F. E. Young. Unpub-
lished data.

30. Wilson, G. A., R. Roberts, and P. E.
Young. Unpublished data.

31. Wilson, G. A. and F. E. Young. Re-
striction and modification in bacilli. la D.
Schlessinger (ed.), Microbiology 1976, In
press.

APPSNOIX B TO APPENDIX D

POLYOMA AND sv40 YIRU.5

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. Sot.
Exp. Biol. 88, 362568. 1965; 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 ls induced (Gross, L., Oncogenic
Viruses, Second Edition, Pergamon Press,
NY) .

Polyoma virus grows lytfcally in mouse
cells in tlssne culture. Thus mouse cells in
rulture 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. Viral., II, 42&-431.
1973). The virus does not replicate to a
signlflcant extent in human cells in tissue
culture (Eddy, B.E., Viral. Monogr., 7, l-114,
196% Pollack, R. E., Salas, J.. Wang R. Ku-
San0 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 DN.4 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 expressjon 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 infeot humans (Hartley, J., Huebner. R..
Parker, J. and Rowe, W. P., unpub&hed
data). `Thus no antibodies to the virus have
been detected in people living ln bulldings
that are infested with virus-infected mlop.
nor ln laboratory workers who have been ex-
posed to the virus for a number of years.

At most, a small segment of polyoma virus
DNA shcvws weak homology with a portion of
the late region of SV40 DNA (Ferguson, J.
and Davis. R. W.. J. Mol. Biol., 94, 135-150,
1976). However, there appears to be no
genetic interaction between the two viruses
and there fs no immunological cross-reaction
between the gene products of the two viruses.

SV40 causes perisitent but apparently
harmless infections of the kidneys of vir-
tually all adult rhesus monkeyi (Hslung,
G. D.. Bact. Revs. 32.185-205. 19881. it causes
tumors when inje&ed lnto~ n&&n ham-
sters (Glrardf A J, Sweet, B. H, Slotnick.
V. B. and Hillemann, M. R.. Proc. Sot. Exp.
Biol. Med., 205, 42042'7, 1964) and trans-
forma cells of several mammalian speciea
(fnoludlng human). SV40 is able to infeot
humana eince antibodies to the virus are
found in a small proportion of the hu-
population (Shah, K. V, Goverdhsn, M. K.
aad Ozer$ H. L., Am. J. Epld. 93, 291-298,

1X0) and serum conversions have been
noted in many laboratory personnel who
have been exposed to the vlrua (Horvath,
L. B., Acta Mlcrobiol. Acta Scl. Hung. 22,
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, 0.. Johnson, R. T,
Shah, K., Rubinstein L. G. Preunlsl 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 (Soriano,
F., Shelburne, C. E. and Gokcen, M., Nature.
249, 421424, 1974). In other studies a non-
structural antigen characteristic of papova-
viruses. T antigen, has been detected in t,he
nuclei of cells cultured from 2 meningioma.!.
while another SV40-specific  antigen, U
antigen, has been found In the cells of
a third tumor Of the same type (Weiss, A. F I
Port.man. R., Fisher, H., Simon. J. and Zaz:~
K. D., hoc. Nat. Acad. Scl. USA 72, 609-613.
1975). Furthermore new papovaviruses have
been isolated from the brains of patients with
PML (JC virus-Padgett. B. L., Walker, D. L.,
zuRbein, G. M., Eckroade, R. I. and Des+%
B. H., Lancet 1, 1257-1260, 1971), from the
urine of a patient carrying a renal allograft
(BK virus-Gardner, S. D., Meld, 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
(Tskemoto, K. K., R&son, A. S., Mullarkey.
M. F., Blaese, R. M. Garon, C. F. and Nelson.
D. J., Nat. Cancer Inst., 53, 1205-1207, 1974)
All of these viruses which are dlstrihuted
widely throughout human populations share
sntigenic and biological properties with 8V
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 lntrace!-
lular T antigen, which appears to be coded by
the A gene of SV40 cross reacts extensively
with antigens found ln cells infected (?r
transformed by BK or JC viruses: both Ji'
and BK viruses induce tumors in newborn
hamsters (Walter, D. L., Padgett, B. L., zli-
Rheln, B. M., Albert, A. E. and Marsh, R. F
Science 181. 674-676, 1973: Shah, K. V.,
Daniel, R. W. and Strandberg, J.. J. Nat. Can-
cer Inst. 54, 945-950, 1976): BK virus cause%
transformation of hamster cells in culture
(M&jor, E. D., and Dihlayorca. G., Proc. Nat.
Acad. Scl. US 70, 3210-3212, 1973: Portolani.
hf., Barbanti, A., Brodano, G. and Laplaca.
M.J., Vlrol, 15, 420-422. 1975) and is able ti
complement the growth of certain tempera-
ture-sensitive mutants of SV40 (Mason, D. I%
and Tak~.motn. K. K., submitted for publjca-
tlon!,

k`VRTHEIC WOBK

At pr@e.nt, a potential eukaryotlc cecmr
of choice is polyoma virus. And while avail-
able information indicates that. It fulflile
all the necessary criteria. we recommend
that the following subfecta be further fn-
vestigated :

1. The moleculsa mechanism of resis&r.re
of human cells to the virus.
2. The extent of homology between polg-
oma virus DNA and the DNA.9 of humKli
papovaviruses.

3. The ability of human papovavlruses t.3
complement   defective   PolYo-   virus
genomee.

Report of a Working `Group Conslstlng of:
Dr. Bernard Fields, Harvard Unlverelty
School of Medicine.

Da. Thomae J. Kelly, Jr.. Johns Hopkins
University School of Medicine.
Dr. Andrew Lewis, National Institute c.f m-
lergy and Infectious D&ease&
Dr Malr-olm Martin, National Instituk @

FEDERAL REGBTER, VOL 41, NO. I76--THURSDAY, SEPTEMBER 9. 1976


Allergy and Infectious Diseases.
Dr. Robert Martin. Natloual Institute of
Arthritis, Metabolism, `and Digestive Dis-
Drymer Pfefferkoan. 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 Instl-
tute.

Rapporteur: Dr. Joe Sambrook, Cold Spring

Harbor Laboratory.

APPENDS C TO A~p~r~nrx D

mMMAIIY OIr THE WOBKSHOP ON THE DESIGN

AND TESTING OF S`4FEP PaOKARYOTIC VEHICLES
ANIl BACTERIAL HOSTS FOB RsssARCH ON RE-
COMBINANT DN.4 MOLECULES

Torrey Pines Inn, La Jolla, California

The development of techniques for the
cloning of DNA from both prokaryotic and
eukaryotlc 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
a potential biohazard. One of the requlre-
merits 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 cn-
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 Prokarvo-
tic Vehicles-and Bacterla~Hosts for Reseakh
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 testine 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 flrst session of the workshop, chaired
by W. Szybalski (University of Wisconsin),
was devoted to bacteriophage vectors. Szy-
balskl outlined the main safety features of
the two-component, phage-bacterial 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 fore&n DNA carried bv the Dhane vector:
(1) Est~bllshment of a stable prgphage 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 tc ensure safety, both routes should
be blocked by appropriate genetic modifica-
tions. For phage A, route (1) can be blocked
by phage mutations that interfere with lyso-
aenieation (ati-. fat-. cI-. cIII-. tir) and
plasmid formation (i+, ?&R. US. ril, clT,
Ots, trots) , and by mutations on the Mch.eri-
chta coli host that affect these processes
fottB-. dncAts1 and host survival. Route 12).
(which is of low probability since A phages
do not survive well in natural environments
(no Xc1 phage was recovered after ingestion
of lDs-10" particles), are kllled by deslcca-

tion, and have a low chance to encounter a
naturally sensitive hosi) 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-
sianed for Dhme nronaeation in the labora-
t&y (e.g., highcdnc&t;ations 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-
stabilitv of the Dhaee would minimize the
possibihty of tr&f& 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
a 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 10" 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., Xdvcrots,
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, e.g., 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. Wflliams (Universiy of Wisconsin)
described four specially constructed A-@36
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 lp parti-
cles/ml.~R. Davis. J. Cameron and K. Strllhl
(Stanford University) found that X 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 exDreSSed in E. coli. nartiallv comnensat-
ing for deficiencies in the hi&line pathway
or in poZA or Zig functions. These investl-
gators surveyed over 1000 strains of E. cdi
isolated in the natural environment and
did not find a sinale strain that could SUD-
port propagation of the Xvii' vector.    -

V. Bode (Kansas State University) dls-

cussed the possibility of growing tall-free
X heads. Such heads, which are packed with
DNA, are very fragile, unless stored in 0.1 M
putrescine buffer. Head yields close to lw:ml
could easily be attained and, when required,
heads could be quantitatively rejoined with
separately supplied tails under special lab-
oratorv conditions. W. Arber. D. Scandella
and J."Elliott (University of B&l) 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 forelgn
DNA.

K. Matsubara, T. Mukai and Y. Takagi
(University of Osaka and Kyushu Unlver-

sity ) 9  and 0. Hobom and P. Philllppsen
(University of Frelburg and Stanford Univer-
sity) described various defective A plasmids
(Xdv) t.hat could be used as efeclent vectors
Matsubara has shown that temperature-
sensitive cro mutations permit obtaining be-
tween 1000 and 3000 cloned molecules ner
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. Phlllippsen described
many new Adv plasmlds created by cutblng
X DNA with HindIII and Barn1 restriction
endonucleases followed by ligation. The final
talk by F. Young, Cl. Wilson and M. Williams
(University of Rochester) summarized the
progress  on the development of safer
BaciElus subtilis host mutants and Dhaees.
especially +3, as vectors. New res&lct&
nucleeses, Bgl-1 and Bgl-2, were also de-
scribed.

The morning session on bacteriophage
vectors wss followed by a session on plasmid
vectors that was chaired bv D. Helinski (Uni-
versity of California, S& Diego). He&ski
presented the following properties as highly
desirable characteristics of a safer plasmid
vehicle:  (a) Non-conjugative; (b) non-
mobilizable or poorly moblllzable by a con-
jugative plasmtd: (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 fllniversitv
of California, San Diego) described the prop-
erties of a variety of derivatives of the ColEl
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. culi 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-ColEl plasmld and
derivatives of this plasmid (mini-ColEl-kan
and mini-ColEl-trp) as cloning vehicles.
Finally, she described the temperature-sensl-
tivity properties of trp and km derivatives
of a temperature-sensitive replication mu-
tant of ColEl isolated by J. Collins (Moleeu-
lar Biology Institute, Stockhelm) and hybrid
ColEl plasmids carrying the EcoRI generated
Cts fragment of bacteriophage X-trp61.

J. Carbon (University of California. Santa
Barbara) described a replica plating method
that greatly facilitates the detection of E. coli
clones bearing ColEl plasmids. The proce-
dure, which utilizes the F, plasmid to pro-
mote the transfer of a hybrid ColEl plasmid
to a suitable auxotrophic recipient, was suc-
cessful in identifying clones bearing hybrid
plasmids carrying a number of different re-
gions of the E. 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-coniuestive
pls.sn&ls carrying foreign DNA of a poten-
tially hazardous nature. Clark described the
variations in transmission frequencies be-
tween the nonconjugative plasmids pSC101,
pML31, pSC138 and a number of pSClO1 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 Illinois) and A. Chakrabarty (General
Electric Research and Development Center)

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


NOTICES

described the properties of a variety of plas-
mids isolated from Pseudomonas putida.
These contributions were followed by a dis-
cussion on the merits of developing plasmid-
host systems involving Pseudomonas strains
that naturally exhibit unusual growth re-
quirem.?nt.s. Similar studies with plasmids
Isolated from Bacillz~ megaterium by 33.
Carlton (University of Georgia) from B. sub-
tilis by P. Iovett (University of Maryland)
am  other naturally occurring  BCU?iZZus
species by W. Goebel and K. Bernhard
(Microbiology Institute, Wurzburg) were dfs-
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 plasmlds described
show considerable promise as plasmid cloning
systems involving a host other Bhan 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 Coniam-
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 currentlv available in-
__--- .- ~~
formation on the ecology and epidemiology
of E. coli and related bacterial species Since
it was recognised that E. coli K-12 would be
the prokaryotic host most commonly em-
ployed in the cloning of DNA molecules in
the immediate future. F. (Drskov (Escherichia
Reference Center, Copenhagen) reviewed the
sPate of E. cold serotyping and what has been
learned about the distribution of E. coli types
in health and disease. Only certain E. COZi
types a,re generally recognized as good colo-
nizers of the human aut and such strains
come from a handful 2 the 160 well defined
0 (lipopolysaccharide) antigen types and in-
variably possess K (acidic polysaccharide
capsule) antigens. Some serotypee 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 0 or K antigens and is
considered to be rough. This may account,
CLt least in part, for ite demonstrate poor
ahilitv to colonize the human or animal aut.
However, R. Freter (University of Michigan)
pointed out that we still remain largely
ignorant of the factors which control in-
testinal E. coli populations. Freter also noted
that while adherence to the mucosal surface
of the small lnteetine is imoortant in the
pathogenesls of E. coli diarrhkal disease, the
`normal' long-lasting symbiotic relationship
between a mammalian host and bacterium is
established in the cecum and colon. It is in
these locations that factors come into play to
determine whether an E. ooZ4 strain passing
through the intestine will become success-
fully implanted or whether it will be quickly
eliminated in the feoes. 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. colt
previously grown under usual laboratory
conditions fare poorly while cells of the same
strain `me-adauted' in Kh. nH. etc.. often
colonize^ well. %reter has deieloped .a con-
tinuous flow culture model which may be
useful in studylng the mechanisms of im-
plantation. Falkow reviewed the pathogenic-
ltv of B. col. E. co24 Causes diarrheal disease

either by direct invasion of the bowel epl-
thelium or by elaboration of enterotoxln(s) .
While invasive 8. coli appear to owe their
pathogenicity to a constellation of at least

five unlinked chromosomal gene clusters,
toxlgenic E. coli species generally owe their
pathogenicity to the  possess lon  of two
aaecies. Ent and K. The introduction of Ent
aid K `plasmids may be sufficient to convert
a normal wild-type E. coli into a strain now
capable of causing overt clinical disease.
However, the introduction of these plaemids
into E. coZi K-12 sublinas had no discernible
effect on their ability to cause disease, al-
though the K-12 strains could now better
colonire calves. Despite the observat.ion that
E. coli K--l2 did not appear to offer a signifl-
rant hazard as a potential enteric pathogen
even when it possessed well-defined deter-
minant 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 vioo and still posed a significant
problem requiring control. E. Geldreich (U.S.
Enviromnent,al Protection Agency, Cincin-
nat,i. Ohio) discussed the possible outcomes
of the release of E. 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-puriilcation capacity of re-
ceiving waters to limit potential hazards.
While these are realistic barriers to the dis-
semination of E. co2i and associated fecal
organisms via the water route, they are not
infallible because of technological limita-
tions, improper operational practices an's
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 E. coli K-12 ss 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-
jugate with E. coli, the potential dissemina-
tion of recombinant DNA molecule includes a
greater spectrum of mimoorganisms 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
ecoloaical niches: (bl nreclude cell wall bio-
synthesis except .in spkcially 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 B. coli K-12,
although F. Young described a B. s-ubtilis
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
transf matlon,  however,  and therefore
might Tie useful only with a phage vector
which has yet to be developed and/or dis-
covered.

A. I. Bukhari (Cold Spring Harbor Labora-
torv) described the use of the daaD8 mum-
tlon'in E. calf K-12 to block celi wall blo-
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-a to 10-O.

Tbr mutation conferring bile salts 6ensivit.y
was obtained after Mu-l infection of an Hfr
strain and, although exhibiting the theoret-
ioallv useful nronerties of ease of DNA hola-
tion"and inadilit'g to survive in the intestinal
tract, might be due to Mu insertion which
would compromise its use for safe strain
tonstruct10a

Curtis6 reported on the work performed by
him and his coworkers in constructing and
`%?stlng numerous strains with different mu-
tations, Survival of strains in vice was tested
by feeding rats 10'0 cells in milk by stomach
tube. Spur mutations did not reduce strain
t,it.ers m feces whereas AthyA: AthyA drm,
and Ltf1y.4 dra mutations gave lOa-fold, 109-
fold and Iw-fold reductions, respectively, in
strain t.iters in feces. Strains with AthyA
mutations also exhibited thymineless death
in in cifro tests. Since strains with dapD8
s!lele can revert to Dap+, strains were con-
structed with both dupD8 and AbioH-usd
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 diaminopimellc acid
but containing NaCl and 0.6% usable car-
bon ljources. This survival was due to the
production of the mucopolysaccharlde, col-
anic acid, which permits many of the cells
to grow and survive as spheroplasts. A Agal-
c?L~' mutation (also deletes ktt, bio and
uurB aeileSl w&5 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 AbioZf-asd AgaZ-
chZ st,rains are more readily lysed, trans-
form at higher freauencies and are coniu-
gation-defective in- matings with donors
possessing conjugative plasmids in the
P, W and 0 incompatability groups but
Con+ as recipients for F, I and T group
nlasmids when COmDared to the da,w+ oaz*
parent strain. -
        Strains with endA `m<Ce.-
tions were also observed to exhibit in-
creased transformation frequencies. Attempts
to introduce temperature-sensitive pold
alleles into st,rains to block replication
of ColEl cloning vectors at elevated temper-
atures and to cause DNA degradation at ele-
vated temperatures in the presence of recA
and AthyA alleles often do not have the same
properties in the constructed strains as in
the strains in which the allele was origlnal-
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
microoreanisms. 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 SuII+
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 AtZzy.4 and dna mutations into
their daeD8 AbioH-asd Aoal-chl'-u.L;rB h.?it

naZAr (for esse in monitoring) Su+ ir Q80r
strain to accomplish this objective.

The final session involved a general dls-
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 phsge-host systems have
been developed that should meet the criteria
of an EK2 system specified by the National
Institutes of Health guidelines for resea.r&
on recombinant DNA molecules. Additional
testing is required to conArm the EK2 prop-
erties of these available systems, but it is
anticipated that these vector-host svstems
will meet thase tests.

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


NOTICES

38163

Dr. Donald R. Helinski, University of Cali-
fornia, San Diego.
Dr. Stanley Falkow, University of Washing-
ton.

or. Roy Curt&s III, University of Alabama.
Dr. Waclaw Ssybalski. University of WiSCOn-
sin.

APPENDIX D TO
APPENDIX D

SUPPLEMENrARY INFORMATION ON PHYSICAL
         CONTAINMENT

CONTENTS

I. Biological safety cabinets.
Table I.

II. Universal biohazard warning symbol.
III. Laboratory techniques for biohazard
    control.
  A. Pipetting.
  B. Syringes and needles.
  C. Opening culture plates, tubes, bot-
     tles, and ampoules.
  D. Centrifuging.
  E. High-speed centrifuges.
  F. Blenders, ultrasonic disintegrators,
     colloid mills, ball mills, jet mill%
     grinders, mortar and pestle.
  G. Miscellaneous precautions and rec-
      ommendations.

N. Personal hygiene, habits, and practices.
V. Care and use of laboratory animals.
  A. Care and handling.
  B. Cages housing infected animals.
  Cl. General guidelines that apply to
      animal room maintenance.
  D. Necropsy rules for infected animals.
VI. Decontamination and disposal.
  A. Introduction.

B. Decontamination methods,
C. Laboratory spills.
D. Disposal.

E. Characteristics of chemical decon-
   tamlnants in common use in labo-
  ratory operations.
F. Properties of some common decon-
   taminants.

G. Vapors and gases.
H. Residual action of decontaminanta
I. Selecting chemical decontaminants
   for research on recombinant DNA
   molecules.
Table II.

VII. Housekeep%g.
   A. Introduction.
   B. Floor care.
  C. Dry sweeping.

   D. Vacuum cleaning.
   E. Selection of a cleaning solution.
   F. Wet mopping-two-bucket method.
   G. Alternative floor cleaning method
      for animal care areas and are88
       with monolithic floors.
VIII. Clean-up of blohazardous spills,
  A. Biohazardous spill in a biological
      safety cabinet.

B. Biohazard spill outside a biological
  safety cabinet.

  C. Radioactive biohazard spill outside a
     biological safety cabinet.
IX. A secondary reservoir and flltration
   apparatus for vacuum systems.
X. Packaging and shipping.
  A. Introduction.
  B. Packaging of recombinant DNA
      matfrials.

C. Labeling of packages containing re-
  combinant DNA m&&&.
D. Additional ahipping requirementi
  and IimItations for recomblnan$
   DNA materials.

Table III.
Table N.

XI. Training aids, materials and courses.
  A. Slide-tape cassettes.
   B. Films.
   C. Courses.

XII. Outline of a safety and operation

manual for a P4 facility.
References.

I. BIOLOGICAL 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-
ohere. 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: fil With an eight-inch hieh.
full-width open` front; (ii) with an lnstalied
front closure panel (having four, eight-inch
diameter openings) without gloves; and (iii)
with an installed front closure panel
equipped with arm length rubber gloves. See
Table I for ventilation reauirements. agent

I

use limitations, and minimum performance
requirements.

2. Class II. A ventilated cabinet for per-
sonnel and product protection having an
onen front with inward air flow for ner-
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

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 a toxic,
radioactive, or  flammability  standpoint
should not be used in this cabinet because of
the high quantity of recirculated air.

(ii) 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 aastiaht construction nrovidine total
protecti& for personnel and* produci 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 (i.e.. 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.

                                                            c
a&ax           El-P3 l-3        n     00   300     Not opplfcablc   99.971    .z
                                                            Q
                                                            E
                                                            P
Pass XI, * 1      h-P3  l-3        n     a60 436     CM tfght#            J
                                Ied-gate c    99.973
                                            1x10 cc/M          x

                                            at a-q prassun      z

fEDERA REGISTER, VOL 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976


38466                              NOTICES

The biological hazard warning symbol
(biohazard symbol) specified herein shall be
used to signify the actual or potential pres-
ence of a biohazard 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:

!

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
afaxed, provided the proportions shown
above are maintained. and. in anv case, that
the symbol can be eaiily seen from as many
directions as possible.
Except, when circumstances do not permit,
the symbol shall be oriented with one df 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
oolor is optional as long as there is sufficient
contrast for the symbol to be clearly defined.

The biohazard symbol shall be used or
displayed only to signify the actual or
potentlti presence of biological hazard.

Appropriate wording may be used ln es-
sociatlon with the symbol to indicate the
nature or identity of the haeard. name of
individual resDonsible for its control. Dr&
cautionary i&x-m&ion, etc., but -never
should this information be superimposed on
the symbol. (See next page)

., -,

A. Pipetting. 1. No infectious or tOXiC ma-
terials should be Dinetted bv mouth (2.3.4).

2. No Infection's -mixtures should `be tire-
pared by bubbling expiratory air through a
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 nioettes should be DlaCed
horizontally In a p&i containing enough
suitable disinfectant to allow complete lm-
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 shodld not be mixed
by alternate suction and expulsion through
a pipette (2,3,4).
8. Mark-to-mark pipettes are preferable
to other types, es 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, aeros
production or spills, avoid unnecessary use of
the svringe and needle. For instance:
(i) -Use-the needle for parenteral injec-
tions but use a blunt needle or a cannula on
the syringe for oral or int;anasal inocula-

tions.

(ii) Do not use a syrir.ge and needle as a
substitute for a pipette In making dilutions

of dangerous fluids.

2. Use the syringe and needle in a Blologl-
cal Safety Cabinet only and avoid quick snd
unnecessary movememt3 of the hand holding
the syringe.

3. Examine .$z:s syr:n;es for clilps and
cr<acks, and needles for barbs and plugs.

NOTE: This should be done prior to stcri!-
ixation before use.

4. Use needle-!ockl::g cLLuer-Lok R trnc)
                           . L
syringes on:g, and be bui'e That rhe needie is
locked securely into tine barrel. A disposable
syringe-needle unit (where the needle is an
integral part of the unit1 is preierred.

5. Wear surgical or orher rj-pe rubber gloves
for all nlanipulntions v:i!h needles and
syringes.

6. Fill the s>-ringe carefully to ulinimiLe
air bubbles and frothing of the inoculum.

7. Expel excess air, liquid and bubbles
from a syringe vert,ically into a cotton pledget
moistened with the proper disinfectant, or
into a small bottle of sterile cot.ton.

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 fs 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 pledget mois-
tened with the proper disinfect.ant. 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 u?lexpectz.i 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 sor:ing problem in the
service area.

16. Do not, discard syringes and needles
into pans containing pipettes or other glass-
%-are that must be sorted out from the
syringes and needles.

C. Opening Culture Plates, Tubes, Bottles.
and AmpouZes. 1. Plates. t.ubes and bottles of
fungi may release spores in large nur hers
when opened. Such cultures should be ma-
nipulated in a B!o!ogical Safety Cabinet
(6,15).

2. In the abse:m? of defini:e accidents or
obvious spillage, it is not certain that open-
ing of plates, tubes and bottles of other
microorganisms has caused laboratory ln-
fection. However, it !s 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 syner&s In petrl dish cul-
tures is usually infected and forms e film
between the rim and lid of the inverted

FRDERAL REGISTER, VOL. 41, NO. 176THURSDAY, SEPTEMBER 9, 1976


NOTICES                            3SiG7

plate. Aerosols are dispersed w-hen this film
is broken by opening the plate. Vented pIas-
tic petri dishes where the lid touches the
rim at only three points 8re 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 8fter removal from 8n 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
1 Biological Safety Cabinet (8).
  5. Leas obvious is the release of aerosols
when screw-capped bottles or plugged tubes
are opened. This happens when 8 film of ln-
fected liquid which may collect between the
rim and the liner is broken during removal
of the closure (8).

6. Dried. infected culture material mav
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 liquid is broken after nicking with 8 file,
the snapping action creates aerosols. The
following methods have been recommended:
(i) After nicking the ampoule with 8 fiIe,
wr8p the ampoule in disinfectant-wetted
cotton before breaking. Wear gloves (2).
(ii) The bottom of the amnoule should be

held'ln several layers of tlssde 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 8 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 cm3
of broth should be added slowly to avoid
blowing dried materi8l out. The contents
may then be mixed without bubbling 8nd
withdrawn into 8 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 8 bubble. After
allowing this to cool, he breaks the bubble
while holding it in 8 large low temperature
flame: this immediately incinerates any in-
fectious dust which may come from the am-
poule when the glass is broken (16). Prellm-
inary practice with a slmulant ampoule of
the same type actually in use is necessary to
develop 8 technique that will not cause ex-
plosion of the ampoule.

(iv) A simple device has been recommend-
ed consistinn 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 saf&y centrifuge cup (3,7,8.14,22)
may be used to house or safeguard all cen-
trifuging of infectloua substances. When
bench type centrifuges are used in 8 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-
inet (z&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 lnsoect the innside of the trunnlon
cup fdr ro&h walls caused by erosion or ad-
hering matter, and csrefully remove bits of
glass from the rubber cushion (4,lO).

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,lO).

5. Avoid decanting centrifuge tubes. If
you must do so, afterwards wipe off the outer
rim with 8 disinfectant; otherwise the ln-
fectious fluid will spin off 8s an aerosol
(4, 10).
. 6. Avoid filling the tube to t,he 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 8 plug-in closure and the
rim of the tube. Even screw-capped bottles
are not without risk, however; if the Cim 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 mech8nlcally unsound (8).

8. Kitchen foil is often used to cap centri-
fuge tubes. This creates more risk then the
screw cap. Boll caps often become detached
in handling and centrifuging (8).

9. The balancing of buckets is often mis-
managed. C&xre 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
st.ains can be aDDlied 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 eouidistant from the axis of rota-
tion. To lllistrate 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
t.runnion cups in 8 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 8 bre8kage or
accidental dispersion of infected particles the
pump 8nd the oil in it will become con-
tamin8ted. A high efficiency 5lter should be
placed between the centrifuge and the
p-p 03).

2. High speed rotor heads are prone to
metal fatigue, and where there ia a chance
that they asy be used on i&n-e than one
machine each rotor should be accompanied
by its own log book lndloatlng the number of
hours run at top or de-rated speeds. Failure
to observe this precaution can result in
dangerous and expensive dlsintegr8tion. Fre-
quent inspection, cleaning snd 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 dlf-
ferent mate&& are provided (e.g., celluloid,
polypropylene, stainless steel), care must be
taken th8t the tibe closures designed
specifio8lly for the type of tube in use are
employed. These caps 8re often similar in
appearance, but are prone to leakage if ap-
plied to tubes of the wrong material. When
properly designed tubes r+nd rotors 8re well
maintained and handled. leakine should
never occur (6) .

3. Cleaning and disinfection of tubes,
rotors and other components requires con-

siderable care. It is unfortunate that no
single process is suitable for all items, and
the various manufacturers' recommendations
must be followed meticulouslv if fatigue.
distortion and corrosion are td 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 inilammable and prone to
shrinkage with age and distortion on boiling.
but can behave 8s high explosive in an auto-
clave (5) Large-scale sonal centrifugalion
requires special attention (11).

F.  Blenders,  ultrasonic disintegrators.
colbid mills, ball milLs, jet mills, grinders,
motar and pestk. All these devices release
considerable aerosols during their operation.
For m8ximum protection to the operator
durine the blending of infectious materials.
the f:llowing practices should be observed:
1. Operate blending and cell-disruption
and grinding equipment in a Biological
Safety Cabinet (9 )

2. Use ssfetv blenders desianed to orevent
leakage from the rotor bearing at the-bottom
of the bowl (9).

3. In the absence of 8 leak-proof rotor, in-
spect the rotor bearing at the bottom of the
blender bowl for leakage orlor to oneration.
Test it in 8 prelimin&y run with sterile
aallne or methylene blue solution prior to
use with infected material (9).

4. Sterilize the device and residual lnfec-
tlous contents promptly after use. Use *
towel moistened with disinfectant over the
top of the blender (9).

5. Class 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 (Enalsnd) .  in which material Is

homogeni`zed-in a piastlc bag in a closed con-
tainer, would appear to be safer than some
of the other blenders (8).
7. A heat-sealed flexible mastic film en-

closure for 8 grinder or blender can be used,
but It must be opened in 8 Biological Safety
Cabinet (7).

8. Blender bowls sometimes require sup-
plemental cooling to prevent destruction of
the bearings and to minimize thermal effort.s
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 autaanalyzer stirrer, and the mico-
tonometer, inasmuch as it Ferns that air-
borne transmission of infectious heoatitis

may occur in the laboratory (20).

G. Miscellaneous precauticms and recom-
mendations. 1. Water baths and Warburg
baths used to inactivate, incubate, or test ln-
fectious substances should contain a disin-
fectant. For cold water baths, 70 percent
propylene glycol is recomemnded (4, 10).

2. Deepfreeze, liquid nitrogen, and dry Ice
chests and refrigerators should be checked
and cleaned out periodically to remove any
broken ampoules, tubes, etc., containing in-
fectious material, and decontaminated. Use
rubber gloves and respiratory protection dur-
ing this cleaning. All infectious or toxic
material stored in refrigerators or deep-
freezes should be properly labelled. Security
measures should be commensurate with the
hszards (4,10,21).

3. Freeze-dried culture ampoules should al-
ways be opened in 8 Biological Safety Gabl-
net. The ampoule should be wrapped in 8
disinfectant-soaked swab before bresklne it
open to minimize the risk of cutting ihe
hands, and to a lesser extent of releasing

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


8841%

NOTICES

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 eilluent air from the vacuum
pump is desirable either up (preferably), or
dwcn stream of the pump (5).

4. Ensure that all virulent fluid culttires or
viab!e 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 of the glass vessel (4,lO).
5. All inoculated uetri elates or other ln-

oculated solid media sho;ld be transported
and incubated in leak-proof pans or leak-
proof containers (4,lO).

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 ot!ler 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
carefullv for notential breakage of flasks or
other &ntain&s being shaken:Screw Capped
durable plastic or heavy walled glass flasks
should be used. These should be securely
fastened to the shaker platform. An addl-
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 er-
tremely hazardous operation (4,lO).

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 lees obvious ex-
tent, they can raise the quality of the Iab-
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, ampiification will permit
a better review of the applicability to any
one specific laboratory.

Consenuentlv. 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, IO).

2. Foot-operated drinking fountains should
bc 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 area.e
(5, 10,26).
4. Shaving and brushing of iceth are net
permitted in the laborat.ory. Pazors, 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 t!:orzKsh washing
of the face and bands ( 27).

5. A beard may be undesirable in the lab-
oratory in the presence of actual or potential
airborne contamination, because it retains
particulate contamination more persistently
than clean-shaven skin. A clean-shaven face
$s essential to the adequate facial fit of a

face mask cr respirator when the work re-
quires respiratory protection (10,27,31).

6. Develop the habit of keeping hands
away from mout.h. no% eyes. face, and hair.
This may prevent self-inoculation (10.27).

7. For product pratcction. prrxms with
loxg hair should wear a suitable hair net
or head cover that c.;n bc decontaminated.
Tbis has long beeii a r-~:~iiremcnt in lies-
pita1 operating room% and in t::e mnnufnc-
ture of biologicsl l:hnrnace:!tical producm.
A head cover also ni!! l%rotect the hair from
fluid splashes, from YV. iugi:ig into Bunsen
flames and petri dishes. and xv-i!1 reduce facial
contamination caused by ha!,itual repetitive
manual adjustment of the hair (5).

8. ILvl~`-flowin~
      7     _ hnir xiii ioose-iisi,piilg
clothing are dnc~oroui in the pres?n!?o of
open flnme or moving machinsry. Rings and
wrist watches also are a mechnnical haenrd
during operation of some types of machines
(5, 10).
9. Contnzt :enses do not pr"sizle eye protec-
tion, The capillary spsce between the con-
&act 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 h displaced, muscle spasms ~-ill
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 resaotiSibilitv of suuervisow t0
identify employebs 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 as much as possible (10,27).
13. When change rooms with showers are
provided, the employer should furnish skin
lotion (27).

14. When employees are subJect to poten-
tlal occupational infection, the shower and/
or faceihand-washing facilities should be
provided with germicidal soap (8,27).
15. Personal cloth handkerchiefs should
not be used in the laboratory. Cleansing tls-
sue should be available instead.
16. Hand n-ashing for persomtl protec-

tion :

(i) This should be done promptly after
removing protective gloves. Tests show it i.e
not unusual for microbial or chemical con-
tamination to be present despite use of
gloves, due to unrecognized small holes,
abrasion.% tears, or entry at the a-rlst.

(ii) Throughout the dsy, at intervals
dictated br the nature of the worl:. the
hands sho;d be washed. Pre:+nce cf a~wrist
watch discourages adequsto vxhing of the
wrist (1025).

(iii) Hands should be washed after re-
moving soiled protective clothing, before
leaving the laboratory area, before eating,
and before smoking. The provision of hand
cream by the employer encourages these pr`ac-
tices (5.8.19).

(iv) A disinfectsnt xvaslsh cr dip msy be
desirable in some cases, but Its uce must not
be carried to the point of causing roughen-
ing, desiccation or selwitizat:z,n of the skin
17. Anyone -wlt,h a fresh or healing cut.
abrasion, or skin lesion should no7; work with
infective material unless t.he injured area is
completely protected ! 6.25).

13. Persons vac,nilis ted for smallpox may be
shedders of vaccinia vu'us during the phase
of cutaneous reaction. Therefore, vaccination
requires permi,&on of the appropriate super-
visor, because t-v:o u-eeks' absence may be
necessary before returxing to work v;ith nor-
mal cell cul?urcs or with susceptible animals,
especially the normal mouse colony (25).

19. The surgeon's mask of gauze or filter
paper is of little value for personal respira-
tory protection 129). It is designed to prevent
escape of droplets from the nose or mouth
(23G). I:  biohazards demand respiratory
protection, then nothing but a full face res-
pirnbr or ventilated hood will suffice. A half-
mtik respirator does not protect the eyes,
which are an unevaluated avenue of ixfec-
tion through the conjuctiva and the naso-
lacrimal duct (5.8)

20. Nompecific cont.aminstion by environ-
mental organisms from humans, animals,
equipment, containers for specimens or 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,OOO 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.

(ii) 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 u.sed
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 accomulished bv
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 occilrred 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 contaminatton (9)
(ii!) Prolific dispersal of bacteria occurs

from infected abrasions. small pustules,
bolis, and skin disease (23F, 24B). Wa.sh!ng
the lesions with aermicidal sono will sreatlv
decrease the number of orcar;isnu on the
skin and dispersal into the a&. Healthy nasal
carriers who gem=rate aernsoli7ed staphp-
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 source-of
staphylococcal contamination of ceil ii:ies.

(iv) Footwear. In moderate and high risk
situations, shoes reserved for only laboratory
use have been recommended as a preeau-
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 mi~rnbial load brought into the

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


NOTICES

38469

laboratory each day by street shoes. Shoes
are efllcient transporters. In one study, there
were 4 to 850 times as many bacteria per
squars centimeter on the laboratory foot-
wear 88 on the floor itself (30).

A. Care and handling. 1. Special attenbon
must be given to the humane treatment of
all laboraturv anfmat in accordance with the
Animal Welfire Act ti 19'70. The implement-
ing rules and regulations appear in the Code
of Federal Regulations (UFR) Title 9, chap-
ter I, Subchapter A, Parts 1, 2,o 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 fu`e specific minimum requlre-
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
coap; yf 293CFR Chapter I, Subchapter A,
    . . .
3. Each laboratory should establish proce-
dures to ensure the use of animals that are
frse of diseases prefudicial to the proposed
experiments and free from carriers of disease
or vectors. such as sctoparasites, which en-
danger other experimental animals or per-
sonnel (10).

B. Cages housing infected animaLs (lo).
1. Careful handling procedures should be
employed to minimi= 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 sterilira-
tion.

3. All watering devices should be of the
"non-drip" type.

4. Cage5 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 fesd-
1% watering, handling, or removing in-
fected snlmals. Bare hands should NEVER
be placed in the cage ta 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 bs properly restrained to
avoid accidents that might result In dissem-
inating biohasardous material, as well as to
prevent injury to the animal and to per-
sonnel.

`7. Animals exposed to biohasardous aerosols
should be housed in ventilated cages, in gas-
tight cabinet systems, or in rooms designed
for protection of personnel by use of ventl-
let&d suits.

8. Animals inoculated by means other than
by aerosols should bs 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 svi-
dence indicates that infectious hepatitis may
be transmitted from non-human primates
(typically chimpanzees) to man. Newiy 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

for each newly imported animal. A sign
should be posted at rooms housing these ani-
mals to warn that the animals are potentially
infectious.

C. General Guidelines that Apply to Anlmal
Room Maintenance (10). 1. Doors to animal
rooms should be kept closed at all times BX-
cept for necessary entrance and exit.
2. Unauthorized persons should not be per-
mitted to enter animal rooms.
3. A container of disinfectant should be
kept in each animal room for dlsinfe&ng
gloves and hands, and for general decon-
tamination, even though no infectious ani-
mals are present. Hands, floors, walls, and
cage racks should be washed with an ap-
proved disinfectant at the recommended
strength as @equently as the supervisor
directs.

4. Floor drains in animal rooms, as wsli
as floor drains throughout the building
should be flooded with water or disinfectant
periodically to prevent backup of sewer gases-
5. Shavines 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 storaee areas.

7. Special care should 6;? taken ta prevent
live animals, especially mice, from finding
their way into disposable trash.
D. Necromu rules for fnfected animals
(10). 1. Ne&opsy of i&ct.&i `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 necropaies.
3. Rubber cloves should be worn when ner-

forming ne&psiea               .

4. The fur of the animal should be wetted

with a suitabls 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 biohasardous 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 suie
able germicide.

9. 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. DECONTAMlNATION *ND DISPOS.4L
         (7. 10. 38-42)
A. Introduction. Available data on the

efllcacy of various decontaminsnts 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 oandidate 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 experl-
mental materials by a variable, persistent,
and unwanted background of microorga-
nisms. This additional factor should be con-
sidered In selecting decontamination mate-
rials and met.hods.

B. Decontaminatfon Methods. Physical and
chemical means of dscontaminatlon fall into
four main categories: Heat: Liquid De&n-
taminants; Vapors and Gases; and W Radia-
UOll.

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 sterility. Dry heat at 160 to 170 C for
periods of 2 to 4 houm is suitable for deatruc-
tion of viable age ts on impermeable non-
     P
organic material uch as glass, but is 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 efllcient means for disposal.

2. Liquid Decontaminunts. In general, the
liquid decontaminants find their most prac-
tical use in surface decontamination and, at
sufacient concentration, as decontaminanta
of liquid 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 stats of dispersion, penetrability
and reactivity of organic material at the site
of application. Small variations in the above
factors may make large ditrerences in effec-
tiveness of decontmination. For this reason.
even when used under highly favorable con:
ditlons, complete reliance should not b-e
placed on liquid decontaminants when the
end result must be sterility.

There are many liquid decontaminants
available under a wide variety of trade
names. In general, these can be categorized
as halogens, acids or alkalies, heavy metal
salts. auaternar7 ammonium comwunds.
phenoli& compoiids, aldehydes. ketones.
alcohols and &mines. Unfortunately, the more
aotive the decontaminant the more likely lt
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 gssee 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-
trolled conditions of temperature and hu-
miditv. excellent decontamination can result.
Vap& and gas decontaminants are primarily
useful in decontaminating: (1) Biological
Safety Cabinets and associated effluent alr-
handling systems and air filters: (ii) bulky
or stationary equipment that resists pentra-
tion by liquid surface dscontaminants; (iii)
instruments and optics that might be dam-
aged by other decontamination methods; and
(iv) rooms and buildinss and associated air-
handling systems. -

4. Radiation. The usefulness of ultraviolet
(W) irradiation as a decontaminant is
limited by its low penetrating power. No ln-
formation is available regarding the effect.lve-
ness of W irradiation for decontaminating
microorganisms  containing   recombinant
DNA molecules. Dependence on W must be
based on the results of experiments imitating
particular anticipated environmental condi-
tions and appllcatlons. Ultraviolet light is
generally of limited application and is`pri-
marlly useful In air locks and animal hold-
ing areas for controlling low levels of air-
borne contaminants.

No one procedure or material will solve a11
decontamination problems. The only method
of assuring the efficacy of selected method-
ologies is to critically examine the results

FEDERAL REGISTER, VOL. 41, N6. 176--THURSDAY, SEPTEMBER 9, 1976


34`m.l

obtained in practical testi with the mlcro-
organism(s) of interest.

C. Laboratiy spills. A troublesome prob-
lem that may occur In the laboratory is the
decontamination of an overt biological spilt
The occurrence of a spill pose5 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 a&
cident, research materials such a5 tissue cul-
tures, media, and annnals 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 ecec-
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 disaosal 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 phrase 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-
ln 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 materials
from laboratories dealing with potentially
infectious microorganisms or animal tissues
are:

1. Hare 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. t0
personnel either: (1) Those carrying out the
immediate disposal procedures or (ii) ThOSe
who might come into contact with the
objects or materisIs outside the laboratory

Gnplex?

Laboratory materials requlrlng disposal
will normally occur as liquid, solid, and
animal room wastes. The volume of these

NOTJCES

can become a major problem when there
ls 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 kind5 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
good5 are delivered, disposable carton-cages
for transport of animals, and large carboys
or tanks of fluids which can be left outside
and drawn from a5 required. Reduction of
this bulk will free autoclaves and other de-
contamination and disposal processes within
the laboratory for the more rapid and effi-
cient handling of materials known to be
contaminated.

Inevitably, disposal of materials raises the
quest.ion. "How can we be sure that the ma-
terials have been treated adequately to as-
sure that their disposal does not constitute
a hazerd?" In the small laboratory, the prob-
lcm is often`solved by requiring that each
investigator decontaminate all contaminated
materinls 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-
uosal 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 dwsiznatinz the state of materials
to be disposed if. ThiYs 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 area5 will be required for
research projects ranging in size from an ln-
dividual researcher to those involving large
numbers of researchers of many disciplines.
Procedures and facilltie5 ti accomplish this
will range from the simplest to the most
elaborate. The primary consideration in any
of these ki to dispel the notlon that labora-
tory wastes can be disposed of in the same
manner and with as little thought &9 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 laboratories studying recombinant
DNA molecules. Examples are combinations
of common flammable solvents, chemical car-
cinogens, radioactive Isotopes, and concen-
trated virus@ 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 decontamf-
nants in common use in laboratory opera-
tions. Every person actively working with
viable mlroorganisms, 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 equlp-
ment. Moreover,  high temperatures and
moisture often damage delicate instruments,
particularly those havihg complex optical
and electronic components.

Chemicals with decontaminant properties
are, for the most part, available as powders,
crystals, and llquld concentrates. These may
be added to tap water ior application cs SW-

face decontamlnants, and M)me, when added
in sufficient quantity, flnd use as decontam-
inants of bulk liquid wastes: Chemical de-
contaminants that are gaseous at room tem-
peratures are useful Ss space-penetrating
decontaminants. Others become gases at rea-
sonably elevated temperatures and can act
as either aaueous surface or easeou5 soace-
penetrating- decontaminants. -

Inactivation of microorganism5 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. Deuendina
upon how these factors are manipulated,
the degree of 5ucce5s achieved with chemi-
cal decontaminants may range from mini-
mal inactivation of target microorganism5
to an indicated sterility within the limits of
sensitivity of the assay systems employed.

There are dozens of contaminants arail-
able under a wide variety of trade names.
In general, these decontsminants can be
classified as halogens, acids or alkalies, heavy
metal salts, quaternary ammonium com-
pounds, phenolic compounds, aldehydes. ke-
tones, a!cohols, and amines. Unfortuatelp.
the more active the decontaminant the
more likely it will posse55 undesirable char-
acterbtics. For example, peracetic acid 15 a
fast-acting, universal decontaminant. How-
ever, in the concentratted state it 1s a hazard-
ous compound that can readily decompose
with explosive violence, When diluted for
use, it has a short half-life, produce5 strong,
pungent, irritating cdors. 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 under&able characteristics.

The halogen5 are probably the second most
active group al decontamlnants. Chlorine,
iodine, 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 hlgh temperature coefa-
cients). The halogens have several undesir-
able features. They readily combine with
protein, 50 that an excess of the halogen
must be used if proteins are present. Also.
the halogem are relatively unstable so that
fresh solutions must be prepared at frequent
fntervals. Finally, the halogens corrode
metals. A number of manufacturera of de-
contaminant5 have treated the halogen5 to
remove some of the undesirable features. For
example, sodium hyp+chlorite reacts with p-
toluenesulfonamlde to form Chloramine T,
and iodine reacts with certain SurfaCe-active
agents to form the popclar iodophors. These
"tamed" halogens are stable, non-toxic,
odorless,  and relatively  ncncorrosive to
metals. However, the halogen5 are highly
reactive elements, and, because they are
reactive they are good germicides. When a
haolgen acts a5 a decontamlnant, fret halo-
gen ls the effect,lve agent. Raking the pH or
combining the halogen with other com-
pounds to decrease the corrosive effect will
also decrease the germicidal power. -4 trade-
off situation occurs.

Ineffectiveness of a decontaminant LY
due primarily to the failure of the de-
contaminant to contact the microorga-
nisms rather than failure of the decon-
taminnnt to act:If one places an item in

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


NOTICES

38471

a liquid decontaminant, one can see that
the item is covered with tiny bubbles.
of course, the area under the bubbles
ls dry, and microorganisms in these drY
areas will not be tiect&by the decon-
taminant. Also, if there are spots Of
grease, rust or dirt on the object, micro-
organisms under these protective coat-
lngs will not be contacted by the decon-
taminant. Scrubbing an item when im-
mersed ln a decontaminant is helpful,
und a decontaminant should have, and
most do have, incorporated surface-
active agents.
F. Propertfes 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 3'7 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-
taminatlng rooms or buildings, but in
the vapor state with water it tends to
polymerize out on surfaces to form para-
formaldehyde, which ls persistent and
unpleasant Formaldehyde gas can be
liberated by heating paraformaldehyde
to depolylplerize it. In the absence of
high moisture content in the air, formal-
dehyde released in the gaseous stats
forms less polymer&d residues on sur-
faces and less time is required to clear
treated areas of fumes than formalde-
hyde released in the vapor state.

4. P7~enoZ. Phenol Itself is not often used
BY n decontaminant. The odor la somewhat
unpleasant and s sticky. gummy residue
remnine on treated surfnces. This is espb
clally true during steam sterilization Al-
though phenol itself may not be in wlde-
spread use., phenol homologs and phenollc
compounds are basic to a number 6f popular
decontamlnant.8. The phenollc compounds
nre effective decontarnlnanta against some
viruses. rlckettslae, fungl and vegetative bat-
terla. The phenollca are not effective in ordl-
nary usage against bacterial spores.
5. Quaternary Ammonium Compound8 or
Quats. After 30 years of testing and use,
there ls still 8 considerable controversy about
the efficacy of the Quats as decontamlnants.
These catlonlc detergents are strongly sur-
face-active and BPB effective against llpld-
containing viruses. The Quata will attach to
protein so thet dilute solutlone of Quata will
quickly lose effectiveness in the presence of
protelm The Quatd tend to clump micro-

orgsnlsms and are neutralized by anlonlc
detereents. such 88 8080. The Qusts have the
advantageS of being n&toxic, odorless, non-
staining, noncorrosive to metals, stable, and
inexpensive.

6. Chlorine. Thb halogen ls a universal
decontamlnant active against all microor-
ganisms. including bacterial spores. Chlorine
combines wlth protein and rapidly decreasea
in concentration in its presence. Free, avsll-
able chlorlue ls an active element. It is a
strong oxldizlng agent, corrosive to metals.
Chlorine  solutions  will gradually lose
strength so that fresh solutions must be pre-
pared frequently. Sodium hypochlorlde ls
usually used as a base for chlorine decon-
tamlnants. An excellent decontamlnant can
be prepared from household or laundry
bleach. These bleaches usually contain 5.25
percent available chlorine or 52,500 ppm. If
one dilutes them 1 to 100, the solution will
contain 525 ppm of available chlorine. and,
lf a nonlonic detergent such 88 Naccanol la
added in a concentration of about 0.7 uer-
cent, a very good decontamlnant ls crer;ted

7. Iodine. The characteristics of chlorine
and iodine are similar. One of the most
popular groups of decontamlnanta used ln
the laboratory ls the iodophors, and Wes-
wdyne is perhaps the most popular. The
range of dllutlon of Wescodyne recommend@
by the manufacturer ls 1 oz. ln 5 gal. of water
glvlng 25 ppm of available lodlne to 3 oz. in
5 gnl. giving 75 ppm. At 75 ppm, the con-
centratlon of free iodine ls .0075 percent. This
em&l 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 dlfficultles may
be experienced lf any appreciable amount of
protein ls present. For bacterial spores. a
dilution of 1 to 40 ~lvlne 750 Darn is recum-
mended by the m&uf&turer-. -For washing
the hands, It ls recommend@ that Wescodyne
be diluted 1 to 10 or 10 percent in 50 percent
ethyl alcohol (a reasonably good -d-n-
tnmhnnt itself I which wlll elve 1.500 rmm of
avsllable iodine: at which c&cent&i& reb-
tlvely rapid lnaotivatlon of any and .sll mlcro-
orgnnisme will occur.

0. Vapor8 and gases. The use of fcumalde-
hyde as a vapor or ges hss already been dls-
cussed. Other chemical decontamlnanta
which have been used thla way Included
ethylene oxide, peracetlc acid. beta-proplolac-
tone (BPL), methyl bromide, and ethylene
amine. When these oan be used in closed
systems and under controlled conditions of
temperature and humidity. excellent deam-
tamln&.lon can be otbalned. Residues from
ethylene oxlde must be removed by aeration;
but otherwise it ls convenient to use.
versatile, and noncorrosive. Paracetic acid h
corrosive for metals and rubber. BPL ln the
vapor form ncte rapidly against bacter&
rlckettslae. and viruses. It has a half-life of
3.5 hours &hen mlxed with water, la easily
neutralized wlth water, and lends itself to
removal by aeration. The National Institutes
of Health does not_ recommend BPL as a
deoontamlnant because it has been identified
s a suspect carcinogen.

H. Residual action of &contaminants. Ae
noted ln the preceding discussion of decon-
tamlnant DroDertles. manv of the chemical
decontamihadts often have residual proper-
ties that may be considered a desirable fea-
ture in terms of aiding ln the control of
background contamlnatlon. One ls cautioned,
however, to consider residual properties care
fully. Ethylene 6xlde used to sterlll%.e labora-
tiry shoes can leave residues which cause
skin irritation. Animal cell cultures, as well
88 viruses of interest, nra also lnhiblted or
lnactlvated by decontamjnants persisting af-
ter routie cleaning procedures. Therefore,
reusable Items that are routinely held ln
liquid decontamlnant prior to sutoclavlng

and cleaning should receive particular atten-
tion in rinse cycles. Slmliarly. during gen-
eral area decontamlnatlon wlth 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 gastlght bags
or by insuring adequate aeration following
decontamination.

I. Selecting chemic&l decontaminanta fM
research on reclombinant DNA molecules. No
single chemical decontamlnant or method
will be effective or practical for all situations
in which decontamination ls required. Selec-
tion of chemical decontamlnants and proce-
dures must be preceded by practical consld-
eration of the purposes for the decontamlna-
tlon and the lnteractlne factors that will ul-
timately determine ho& that purpose ls to
be achieved. Selection of any glven procedure
wlU be lnliuenced by the information derived
from answers-to the following questions:

1. What is the tareet mlcrooreanlsm~s~ ?
2. What decont&lnanta in ihat form are

known to. or can be expected to, lnnctivate
the target microorganism(s) 7
3. What degree of inactivation ls required?
4. In what menstruum ls the mlcroorga-

nlsm suspended; i.e., simple or complex, on
solid or porous surfacea, and/or airborne?

5. mat ls the highest concentration o!
cells anticipated to be encountered?

6. Can the decontamlnant either aa an
squeous solution. a vapor, or * gas reason-
ably be expected to contact the mlcroorga-
nlsms, 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 avallablllty of an effective
concentration of the decontamlnant or wlll
auf&lent time be available for preparation
of the worklng concentration shortly before
its anticipated use?

The primary target of decontamination ln
the lnfectlous disease laboratory is the
microorganism under active lnvestlgatlon.
Laboratory  preparations or   infectious
agents usually have titers grossly ln excess
of those normally observed ln nature. The
decontamlnatlon of theea h&h-titer ma-
terlals presents cer%ln problems. Malnte-
nnnce systems for bacteria or vlrusea rue
speclflcally selected to preserve viability of
the agent Agsr, protelnacwue nutrients,
and cellular materials can be extremely ef-
fective lm physically retarding or chemically
blndlng active moletles of chemical decon-
tamlnants. Such lnterferencea with the de-
sired action of decontamlnanta may requlre
the use of decontamlnant concentrations
and co&act tlmea in excess 02 those shown
to be effective in the teat tube. SlmiMulv. a
major portion of  d-tamlnan t wnkct
time required to achieve a given level of
agent inactivation may be expended ln in-
actlvatlng a relatively small number of the
more resistant members of the population.
The current state of the fut provides llttle
information on wblch to predict the prob-
able virulence of these survivors. These
problems are, however, common to all po-
tentially psthogenlc agents and must alwtrya
be considered ln selecting decontamlnanta
and procedures for thelr use.

Microorganisms exhibit a range of reslst-
ante to chemical decontamlnants. In terms
of practical decontamlnatlon. most vegeta-
tive bacteria, fungi and llpld-containing vl-
ruses, are relatively susceptible to chemical
decontamination. The non-lipid-containing
viruses and bacteria with a waxy coating
such sa tubercle bacillus occupy a mld-range
of resistance. Spore forms are the most re-
slstnnt.

A dewntamlnant selected on the basis of
its effectiveness against mlcroorgnnlsms on"
any range of the reslstanoe scale will be ef-

FEDERAL REGISTER, VOL 41, NO. 17&THURSDAY, SEPTEMBER 9, 1976


38472

NOTICES

fective 8gainst microorganisms lower on the
scale. Therefore, lf decontamlnants that ef-
fectively control spore forms 8re selected for
routine laboratory decontamiaation, lt c8n
be assumed that any other microorganisms
generated by laboratory operations, even in
high concentrations, would also be lnacti-
vated.

An additional area that must be considcrcd
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-
dlzers. strong acids and bases, and either
gaseous or aqueous formaldehyde should re-
act readily with nucleic acids. Their ability

-.

to destroy the nucleic acid being studied,
however, should be confirmed in the experl-
menter's laboratory. Because of inns& dlf-
ferences in the chemlstrv of RNA end DNA
the effectiveness of 8 decbntamlnant 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-
droxvl erouo in the 2' nositlon. whereas DNA
mole&yes dre not susceptible.to mild alks-
line hydrolysis.
Table II summrtrizes 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-
mend&ions of manufacturers of proprietary

VII. HOUSEKEEPniG

A. Introduction. Well-defined housekeep-
ing procedures and schedule8 are essential
in reducing the risks of working with etio-
logic agents and in protecting the integrity
of the research program. This is p8rticul8rly
true in the biological Laboratory operating
under less than totsl containment concepts
mui in all are&s used for the housing of 8ni-
m8ls. whether or not they have been inten-
tiormlly infected. A well-conceived 8nd well-
executed housekeeping program 1imlt.a physi-
Cal clutter that could distract the attention
8nd interfere with the activities of labora-
tory personnel at 8 critical moment in 8 po-
tentlslly h8z8rdous procedure, provide8 a
work are8 th8t will not in itself be 8 source
of physical ln]ury or contamination, and pro-
vides 8n 8re8 that promotes the efllclent use
of decontamlnants-in the event of the In-
sdvertent release of 8 harmful egent. Less
immediately evident are the benefits of es-
tablishing. among personnel of widely vary-
ing levels of education. an appreciation of
the nature 8nd sources of biological con-
tamination.

Housekeeping is an omnibus term that can
be interpreted 8s broadly or 8s narrowly 88
one choose8 It c.8n be seen that many of
the procedures found under special headings,
such ss decont8minatlon, disposal, and ani-
mal care. are. in reality. specific instructions
for safely accomplishing otherwise routine
housekeeping chores. In these safety sug-
gestions for resesrch on recombinant DNA
molecules, It has been elected Ito address
8pecl5c8lly only t88ks of 8 janitorl8l nature
under the subject of housekeeping.

products are suggested. It has been assumed
that micoorganlsms will be afforded 8 high
degree of potential protection by organic
menstruums. It has not been assumed that
8 sterile state will result from application of
the indicated concentrations and contact
times. It should be emphasized that these
data 8re 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
d&advantages 8s well 8s 8 range bf potentla!
for inactivation of 8 diverse mlcroflor. Eausl-
ly evident is the need for compromise &. an
8lternative to maintaining 8 veritable "drug
store" of decontaminants.

-

-          -.
-.-.-am

The objectives of*housekeeping In the blo-
logical 18bor8tory 8re to:

1. Provide 84 orderly work 8re8 conducive
to the  8ccompllshment of the research
pmgrsm.
2. Provide work 8re8s devoid of physical
hazsrds.

3. Provide 8 clean work are8 with back-
ground contamln8tlon ideally held to a xem
level but more reallstlcslly to 8 level such
that extr8ordin8ry measures in sterile t&n-
nlques 8re not required to nalnterln lnteg-
rlty of  the biological  systems  belng
researched.

4. Prevent the 8ccumul8tion of materhIs
from current and pest experiments that con-
stitute 8 hazard to laboratory personnel.

6. Prevent the cm&ion of aerosols of h8.z-
8rdous materials as a result of the housekeep-
ing prwedures used.
Procedures developed in the 8re8 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 8n
appm8c.h avoids the confusion of multiple
praoticee and retmining of personnel. The
prlm8ry function, then. of routine honse-
keeping procedures is to prevent the ~cumu-
lation of organic debris th8t (1) may harbor
microorganisms that are 8 potential threat
to the integrity of the biologlaal systems un-
der investlg8tion, (ii) may enhance the 8ur-
vlval of microo~ inadvertently re-
leased In experimental procedures, (iii) m8y
retard penetration of deeont8mlnants, (iv)
may be tm.ns.fer8ble from one 8re8 to 8n-
other on clothing and shoes, (v) may, with
sufliclent buildup. become 8 bloh8z8rd 88

8 consequence of secondary aerosolization by
personnel and 8ir movement. and (vi) m8y
cause allergenic sensitlz8tlon of personnel,
e.g., to anlm81 dander8
Housekeeping ln animal c8re unit&i has
the same primary function a9 that stated
for the laboratory and 8hould, $ addition,
be as meticulously carried out in quarsntine
and conditioning areas 8s in area.5 used t.o
house experlment8lly infected animals. No
other are&s in the laboratory have the con-
stant potential for creation of significant
quantities of contaminated organic debris
than do animal 08re facilities.

In 8ll laboratories. efforts to achieve f&&l
decontamination and to conduct 8 major
cleanup of the biological complex are nor-
mally undertaken at relatively long time ln-
tervals. Routine housekeeping must be relied
on to provide a work 8rea free of slgnlfioant
sources of background contamination. The
provision of such 8 work area is not simply 8
nmtter of indicating in 8 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 ,bioh8rard
involved. decide on 8 detailed procedure for
its accomplishment, and provide lnstructlons
to 18boratory personnel in 8 manner that
minimizes the opportunity for misunder-
stsnding.

The following check&t outlines a portion
of the items requiring critic& review by the
laboratory supervlsor. It is not intended to
be complete but is presented es 8n example
of the detailed manner ln which housekeep-
tng in the blologicsl laboratory complex must
be viewed.

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


Administrat~ion Areas
Aisles

Animal Food Storage
Animal Bedding Storage
Biological Safety cabin&3
Bench Tops and Other Wc& Surfaces
Ceilings
Change Rooms
Cleaning Solution Disposal
Cages and Oage Racks
Dry Ice Chests
Deep Freeze Chests
Entry and Exit Ways
Equipment Storage
FlOOIX
Glassware
General Laboratory Equipmetit Cleanup
Hallways
Incubators
Instruments

Insect and Rodent, Control
Light Fixtures
Mechanical Equipment Areas
MOPS
Pipes-Wall and Ceiling 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 DAY personnel
who are knowledgeable of the research pro-
gram and special hazards of the research en-
vironment. The recommended approach to
housekeeping iii the assignment of house-
keeping tasks to the research teams on an ln-
dividual basis for their immediate work areaa
and on a cooperative basis for areas of com-
mon usage. Similarly, animal caretaker per-
sonnel should be responsible for houeekeep-
lng 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 aesure compliance. This
approach assures that research work flow
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 will be
kIlOWL

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 hlgh-effi-
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 gro.ss
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 selutlons can be
accomplished by a nu&ber of methods.
Among them 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
floe squeegee.

After cleaning up a spill of infected mate-
rial. the residual solution should not be

NOTICES

discharged io a sanitary sewer until it has
been autoclaved or given further chemical
treatment, such as by the addition of sodi-
um hypochlorite suiiicient to pro\-ide a fitlal
concentration of 500 ppm chlorine. Most
household bleaches are marketed with a
chlorine content of 5.25%. These in a final
dilution of 1 :lOO, yield 5i5 ppm of a\,ailable
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
aerosollzation of dust should be used.

Sweeping compounds available from the
usual janitorial supply firms fall in three
categories:

Wax-baaed compounds used 011 vinyl floors
and waxed floor ooverings.
oil-based compounds for concrete floors.
Oil-based compounds with abrasives (such

as eand) to achieve a dry scouring action
where much soil is present.
Dry-dust mop heads can be purchased as
treated disposable units or as reusable, wash-
able heads that must be treated with appro-
priate 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 unlt. however, can negate
this factor because of its ability to pass huge
volumes of exhaust air while retaining par-
ticles with a mlnhnum efeciency of 99.9'7 per-
cent. Wet and dry unlta ticorporatLng a
HEPA filter on the exhaust are available from
a number of manufadurers.

There are no particular requirements with
respect to the manner in which the dry
vacuuming is accomplished other than to
emphasize that the objective is to remove alI
debris and particulate matter. The manufac-
turer's directions adequately detail the fre-
quency of bag changes, fflter changes, and
mechanical adjustments.
Drv material vacuum-collected during these
floor-cleaning ectivlties is potentially con-
taminated, but the nature of the risk in
probably greater to the experiment than to
the experimenter. It Is wise to effect bag and
fflter changes and to clean out Eollection
tanks in a m&nner that will avoid or mini-
mize aerosolizlng 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 fllter. Even though it
may serve as a primary fflter. the disposable
bag must be removed with caution A bel-
lows effect may pump dust out of the bag
if its intake onenina is not sealed before
moving it to a &tic-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 avoid contaminating experimental ma-
terials, the emptying of vacuum collection
tanks and chaneina of baas and fllters 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
glwd k present. After making the fllter
changes, all external surfaces of the imme-

3&73

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 Is recommended that gaseous ster-
ilization may be used to minimize aerosoll-
zation of microorganisms before waste is
emptied from the vacuum container. Be-
cause complete penetration of sterill+g
gases into the collected dry dust may be B
problem, all wastes should be Dlaced 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 ln an attached
double-door  carboxyclave  (an  autoclave
equipped with an ethylene oxide gas sterili-
zation system) to allow for removal and
emptying of the collectton tank.

If a wet vacuum is to be used for pickup
of the detergent-germicide solution from
the floor, the manufacturer's reconunenda-
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 fsllure. Liquids collected in the vac-
uum cleaner after floor mopping will con-
tain decontamlnant materials. These liquids
may be poured down a convenient floor
drain, except in the case of cleanup wastes
from an overt spill. The collected liquid
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 0 cleaning solutton. The
selection of a detergent-&contaminant corn-
binatlon 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. Wlth rare exception. this
will be identl6ed as the animal holding area
and the expected microorganisms may WI1
include fungi. virwes. and the vegetative
and spore forms of bacteria A decontamlnat-
Ing solution for such a range of mlcroorga-
nkms would, however, be expensive and ex-
cessively wrroslve for routine use. Except
in those rare instances where it can be as-
sumed that pathogenic sporea 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 debils from
bedding and food. thus offering potential
for removal or at least a large initial reduc-
tlon in their numbers by vacuum cleaning. A
wide range of cleaning solutions that am
mildly sporicidal, reasonably residual. and
are not destructive to the physical plant am
available. Phenol derlvatlves In combination
with a detergent have these characteristics
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 blocidal substan-
that include the mercuriale, quaternary
ammonium compounds, chlorine compound&
lodophores. alcohols, formaldehyde, glutaral-
dehyde, and combinations of alcohol with
either iodine or formaldehyde. These havm
been discussed in Section VI.

The laboratiry supervisor should make a
selection from those types most readily wuU-

FEOERAL REGISTER, VDC 41, NB. 17~THURSOAY, SEPTEMBER 9, 1976


3YP74

NOTKES

able which meet the gerleral criteria of effec-
tiveness, residual propert,&, and low corro-
siveness.

F. Wet mopping-&Go-bucket ?nen:od. Wet
mopping of floors in laboratory and animal
care areas is, from a safety standpoint, most
conveniently and etaclently accomplhhed
using a two-bucket system. The principal
feature of such a system is that fresh detor-
gent-decontaminant solution is always ap-
plied to the fl@X from one bucket, while all
spent cleaning solution wrung from the mop
is collected in the second bucket. Compact
dolly-mounted double-bucket units with
foot-operated wringers are available from
most janitorial supply houses. A freshly
laundered mop head of the cotton String
type should be used daily. This requires that
a mop with removable head be provided as
opposed to a fixed-head type. In practice, t.he
mop is saturated with fresh solution, very
lightly wrung into the second bucket and
applied to the floor using a figure eight mo-
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-
matelv 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-
t.inued in 100 it? increments untfl the total
floor 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
lea& potential contamination to those of
greatest potential. Before a mop head is sent
lo a laundry, it should be autoclaved. Spent
cleaning fluids are disposed of by flushing
down tl?e drain.

If the cleanun follows an overt sl)ill of in-
fe&ous materlrll, the spent cleaning solution,
titer removal from the floor, should be auto-
claved or treated with chlorine solution.
Chlorine (as household bleach) should be
added to give 600 ppm and held for a contact
time of 16 minutes before dumping in the
sanitary sewer.

0. Alternatizie floor cleaning ?nethod for
animal care areas and areas wills monolithtc
floors. The absence of permanently placed
laboratory benches and fixed equipment,
coupled with the mobility of modern cage
rs&ks, makes possible alternate floor-cleaning
procedures in animal care facilities. As in all
considerations of methodologies in blomedi-
ti laboram facilities. M, is necessary to
aseeea the compattbillty of procedures and
facilities from the hazard point of view. The
alternative floor-cleaning procedure to be
d&cussed requires that floom are completely
sealed or of monolithic con.struction so that
liquid leakage to adjaent arlreas does not
oemr and that, floor drains or Wet vacuum
cleaners are available.

Subsequent to the removal of all debris by
dry vacuum, move the cage racks to one sfde
of tme room. Cover the floor of the remaiting
cleared portion of the - wim detergent-
decontamlnant solution applied at a rate of
approximately one gallon per 144 ft? from a
one-gallon tank sprayer, using a setting on
the nozzle which will cause the solution to
flow on and not create a spray, The nozzle is
pl,ac%d close t.o 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 1% up with a wet vat-
uum. Nlow the floor to air dry; move the
csge racks into the cleaned area, and repeat
the process for the remaining floor area. Floor
drains in these areas should be rim-fish, at
lea& stx inches in diameter, and fitted with
a screen or poreus trap bucket ta catch large
debris that es&spas the initial dry cleaning.
such screens and mket.6 should be emptied
after treatment with a decontaminant. If
qaee ut.ilWion doea MA. require frWUent
Iloo? WaShdoWn, pour I) half-gallon of deter-

gellt-dccollt;tnlll~al~t solution into the drain
each week to keep the t.rap in the waste line
filled against backup of sewer gases.

A. Biokacardo&s spill in a biological sa,@Cy
cabzrLet. Chemical decontamination pme-
dures should be initiated at once while the
cabinet continues to operate to prevent
escape of contaminants from the cabinet.

1. Spray or wipe walls, work surfaces, and
equipment with a 2 percent solution of an
ivdophor-decont.aminant   ( Wescvd yne   or
equivalent). A decontaminsnt detergent has
tile advantage of detergent activity, which is
import.ant because extraneous organic sub-
stances frequently interfere with the reaction
between the microorganisms and the active
agent of the decontaminnnt. Operator should
wear gloves during this procedure.

2. Flood the ton work surface tray. and. if
a Class II cabined, the drain pans aiid catch
barilns below the work surface, with a decon-
t.aminant and allow to stand IO-15 minutes.

3. Remove excess decontaminant from t.he
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
wit.h a sponge or cloth soaked in a decon-
taminant. Then replace in position and drain
recontaminant from cabinet base into ap-
propriate coutainer and autoclave according
to standard procedures. Gloves, cloth or
sponge should be discarded in en autoclave
pan and autoclaved.

B. Biohazard spill outside a biological
sajety cabinet. 1. Hold your breath, leave the
room immediately, and close the door.

2. Warn others not to enter the contami-
nated area.

3. Remove and put into a container con-
taminated garments for autoclavlng and
thoroughly wash hands and face.

4. Wait 30 minutes to allow disslpatioil of
aerosols created by the spill.

5. Put on a long-sleeve gown, mask, and
rubber gloves before reentering the room.
(For a high risk agent, a jumpsuit with
tight-flttlng wrists and use of a respirator
should be considered).

6. Pour a decontaminant solution (670
lodophor 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 auticlavable dust pan and
squeege, transfer all contaminated materials
@aper towels, glass, liquid, gloves, etc.)
into a deep autoclave pan. Cover the pan
with aluminum foil or other suitable cover
and sutoclave according to st.andard direc-
tions.

s. 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 dim-
cult.

C. Radioactive biohazard spill outside a
biologfcal safety cabinet. In the event that a
biohazardous spill also involves a radiation
hazard, the clean-up procedure may have to
be modifled, depending on an evaluation of
the risk assessment of relative biological and
radiological hazard.

Laboratories handling radioactive sub-
stances must have the services of a deslgnat%d
radiation protection ofacer available for con-
sultatlon.

The following procedure indicates suggest-
ed variations from the bloheuard spill pro-

cx?dure (above) that should be considered
when a radioactive biohazard spill occurs out-
side a Biological Safety Ca13lnet.l
1. Holding your breath, leave the r@zm im-
mediately and close the door.
2. Warn others not to enter the con&ml-
nated area.

3. flemove and put In a container con-
tanilnated garments for autoclaving ad
thoroughly wash hands and face.
4. Wait thirty minutes to allow &&pa-
tion of aerosols created by the spill.
*Before clean-up proceaures begin, (I radiu-
tion protection o#irer sho?rld survey the spill
for External radiation ilacard to determine
the relaiive 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-
fit,ting slce~;?~ nlld a respirator should be ccn-
sidered).
6. Pour a decontanlinant solutjon (5 `> lodc-
pho: or 5',;) hypochlorite are recommended)
around the soil1 and allow to flow Into the
spill. Paper tawels soaked with the decon-
tam~uant. may be used to cover the area. Tc
minmize aerosolization, avoid pouring the
deaont~millnnt solution directly onto t?i?
:,p111.

7 l.et .-lxnd 30 lninutes to allow adequaie
dlsinfectwlt contact tinle.

8. *In niost cases, the spill will Znuolve
`,C or :H, whtch present tu) external haard
Hou:ei:er, if more energetic beta or gamma
emitters are inrolved, care must be taken to
prevent hand and body radiation exposure.
The radiation ,protection officer must make
ihis determinalfon before the clean-up opera-
tion is begun.

If the radiation protection officer approvee.
the bio-hazard-handling procedure may be-
gin: Using an autoclavable dust pan and
Yqueegee, transfer all contaminated materials
(paper towels, glass, liquid, gloves, etc.) into
a deeg 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 tha&radioactice vapors may be re-
leased and thereby contaminate the auto-
clut'e, the material must not be autoclaved
In that case, suflcfent deccmtaminant solu-
tion to immerse the contents should be added
to the wastes contafner. The cover should be
seaZed wiih waterprooj tape, and the con-
tainer stored and handled /or disposal a.*
radioarttive taaste. Radioactive and bioha.zard
warn&g symbols should be affixed to the
waste container. As a general rule', autoclar-
fngf should be avoided.

9. If autoclavlng has been approved, ths
dust pan and squeegee should be placed in an
autoclavable bag and autoclaved according
to standard directions. Contact of reusable
Items with plastic bags should b% avoided-
separation of the plastic after autoclaving
can be verl difficult.

*A final radioactive survey should be made
ot the spell area, dust pan and squeegee wfth
a Geiger counter, or a smear should be taken
and counted in a liquid scintdllation counter.

The sspiratlon of tissue culture medih
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 aerosola or
fluids of house vacuum systems or labora-
tory pumps, some investigators have in-
stalled side arm flasks containing cottos
sulfuric acid or decontamunt between abbe
reservoir and the vaculim line. Cotton 1%
not completely effective 85 a Altering sg%nt,

1 wp in procedures have been starred
and itallciz&.

FEDERAL REGISTER, VOL. 41, NO. 176lHURSDAY, SEPTEMBER 9. 1976


NOTICES

3847&

sulfuric acid will corrode pipes, and con-
taminants may lose their inactivating abil-
ltv unon standing. The introduction of a
c&ridge-type filter that is moisture resist-
ant and has a rated capacity to remove
particles 350 nm (0.35u) or larger in size
provides an  &ective barrier to ilrus
aerosols.

The secondary reservoir and filtration ap-
paratus can be assembled from readily avail-
able units as shown in Figure 1. A length
of plastic tubing `/ inch 1.D x I:16 inch
wall is attached at one end of the reservoir
and at the other end to the lowrr arm of
a filtration and media storage flask. These
flasks vary in capacity from 250 to 4000 ml.
the choice of flask depending on available
space and amount of fluid that could be
accident.ally aspirated. A second tube `of
the same dimensions is 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 t.uhes 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
liquid can drain back into the reservoir by
gravity if the connection at the vacuum line
is broken. This prevents t,he loss of fluid
which the investigator needs to retain.

Should the flask be used onlv for the re-
covery and storage of waste fluids, then the
addition of a few grams of Dow Cornlng
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 flnal 5~~ concentration
of an iodophor or other appropriate decon-
taminant. holding for 30 minutes and drain-
lng as above.
If the filter becomes contamlnat,ed or re-
quires changing, the Alter 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 fllter can t.hen be Installed
and the assembly replaced.

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 etiologtc agent l&ted in
paragraph (c) of 42 CFR 72.25 (which is
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 mnt~,-
rials. 1. Volume less than 50 ml. Material
shall be placed in a securely closed, water-
tight container [primary container ( test
tube, vial, etc.) ] which shall be enclosed in
a 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 er-
teed 50 ml. The space at the top, bottom
and sides between the primary and secondary
containers shall contain sufficient nonpar-
ticulate absorbent material to absorb 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 corrueated 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),
W(S).

Descriptions of thls packaging method a?e
giren in Table III.

2. Volumes of 50 ml or Greater. Material
shall be placed in a securely closed, waier-
tight container (primary container) which
shall be enclosed in a second, durable wat,er-
tight container (secondary container). Single
prhrn~ry coniainers shall not contain more
i!~r\n 500 ml. of material. However, two or
more primary containers whose combined
voilunes 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 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, 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-
plng 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

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 out&de 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.

Dcscrintions of nackagee which comnlv
with the regulntiot;? of Ithe Department- of
Transportation (DOT) are given in Table IV.

C. Labeling of packages containing re-
combinatat DNA n~nterials. 1. nlaterlals which
do not cont.ain 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 addre-s 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 et.iologic agent listed in paragraph (c)
of 42 CFR 72.25.

Material data forms, letters, and other
information identifvina or describing the
material should be piaced around the outside
of the secondary container. In addition to
the address label, the label for Etiologic
Agents:Biomedical hZateria.1 must be affixed
to t.he outer shipping container. This label l.9
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 bv the USDA as oart 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.  Ddmestic  Transportation.  CiVil
Aeronautics Board Rule No. 83 (Air Trans-
port Association Restrlcted Articles TarifY
6-D) requires that a Shipper's Certificate,
depicted below, be completed and arllxed to
all shipments which bear the E'I'IOLOGIC
AGENT;BIOMEDICAL MATERIALS label re-
quired under the provisions of the Inter-
state Quarantine regulations (42 CFR
72.25(c) 1.  The Certiilcate must be com-

pleted in duplicate and affixed to the outer
shipping container.

.A. Introduction. Federal regulations and
carrier tariifa have been promulgated to
ensure the safe transport of hazardous blo-

Tbls L to cettifr that the contents ol this cansidnment are Ptonertv classified. described bp P~OFEC
shipDing name end are Dncked. marked and Isbelled and are ia proocr condition for car&v+? by +Ic
according to 811 eo~licabk carrier and Rovrrnmeat regulations. (For intcrnationnl rhirmenrr add
"and to the TATA Restricted Articla Reauhtion.s'*J This consxnment 1s within the limitutio~
prescribed for: PASSENGER AIRC&WTICARGO ONLY Icross out nonapolicnblel.

                                     .
Number d    Specifr Each Article Senaretely
  Pnchkxa        fl'roncr 8hlDDinP Name)       Cleuificrtion

           ETIOLOGIC AGENT. r1.0~
.      I I NG Ch~ct!$'

                                 ETIO. AC.

FEDERAL REGISTER, VOL 41, NO. I76THURSDAY, SEPTEMBER 9, 1975


.38476

NOTICES

Bhlpments of recombinant DNA Materials
exceeding 60 nd in volume and containin.g
any portion of an etiologic agent listed in
paragraph (c) of 42 CFR 72.25 an3 restricted,
by DOT regulations, to transport by $argo
only aircraft. When the volume of a single
primary container exceeds the 60 ml limita-
tion, &Is restriction must be indicated on the
Bhlpper's Certificate by crossing out "Passen-
ger AlrcrW.

When dry ice it3 used as a refrigerant an
"OR&-Group A-DRY ICE LABEL" should
be affIxed to the outer shipping container.

The amount of dry ice used and the dale   (2) Parcel Post Customs Declarat.ion (Pe
packed should be designated on the label.
2. International Transportation.-In addi-  296&A) label.

tion to the packaging and labeling require-  (3) International Parcel PosLInstructions
ments of the regulations previously cited, in-  Given by Sender (poD asaa) 1abe'.
ternational shipments of recombinant DNA   (4) Dispatch note (POD 2972) tsrg
materials in which any portion of the mate-    (6) "Violet Label"
rial is derived from an etiologic agent listed
in paragraph (c) of 42 CFR 72.25 must have   (6) Shipper's Certificate specifKed hx the

one or more of the following documents-  current International Air Transport Associa-
depending on the country of destination:    tion Tariff. Individual country requirementa
(1) Parcel POst Customs Declaxation (PS  are listed in "International Postage Ftatea and
m30) tag.                     _ Fees" (USPO Publication 51).

     A.P?ENDIX D, PACE D-83

FEDERAL REGISTER, VOL. 41, NO. 17kTHURSDAY, SEPTEMBER 9, X976


Vlth rlclring WIthout
Rcfripcrant   Rcfrlnermlt

m
J




3




J



.d




d

   Refrinermt
Plbrrbiwd box closely
f:ttJnS the atyroform
box, taped aha

No. 3 erlnp eta1 t1n f14
404 x 700 or . l-6Sll.m
frletion-seal tin cm,
610 x 70.3, top aolderad
or clipped ,f 4 polnte u

Styroloan box shuck-
absorbent inmulatlon

Plbcrbb.rd box closrly
fitting the styrofom
box, taped rhut

V3C cardboard bar
PSI type, 9-l/16-
x 9-3116" x 11-l/&~
hIth O.D. taped
ahuc with 3" typo *
Pi: :ipe

Styrofoam box shock-
rbaorbtnt In.ul.tlon

Ho, 3 crimp Beal t1n cm
404 I 700 or . I-nalIan
friction-se*1 tGc._n,
610 x 708. top soldered
or cltpped at 4 points L/

VIC cardboard box
PS3 type. V-3/16*
x Y-3/16" I 11-114"
high O.D. toped
*hut vlth 3" typ.
PS3 tmpc

VIC cardboard box  5
PSI type. 9-l/16"
x g-1116" x 11-114'  a
hi6h O.D. taped   -,
#hut with 3" typ,
PSI tape     f
            P

VX crrdborrd b&x  g
12-114" I 12-l/4"
I IO-3/16" hinh
D.D. taped ahut
with 1" wide PSI
tape.

No. 3 crlny se*1 tin esa
404 I 700 or . I-gsllon
Jrictian-seal tin cm,
610 x 708, top mldcred DI
Clipped at 4 pulnf# hl

I-pallon rrlctlon-8fal t1n
cm, 804 x 908, top
o oldercd c.t clipped at 4
pohrl b/

?Ibarborrd box &rely
fittIn& the atyrofou
box, iapcd #hut

500 ml Pyr.x gloss
bottla, rubber-sLlrc
.toppcr, taped, 01
so0 ml phtlc*
bottle. iuwrow,oy
wlda mouth. ,erw
cap*, taped

603 x 510 2-S*110,, frtctfo~ ,bwt`wnt inbUt6ttM
maa1 t1n can, SO4 x 901,
top mldered or clipped at
4 polntl a,

box lo ok taped
shut

.- mN'E& voi 4t, NO. 176THURSDAY, SEPTEMBER 9, 1976


APPENDIX D, Page D-85

ATTACHMENT I

OEPARTMENf OF HEALTH, EDUCATJC;N, AND WELFARE

PlJELlC HEALTH SERVICE
CENTER FOR OISEASE CONTROL
  ATLANTA, GEORGIA 30333
IrIsphone: (404) 633.331 I, Ext. lfi8J

TITLE 42-PUBLIC HEALTH

Chaiter I-Fublic Health Service, t)epartrnent of Health, Education, and Welfare
       BUBCHAPrER F-QlJARANTtNE. INSPECTlC&;ilCENSlNG

&&on 72.25 of Pwt 72, TN@ 42, Code
a Federal Regulations, is amended to
&ad as follows:
Q 72.25 Etiologic rg~:en~sf

<a) Definitipns. As used in this see-
tbll:

411 An "etiologic aeent" mean3 a vi-
&& microorganism or its toxin which
*c~wes. or may cause, human disease.

(2) A "dingnostjc specimen" means
8ny human or animal material ix-Au+
ing. but not limited to, excrcta, secreta.
blood and its components. tissue, and
&Issue fluids being sllipped for prrr~0se.s
of diagnosis.

(3) A "biolo3ical piodu'ct" meax a
biological product prepared and manu-
factured In accordance with the provi-
sions of 9 CFR Part :O. Ltccnsed Veteri-
~ryBiologica1 Products, 42 CFR Part 73,
Licensed Human Biological Products. 21
CFl3 130.3. Near &ups for inuestigaCiona2
Wein Irunrans, 9 CFR Part 103. Biolobical
Products for Experimental Treatment of
Animals, or 21 CFR X30.3(a), Nezu drugs
jar invesligafionai. WC in animals, and
w&h. in accordance with such provi-
*on?., may be shipped in interstate traffic.

<b) Transporfation: etiokgic agent
hinimum packaging requirements. .No
person may knorvin$y transport or cause
to be transported in interstate traffic,
directly or indirectly, any material, in-
cluding but not limited to, ciiazncstic
specimens and biblogical products, con-
hiriing, or rezonably believed by such
wrson to contain. an etiolosic agent un-
as such material is packaged to aith-
&an d leakage of contents. shocks, pres-
Wre changes, and other conditions inci-
$;in.o ordinary handling in transpor-
    .
W .Transportation: etiologic agents

mbject fo additionat requirements. No
pzrson may knowingly transport or cause
Lo be transported In interstate tmffic,
Uirectly or indirectly. nny matcrial.l. other
than diamostic specimens and biological
Products. containing, or reasonably be-
lieved by such person to contain, one or
tnorc o! the fo!!winp etiologic agents

PART 724NTERSTATE QUAkANTlNE
Subpart C-Shipment of Certain Things

wlless such material is packaged in ac-
cordance with the requirements spe:ified
. in paragrotih (b) of this section. and
unless. in addition;stichmaterial is pack-
aged and shipped in accordance wit.5 the
requirements specified in subparagraphs
(l)-(b) of this paragraph:

      * BACE~LAL AGEhTS
IctinobocilIus-all speclcs,
Arfro~ hiMha!.?ii-all SerotvDee.

                -.
Eociiius anlhracis. .
Barlonc~la-all species.
BordeIella--al1 sjxcles.
Borrclia recuwentir. 8. ci;tcrM.
Bruce&z--aJJ species.
Clostndium botulinurn, Cf. chouvoef, Cl. hae-
moly~ibn, Cl. hislo!jrlicum, Cl. my!, Cl,
septlcum, Cl. tctani.
Coryncbactcfium diphtherlae C, equt, C. hae-
molyIicum. C. pstudotuDetcuIorlf, C.,jsyo-
genes. C. zenale.

DipZococctu (Streptoc%ccw) pn~un:~fii~~.
Erysipelothrir insidfosa.
Escherfchfo coff. all entero~athoaenk SM.
.t+s.             - -

Francisella (Pasteur@) tutortwit.
Xakmhilus ducreui. X. tnnzenk.
HereZ!ci vaginicola.W .    .

%Iebs-iella--all species and al! scrotypz.8.
Lepto9pirafnterroyans-all serotype.
Lisferia-all species.
Yimc ??oIumorDha.

Mi copias ma-all species.
Neim-na gomhoeac. N. menfnpifidij.
Posteurelt4-8ll snecies.

Ttep'onema cdrhm, T. peliidum, aad 2'.
pertenue.

Yrbrio feluf, V. comma, lncludtng blot&a
El Tar, and V. ~atahemolyticw.
Yerscnia (Pasteurella) pest&

Actinomycetes (1ncludlr.g Nocardio apqclw,
Aclmomyces species and Arochnia ~:opi-
onicn1.

ElaslO&yces dcrmalilidir.
Coccid'oides immiiis.
Ctyplococcus neolormanc
x1rtopzasma Capsufo~lnlL
Parucoccidioider bruri!ieitttr.
  v.Z..rL. RICKIITTTS1AL..ANO CWLhMYDUt
           .hGCNTI

Echoviruaes~all Wues.
Encephaiomyocarditi Citu_.
HCmwrhogic feccr agents, ln@dlng Crimc~n
bemorrlragic fewer (Congo), Juni8s, and
alfuchupo viruscr, `md othcrj .# re'ct .m.
defined,
HepatitiJ-arsDCidtCdntf9Cn.
Nrrpesvirus-all members.
Jn/tclious bronchilis-Ifkc %Ub.
Influen:a rfrffres-811 types.
krsro virus.
ZymphocytiC choriomcnbgiW UlrUt.
Warburg virur.
sfe4fJer uirw.
Mumps uirw.
Poralnflucn3o trru7es-8l1 tYPoa.
PoZioui;uscs--nil typss. *.
Porvirusrs-all members.
PzillacosFc - Ornftho& - Trachorta-&O?kp~o~

granulo~a group of agents.
Rabies virus-all strixlns.
Reooiruses--a!1 types. .
Respfratory )yncytial oirur.
Rhinouiruscc--all types.
Rickettsia-til species.
Rubella tvuf.

Simian t~iruses-41 Lypes.

Tick-borne encephalitis virus COmplex, 1110
cludlng Russtan sprtng-rummer eneel;hz-
litis. Kyasonur forest d:secrre. Omsk hrnm-
thapic few, and Central Etropean enceph-
alftir uiruses.

(1) VoTume !ess thnn 50 ml. Materiti
shall be placed in a secureiy closed. wa-
tertlght container (primary contaiser
<test tube, vial. etc.) 1 nhlch shs?l be en-
closed in a second, durable waterr?sht
container (secondary container). ESY-
eral primary contntners may be enclosed
in a sin&l:: secondary container. if the
total volume of all the primary con+A!n-
ers so enclmed does not exceed 50 IT??.
Tt&&ce at the top, bottom. `XX! L'des
between the primary and secondary ccn-
Wners shall contain suZ.cient nonvr-
ticulatc absorbent matetial ~JI absorb t?e
entire contents of the primnry contain-
er(s) in ca.x of breakage or lezkngc.
Each set of primaryand secosdazz con-
tainers shall then be enciosed in ari OIL~CI
shlpplng container constructed of corrJ-
gated. Aberboard.. cardboard. wood,. Or
other material of eguiAcnt strenrth.

(2) Volume 50 ml. or .qrcc:cr. P-J;.- `f-
Jnp of ma!erlal in vo1wr.x 0: 59 ::.`. a'.'
more shall Include. in addition. a s(iaXr(
aborbcnt material, In volume at lest
equal to that or the sbsorbent XWcd#l

FEDERAL REWS1IER, VOL. 41, NO. 176-TtlURSDAY, SEPTEMBER 9. 1376


between the primary and secondary con-
tainers. at the top, bottom. and sldes.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 shlpplng
container shall not exceed 4.000 ml.)

(3) Dry ice. If dry ice is used as a re-
frlnerant. it must be placed outside the

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 symbol rneasurlng 38 mm.
(lY2 inches) in diameter must be cen-
tered ln a white square measuring 51
mm. (2 inches) on each side.
(iv) Type size of the letters of label
shall be a.5 foililows:

~OL0GIC AOEZTT ________-__  IO pt. rev.
BIOSIEDICAL MATERIAL _____.  14 pt.
IN CASE OF DAh%AOE OR

LEAKAOE _____-_-~-__~~.  10 pt. rev.
NOTIFY DIRECTOR CDC
ATLANTA. GA _______-_-_.  8 pt. TeP.
404 833 5313 _______________-_  10 pt. rev.

sec%ndar$ contolner(sj. If dry' ice is used
between the secondary container and the   (5) Damaged packages. Carriers shall
outer shlppmg container, the shock ab-  promptly, upon discovery of damage to
sorbent material shall be so placed that  the package that indicates damage to the
the secondary container does not become  primary container. isolate the package
loose inside the outer shipping container  and notify the Director, Center for Dis-
as the dry ice sublimates.           ease Control, 1600 CliftorL Road NJ%
($1 Labels. The label for Etiologlc  ~,dant.a, GA 30333 (telephone (404) 633-
Agents/Biomedical Material, except for  53 13 ), and the sender.
size and color, must be as shown:       (6) Registered mail or eqUiL~ak!?If SYS-
                        tern. Transportation of the follontig
                        etlologic agents shall be by registered
                       mall or an equlvalent system which re-
                        qulrcs or provides for sending notlflca-
           tJgvez the shipper immediately upon
                                      :
                               ActftwbacUus mattd.
                                Coccfdiofdes fmmftfs.

FrancbeIla (Pwteuretla) fularensis.
Hemorrhagic fecer crgentr, lncludlng, but not
UmIted to, Crimean hemorrhagic @VU
(Congo), Junfn, Machupo vinrses.
Mcrpuvfrus sfmfae (B rlrus) ,

(i) The color of material on which the  EibtopZwma captutatum.
                              Liwa vfrus.

label is printed must be white and the
symbol and printing in red.      B$~brlg ~Jrus.
                           monae pseudomatlei.

Tick-borne enccghftlfs rirus complex. ln-
chdlng. but not llmlti to. Rusrfan spting-
Summer cncephctttt.7. Kyasanur forest dir-
ewe. Omsk hcmarr)taofc lever. end Centrot
Eur&can enceph&lJ - VUu~seS. Varfola
minbr u;d Varf6Zoimajo-r.
Yersenia lPaJteureZZo1 DCS;

(dl Notice ol d&&y, faiZure to re-
ceive. When notice:of &livery of agents
containing. or sus ected of contalnlng.
     4
etiqlogic agents 1~  d In paragraph tct
\ (6) of this sectfon is not received by the
sender within 5 days folloffing anticf-
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) Requirementj: r&&ions. The Ad-
mlnlstrator may approve variations from
the requirements of this section if. upon
review and evaluation, he llnds that such
varlatlons provide protection at least
equivalent to that probIded by compIl-
`ante with the requirements specified In
this section and makes such flndings a
matter of 05clal record.
(eeC 361, 58 Std. 703: 42 USC. 264)

Effective July 30, 1972

ADERAl REGISTRR, VOL 41, NO. 176THURSDAY, SEPTEMBER 9, 1976


NOTICES

_I--- --

PACKAGING AND
LABELING OF
ETIOLOGIC
  AGENTS

FlGUt?E 2

CROSS SECTION
F PROPER PACKING

The IntersLrte Uuarantine Regirintionr 142 CFIi. P,rrt ii 25
Etiologic Agents) war revrsed krly 31. 1972 to ~TWIIIL for
packaging and labeling requirements for etlolugrc ,rg~nl$ :!n!!
certain other matclials shipped in intctsti!tc trilf!tc.

Figures 1 and 2 chagram the packagillg iIl,d labelmg of atto.
logic agents in volumes of less than 50 ml. m acu~rtlimce with
the provisions of subparagraph IC) (11 of the crterl regulalton.
Figure 3 illustrates the color and size of the label, described
in subparagraph (C) (4) of the regulatwns, which shall he
affixed to all shipments of etlologic agenlr.

For further infrxmation oil ;r~>y pr3: isic!t\ of thl!, IS$<ll!tiC;i~
cOllldC1:
            Center for D~sexe Control
            Aftn: Bioharirrds CWIIUI 0111~~
             1666 Clrfton Road
          Atlanta, Georgra 30333
             Teler)honc: 404  633 3311

FfGUAE 3

BIOMEDICAL
MATERIAL

FEDERAL REGISTER, VOL. 41, NO. 176lHURSDAY, SEPTEMRRR 9, ,974


NOTICES                           38481

i'iPPE4DIX D, Pdqe D-3{

PACKAGING AlWO LABELING OF ETIOLOGIC AGENTS

ABSORBENT PACKING hl$TERlAL

d-- PRIMARY CONTAINER IRorrk, blood /w+I, ck.1
         *NOTE: Single primary contamer\ mJy nut etcccd 500
          ml. of material. Twu or more prrmJry tontJrnrr\ tihlne
           combined volumes do not cued SO0 ml. m.rv ho en.
          clu\cd in a \inglc, secondary cuntJmcr. The maxtmum
         volume of etiulugic Agent which mJy be encloved m 4

single outer shipping container shall nut exceed XNO ml.

SHOCK ABSORBENT MATERIAL

I     SECONDARY CONTAINER (G&cftxfscrewcu~

wtth waterproo? tape or hcrmetrcallv sealed CL&)

- OUTER SHIPPING CONTAINER

-MAILING LABEL

*ETIOLOGIC AGENT LABEL

The Interstate Quarantine Regulation5 (42 CFIJ M
72.25, Etiologic Agents) wds revbed Jul) 31. 1972, to
provide for pa&aging and labelingiequircmcntr for etb
logic agents dnd certain other mJtert,tlr shipped rn mter-
state traffic. The illustration rhdw, acscptable pxhagmg
and labeling ol etiologic .tgentv in xcrudance with ub
paragraphs (c) 12) and 1-t) uf the cited regulation.

For further information on any provision of this regutation contact:
          Center for Disease Control
         Attn: Biohazards Control Office
          7600 Clifton Road
        Atlanta, Georgia 30333

Telephone: 404-633-3311

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


38182

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-

tow (e-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 monev order made savable to Na-
tional Archives Trust Fund-and mall to:
Sales Branch, National Audiovisual Center
(GSA), Washington, D.C. 20409.
8. Research Laboratory Safety.
This shde tape cassette, stock number
176.79, is available for 675 from the National
Safety Council, 425 North Michigan Avenue,
Chicago, Illinois 60611.
B. Films. 1. Air Sampling for Microblologi-
Cal Partlculates (M-926).
2. Handling the Laboratory Guinea Pig
(T2618-X) .
    Handling
(T%617-X) .      the  Laboratory   Mouse

4. Infectious Hazards of Bacteriological
Techniques (M-382).
6. 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) IM-9251.
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 ln-
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,
Otace of Research Safety. Direct inquires as
above. September 89, 1976, Stanford, CA:
September 21-11, 1976, Cold Spring Harbor,
.?TT
I* x

3. Safety in Laboratory. Presented by Ns-
tlonal Institute of Gccunatlonal Safetv 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 Idbboratorles, Center for Dla-
ease ControI, Atlanta, Georgia, September 14
16, 1976 and September 13-16, 1977.

NOTiCES

B. Policy.

C. Responsib!lity and A*rthority. 1. Man-

agement.
2. Supervisor.

3. Each Employee.

4. Facility Safety Officer.
5. Biohazard Safety Committee.
D. Fiacility Assignment Proceclztres.
E. Reporting of Major and Minor Accidents
and Injuries, Exposure to Toxic or Jnfec-
tious Materials, Unsafe Conditions and Prop-
erty Damages, and Rendering First-Aid.
F. General Laboratory Safety. 1. Fire.
2. Equipment.
3. Physical.
4. Chemical.
5. Radiological.
G. Safety Procedwes 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. Zone Classiiication.

5. Facility Monitoring Procedures.
6. Housekeeping.
J. Others. 1. Packaging and Shipment of
Biohaeardous 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 Reservrch Center.

REFERENCES

1. American National Standards Im,titute.
1968. Z 35.1.

2. Wedum, A. G. 1964. Laboratory safety
in research with infectious aerosols. Publio
Health Rep. 79:619-633.

3. Phillips, G. B. 1965. Microbiological haz-
ards in the laboratorv. J. Chem. Education.
Part One-Control, 4i:A43-A48. Part Tmo-
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. 1699204.

6. Chatlgny, M. A. and Clinger, D. I. 1969.
Contamination control in microbiology. In
R. L. Dimmick and A. B. Akers (eds.). An
Introduction to Experimental Aerobiology.
John Wiley & Sons, New York, pp. 194-263.

7. Chitigny, M. 1961. Protection against In-
fection in the microblologlcal laboratory, de-
vices and procedures. In W. W. Umbrelt (ed.) .
Advances in Applied Microbiology No. 3. Aca-
demic Press, New York, pp. 131-192.

8. Collins, C. H, Hartley, E. G., and Pils-
worth, R. 1974. The prevention of labcuatmy-
acquired Iniection. Public Health Laboratory
Service Monograph Series No. 6. Her Majesty's
Stationery Orace, London.

9. Anon. 1968. Good Laboratory Practices
Manual.  National  Cancer Institute, Be-

thesda, MD.

10. U.S. Public Hea1t.h Serrice. 1974. NIH
Biohazards Safety Guide. GPO Stock ,11740-
00383. SUDt. Document.s. U.S. Government
Printing b!?ice. Washington, D.C. 20402.
(83.85) -
ll.-Baldwin, C. L., Lemp. J. F., and Barbe-
lto. M. S. 1975. Biohazards assessment in
large-scale ronal centrifugation. Appl. Mi-
crobiol 29 : 484490.

12. Morris, `6. A. and Everall. P. H. 1972.
Safe disposal of air discharged from centri-
fuses. J. Clin. Path. 25:742.

i3. Hall, C. V. 1975. A bioIogica1 safety
centrifuge. Health Lab. Sci. 12: 104-106.
14. Reitman, M. and Wedum, A. 0. 1966.

hIicrobiologica1 safety. Public Health Rep.
71: 659465.

15. Hanel, E. and Kruse, R. H. 1967. Lab-
oratory-acquired mgcoses. Misc. Publ. 28, AD-
665376. Frederick Cancer Research Center.
P. 0. Box B, Frederick, MD.

16. Smadel, J. E. 1951. The hazard of ac-
quiring virus and rickettsial diseases in the
laboratory. Amer. J. Public Healbh 41:788-
795. -

17. Heckly, R. J. Quoted in A. H. Harris
and M. B. Coleman (eds.). 1963, Laboratory
infections and accidents. In Diaenostic Pro-
cedures and Reagents. Amer. P<blic Health
Assoc., Inc., New York.

18. Grieff, D. 1969. Safe procedure for open-
ing evacuated glass ampoules containing
dried pathogens. Appl. Microbial. 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 61 dangerous
pathogens. Cmnd 6654, ISBN 0 lo 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 Wlnkler, K. C. 1973.
Airborne transmission and airborne infer-
tion. John Wiley & Sons, New York: A. Page
415, B. Page 425, C. Page 429, D. Page 434,
E. Pages 436437, F. Page 461, 0. Page 494.

24. Lidwell, 0. 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 Genera1 Microbiology,
Cambridge Univ. Press, London: A. Page 17,
B. Page 123, C. Pages'127-128, D. Page 130.

26. 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 D&ease Control.
DHEW Publication No. CDC 768118. USPHS.
CDC, Atlanta, GA.
2'7. U.S. Army 1969. Safety regulations,
microbiological,  chemical and industrial
safety. FDR 385-l. Fort D&rick, Frederick,
MD.
28. Lidwell, 0. XI., Brock, B., Shooter, R. A.,
Cooke, E. M., and Thomas, G. E. 1975. IV.
Airborne dispersal of Staphylococcus aurew
and its nasal acquisition by patients.
29. Guyton, H. 0. and Decker, H. M. 1963
Respiratory protection provided by flve new
contagion masks. Appl. Microbial. 11:66-66.
30. Braymen, D. T, Songere, J. R., end Sul-
livan, J. F. 1974. Ef?ectlvenesS of footwear
decontamination methods for preventing the
spread of infectious agenta. Lab. Anti SCL
24 : 868-694.

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


NOTICES

31. Barbeito, M. S., Mathews, C. T., and
Tavlor, L. A. 1967. hflcrobiological laboratory
ha&.rd of bearded men. Appl. Microbial.
15:899-906.

32. U.S. Public Hea!th Sewice. 1974. Guide
for the Care and Use of Laboratory Animals.
DHEW Publication No. (NIH) 74-23. U. S.
Government Printing Office, Washington,
D.C. 20402. Price: ?O$. Stock No. 1740-0343.

33. Code of Federal Regulations, Title 9-
Animals and Animal Products. For sale by
Supt. of Docbments, Government Printing
Office, Washington, D.C. 20402. (In the capi-
tal city of most states, a copy limited to 9
CF'R Chapter 1. Subchapter A, Parts I,23
can be obtained from the Federal Veterinar-

ian in Charge, Anima: and Plant Health
Inspection Service.)

34. Seamer, J. (ed.). 1972. Safety in the
animal house. Laboratory Animal Handbook
5, Laboratory Anima!s. Ltd., London.
35. Gay, W. I. (ed.). 1965. Methods of an:-
ma! experimentation, Vol. 1. Academic Pre-5.
NCiV Pork.

36. Perkins, F. T. and O'Donoghue, P. X;.
i969. 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, II, III. Handbook of Labors-
tory Animal Science CRC Press, Inc., Cleve-
land, OH.

38. Spa;lldin& E. H 1972, Chcmlcal diq-
infection and antisepsis !n the hospital. J,
Hospital Res. 9:5-31.

39. Lawrence, C. A. and Block. S. S. (eds
1976. Disiufec:io::. srer!llzatio:1, and px,-
ervation. Lea & FeSiger, Philndeip!~is, P.1
40. Steelier, P. Q, (ed.). 1968. The hler,.k
Index. Merck & Co., Inc., Rahaay, NJ.
41. Taylor, L. A., Barbeito, hf. S., ai;d
Gremillion. G. G. 1969. Paraformaldehyde ior
surface sterilization and detoxification. App:
Microbial. 17:614-618.

42. Jemskl. J. V. and Phillips, G. B. 1963
Aerosol challenge of animals. In W. I. C3ay
(ed.) Methods of animal experimentation.
pp. 273-341. Academic Press, New York.
[FR Doc.TG-2>512 Filed 9-8%76:8:45 am]

FRDRRAL REGISTER, VOi 41, NO. 176THURSDAY, SEPTEMBER 9, 1976