NOTICES 
49597 
DNA molecules formed from any combina- 
tion of DNA* will not be considered novel 
when all the components are derived from 
genomes known to replicate within the orga- 
nism uasd to propagate the recombinant 
DNA.* 
A major task of this Committee has been 
first to prepare and then periodically to re- 
vise Ouldellnes that will allow the promise 
of recombinant DNA research to be realized, 
while providing the caution that la dictated 
by concerns about the possibility of hazard, 
however remote. 
Tha present revisions take Into account 
many communications from both scientists 
and non-sclentlsta since the original publi- 
cation of the Ouldellnes During this period 
the Committee has also become better In- 
formed about the general ecology and epi- 
demiology of Infectious microorganisms Of 
particular relevance has been the Informa- 
tion received from many medical micro- 
biologists. Including data from experiments 
with tschericMa coll K-13. These experi- 
ments Include a demonstration that strain 
K-13 cannot be made pathogenic even when 
provided, by standard genetic techniques, 
with the genes for known toxins and other 
pathogenic properties. Other relevant experl- 
menu that have been reported show that the 
Incorporation of foreign DNA does not In- 
crease. but rather tends to decrease, the gen- 
eral Otness of microorganisms, this phe- 
nomenon further contributes to the unlike- 
lihood that cells carrying recombinant DNA 
will survive In nature Indeed, everything 
we have learned tends to diminish our esti- 
mate of the risk associated with recombinant 
DNA In t coll K-13 Nevertheless, the re- 
vised Ouldellnes continue to be deliberately 
restrictive, with the InUnt of erring on the 
tide of caution. 
In constructing these Ouldellnes It was 
isceaaary to define boundary conditions for 
he different levels of physical and biological 
ontalnment and for the classes of expert- 
nenU to which they apply We recognlxe 
hat these definitions do not take Into ac- 
count all existing and anticipated Informa- 
tion on special procedures that will allow 
particular experiment* to be carried out 
under different conditions than Indicated 
her* without affecting risk Indeed, we urge 
that Individual Investigators devise simple 
and more effective containment procedures 
and that Investigators and study sections 
recommend changes In the Ouldellnes to 
permit their us*. 
It la urged that all publications dealing 
with recombinant DNA work Include a de- 
scription of the physical and biological con- 
tainment procedures employed, to aid others 
who might consider repeating this work. 
XI CoirranvifxxT 
Effective biological safety programs have 
been operative in a variety of laboratories 
for many years. Considerable Information 
therefor* already exist* for the design of 
physical containment facilities and the se- 
lection of laboratory procedures appllcabls 
to organisms carrying recombinant DNAs 
11-14). The existing programs rely upon 
mechanisms that, for convenience, can be 
divided into two categories: (l) A set of 
standard practices that are generally used In 
microbiological laboratories, and (II) special 
procedures, equipment, and laboratory In- 
stallations that provide physical barriers 
which are applied In varying degrees accord- 
ing to the estimated blo-hazard. 
Experiment* on recombinant DNAs by 
their very nature lend themselves to a third 
containment mechanism — namely, the appli- 
cation of highly specific biological barriers. 
In fact, natural barriers do exut which limit 
either the inactivity of a vector qr 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 In which they 
replicate can be genetically designed to de- 
crease by many orders of magnitude the prob- 
ability of dissemination of recombinant 
DNAs outside the laboratory. 
As these three means of containment are 
complementary, different levels of contain- 
ment appropriate for experiments with dif- 
ferent recombinants can be established by 
applying different combinations of the physi- 
cal and biological barriers to a constant use 
of the standard practices. We consider these 
categories of containment separately here 
In order that such combinations can be con- 
veniently expressed In the Ouldellnes for re- 
search on the different kinds of recombinant 
DNAs (Section HI). 
A. Standard practical and training The 
first principle of containment Is a strict ad- 
herence to good microbiological practices 
(1-10). Consequently, all personnel directly 
or Indirectly Involved In experiments on re- 
combinant DNAs must receive adequate In- 
struction. This should Include at least 
training in aseptic techniques and Instruc- 
tion in the biology of the organisms used 
In the experiments, so that the potential 
biohazards can be understood and appreci- 
ated. 
Any research group working with agents 
with a known or potential biohazard should 
have an emergency plan which describes the 
procedures to be followed If an accident con- 
taminates personnel or environment The 
principal Investigator must ensure that 
everyone In the laboratory Is familiar with 
both the potential hazard* of the work and 
the emergency plan. If a research group la 
working with a known pathogen for which 
an effective vaccine la available, all worker* 
should be Immunized. 8erologlcal monitor- 
ing. where appropriate, should be provided. 
B Physical containment levels — The ob- 
jective physical containment la to confine 
organisms containing novel recombinant 
DNA molecule*, and thus to reduo* the po- 
tential for exposure of the laboratory worker, 
persona outside of th* laboratory, and the 
environment to organlans containing novel 
recombinant DNA molecules Physical con- 
tainment Is achieved through th* use of 
laboratory practices, containment equip- 
ment. and special laboratory design Empha- 
sis Is placed on primary means of physical 
containment which are provided by labora- 
tory practices and containment equipment. 
Special laboratory design provides a second- 
ary means of protection against th* acci- 
dental release of organisms outside th* lab- 
oratory or to th* environment. Special lab- 
oratory design la used primarily In facilities 
in which experiments of moderate to high 
potential hazards are performed. 
Combinations of laboratory practices, con- 
tainment equipment, and special laboratory 
design can be made to achieve different levels 
of physical containment. Pour levels of phys- 
ical containment, which are designated as 
PI. P3. P3. and P4. are described. It should 
be emphasized that the descriptions and as- 
signment* of physical containment detailed 
below are baaed on existing approaches to 
containment of pathogenic organlams For 
example the "Classification or Etlologlc 
Agents on the Basis of Hazard" (3), pre ; 
pared by the Center for Disease Control, 
describes four general levels which roughly 
correspond to our descriptions for PI, P2. 
P3. and P4: and the National Cancer Insti- 
tute describes three levels for research on 
oncogenic viruses which roughly correspond 
to our P3, P3, and P4 levels (3) . 
It Is recognized that other combinations 
of laboratory practices, containment equip- 
ment. and special laboratory design may be 
appropriate for containment of specific re- 
search activities. The Ouldellnes. therefore, 
allow alternative selections of primary con- 
tainment equipment within facilities that 
have been designed to provide P3 and P4 
levels of physical containment. The selection 
of alternative methods of primary contain- 
ment la dependent, however, on the level of 
biological containment provided by the host- 
vector system used in the experiment. Con- 
sideration will also be given by the Recom- 
binant DNA Molecule Program Advisory Com- 
mittee to other combinations which achieve 
an equivalent level of containment. Addi- 
tional material on physical containment for 
plant host-vector systems Is found on page 
UI-34. 
1. Pi level.— a. Laboratory practices. (1) 
Laboratory doors shall be kept dosed while 
experiments are In progress. 
(31 Work surfaces shall be decontami- 
nated dally, and Immediately following spills 
of organisms containing recombinant DNA 
molecules. 
(3) All biological wastes shall be decon- 
taminated before disposal. Other contami- 
nated materials such as glassware, animal 
cages, and laboratory equipment shall be 
decontaminated before washing, reuse, or 
disposal. 
(4) Although pipetting by mouth ts per- 
mitted. It Is recommended that mechanical 
pipetting devices be used When pipetting by 
mouth, cotton-plugged pipettes shall be em- 
ployed 
(5) Eating, drinking, smoking, and stor- 
age of roods are not permitted In the work- 
ing area 
(fl) Persons shall wash their hands after 
handling organisms containing recombinant 
DNA molecules and when they leave the 
laboratory 
(7) Care shall be taken In the conduct of 
all procedures to minimize the creation of 
aerosols. 
(8) Contaminated materials that are to be 
decontaminated at a site away from the lab- 
oratory shall be placed In a durable leak- 
proof container which Is closed before re- 
moval from the laboratory. 
(9) An Insect and rodent control program 
shall be Instituted 
(10) The use of laboratory gowns, coats, or 
uniforms Is discretionary with the laboratory 
supervisor. 
(11) U»e of the hypodermic needle and sy- 
ringe shall be avoided when alternative 
methods are available. 
b. Containment equipment. Special con- 
tainment equipment Is not required at the 
PI level. 
c. Special laboratory design. Special labo- 
ratory design la not required at the PI level. 
3. P2 leveP — a. Laboratory practices. (1) 
Laboratory doors shall be kept closed while 
experiments are In progress. 
(3) Work surfaces shall be decontaminated 
dally, and immediately following spills of or- 
ganisms containing recombinant DNA mole- 
cules. 
(3) All biological wastes shall be decon- 
taminated before disposal. Other contami- 
nated materials such as glassware, animal 
cages, and laboratory equipment shall be de- 
contaminated before washing, reuse, or dis- 
posal. 
•(4) Mechanical pipetting devices shall be 
used; pipetting by mouth Is prohibited. 
(5) Eating, drinking, smoking, and storage 
of food are not permitted In the laboratory 
area. 
(6) Persons shall wash their hands after 
handling organisms containing recombinant 
‘Denotes laboratory practices, contain- 
ment equipment, or special laboratory de 
sign which were not required at the next 
lower level of containment. 
FfDftAl IE0IST1I, VOL 43, NO. 147 — TUISOAY, SEPTEMBER 27, 1977 
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