27914 
NOTICES 
(i) Monolithic walls, floods, and ceilings 
in which all penetrations such as for air 
ducts, electrical conduits, and utility 
pipes are sealed to assure the physical 
isolation of the work area and to facili- 
tate housekeeping and space decontami- 
nation; (ii) air locks through which sup- 
plies and materials can be brought safely 
into the facility; (iii) contiguous cloth- 
ing change and shower rooms through 
which personnel enter into and exit from 
the facility; (iv) double-door autoclaves 
to sterilize and safely remove wastes and 
other materials from the facility; (v) a 
biowaste treatment system to sterilize 
liquid effluents if facility drains are in- 
stalled; (vi) a separate ventilation sys- 
tem which Maintains negative air pres- 
sures 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 system is not encouraged; 
if one is installed, each branch line lead- 
ing to a laboratory shall be protected by 
a high efficiency particulate air filter. 
The following practices shall apply to 
all experiments requiring P4 level physi- 
cal containment; (i) The universal bio- 
hazard sign is required on all facility 
access doors and all interior doors to in- 
dividual laboratory rooms where experi- 
ments are conducted. Only persons whose 
entry into the facility or individual labo- 
ratory rooms is required on the basis of 
program or support needs shall be au- 
thorized to enter. Such persons shall be 
advised of the potential biohazards and 
instructed as to the appropriate safe- 
guards to ensure their safety before 
-entry. Such persons shall comply with 
the instructions and all other posted en- 
try and exit procedures. Under no con- 
dition shall children under 15 years of 
age be allowed entry, (ii) Personnel shall 
enter into and exit from the facility 
only through the clothing change and 
shower rooms. Personnel shall shower at 
each exit from the facility. The air locks 
shall not be used for personnel entry or 
exit except for emergencies, (iii) Street 
clothing shall be removed in the outer 
facility side of the clothing change area 
and kept there. Complete laboratory 
clothing including 1 undergarments, pants 
and shirts or jumpsuits, shoes, head 
cover, and gloves shall be provided and 
used by all persons who enter into the 
facility. Upon exit, this clothing shall 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 air lock. After the outer door 
(opening to the corridor outside of fa- 
cility) has been secured, personnel oc- 
cupying the facility shall retrieve 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 
laboratory 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 m Biological 
Safety Cabinets. 1 All materials, before 
See footnotes at end of article. 
removal from these cabinets, shall be 
sterilized or transferred to a non-break- 
able sealed container, which is then re- 
moved from the system through a chemi- 
cal decontaminated tank, autoclave, or 
after 'the entire system has been 
decontaminated. 
(vii) No materials shall be removed 
from the facility unless they have been 
sterilized or decontaminatel in a manner 
to prevent the release of agents requiring 
P4 physical containment. All wastes and 
other materials and equipment not dam- 
aged by high temperautre or steam shall 
be sterilized in the double-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 
chamber designed for gas sterilization. 
Other materials which may be damaged 
by temperature or steam shall be steri- 
lized by gaseous or vapor methods in an 
air lock or chamber designed for this 
purpose, (viii) Eating, drinking, smok- 
ing, and storage of food are not per- 
mitted in the facility. Foot-operated 
water fountains located in the facility 
corridors are permitted. Separate po- 
table water piping shall be provided for 
these water fountains, (ix) Facilities to 
wash hands shall be available within the 
facility. Persons shall wash hands after 
experiments, (x) An insect and rodent 
control program shall be provided, (xi) 
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 
protected by a filter and liquid trap in 
addition to the branch line HEPA filter 
mentioned above, (xiii) Use of the hypo- 
dermic needle and syringe shall be 
avoided when alternate methods are 
available, (xiv) If experiments of lesser 
biohazard potential are to be conducted 
in the facility concurrently with experi- 
ments requiring P4 level containment, 
they shall be confined in Class I or Class 
II Biological Safety Cabinets 1 or isolated 
by other physical containment equip- 
ment. Work surfaces of Biological Safety 
Cabinets 1 and other equipment shall be 
decontaminated following the comple- 
tion of the experimental activity con- 
tained within them. Mechanical pipet- 
ting devices shall be used. All other prac- 
tices listed above with the exception of 
(vi) shall apply. 
C. Shipment. To protect product, per- 
sonnel, and the environment, all recom- 
binant DNA material will be shipped in 
containers that meet the requirements 
issued by the U.S. Public Health Service 
(Section 72.25 of Part 72, Title 42, Code 
of Federal Regulations), Department of 
Transportation (Section 173.387(b) of 
Part 173, Title 49, Code of Federal Reg- 
ulations) and the Civil Aeronautics 
Board (C.A.B. No. 82, Official Air Trans- 
port Restricted Articles Tariff No. 6-D) 
for shipment of etiologic agents. Label- 
ing requirements specified in these Fed- 
eral regulations and tariffs will apply to 
all viable recombinant DNA materials in 
which any portion of the material is 
derived from an etiologic agent listed in 
paragraph (c) of 42 CFR 72.25. Addi- 
tional information on packing and ship- 
ping is given in a supplement to the 
guidelines (Appendix D, part X) . 
D. Biological containment levels. Bio- 
logical barriers are specific to each host- 
vector system. Hence the criteria for this 
mechanism of containment cannot be 
generalized to the same extent as for 
physical containment. This is particu- 
larly true at the present time when our 
experience with existing host-vector sys- 
tems and our predictive knowledge about 
projected systems are sparse. The clas- 
sification of experiments with recombi- 
nant DNAs that is necessary for the 
construction of the experimeneal guide- 
lines (Section ni) can be accomplished 
with least confusion if we use the host- 
vector system as the primary element 
and the source of the inserted DNA as 
the secondary element in the classifica- 
tion. It is therefore convenient to specify 
the nature of the biological containment 
under host- vector headings such as those 
given below for Escherichia coli K-12. 
III. EXPERIMENTAL GUIDELINES 
A general rule that, though obvious, 
deserves statement is that the level of 
containment required for any experiment 
on DNA recombinants shall never be less 
than that required for the most hazard- 
ous 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. 
Handling the purified DNA will generally 
require less stringent precautions than 
will propagating the DNA. However, the 
DNA itself should be handled at least 
as carefully as one would handle the most 
dangerous of the DNAs used to make it. 
The above rule by itself effectively pre- 
cludes certain experiments — namely, 
those in which one of the components 
is in Class 5 of the “Classification of 
Etiologic Agents on the Basis of Haz- 
ard” (5) , as these are excluded from the 
United States by law and USDA admin- 
istrative policy. There are additional ex- 
periments which may engender such seri- 
ous biohazards that they are not to be 
performed at this time. These are con- 
sidered prior to presentation of the con- 
tainment guidelines for permissible 
experiments. 
A. Experiments that are not to be per- 
formed. We recognize that it can be 
argued that certain of the recombinants 
placed in this category could be ade- 
quately contained at this time. Nonethe- 
less, our estimates of the possible dangers 
that may ensue if that containment fails 
are of such a magnitude that we consider 
it the wisest policy to at least defer 
experiments on these recombinant DNAs 
until there is more information to accu- 
rately assess that danger and to allow 
the construction of more effective biologi- 
cal barriers. In this respect, these guide- 
lines are more stringent than those ini- 
tially recommended (1). 
The following experiments are not to 
be initiated at the present time: (i) Clon- 
FEDERAL REGISTER, VOL. 41, NO. 131 — WEDNESDAY, JULY 7, 1976 
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