49598 
NOTICES 
DNA molecules end when they leave the 
laboratory. 
•(7) Care shall be exercised to minimize 
the creation of aerosols. For example, manip- 
ulations such as Inserting a hot inoculating 
loop or needle Into a culture, flaming an 
Inoculation loop or needle so that It splat- 
ters, and forceful ejection of fluids from pi- 
pettes or syringes shall be avoided. 
(8) Contaminated materials that are to 
be decontaminated at a site away from the 
laboratory shall be placed In a durable leak- 
proof container which Is closed before re- 
moval from the laboratory. 
•(9) Only persons who have been advised 
of the' nature of the research being con- 
ducted shall enter the laboratory. 
•(10) Children under 12 years of age shall 
not enter the laboratory. 
*(11) The universal biohazard sign shall 
be posted on all laboratory access doors when 
experiments requiring P2 containment are In 
progress. Freezers and refrigerators used to 
8 tore organisms containing recombinant 
DNA molecules shall also be posted with the 
universal biohazard sign. 
(12) An Insect and rodent control program 
shall be Instituted. 
*(13) The use of laboratory gowns, coats, 
or uniforms Is required. Laboratory clothing 
shall not be worn to the lunch room or out- 
side of the building In which the laboratory 
Is located. 
•(14) Animals not related to the experi- 
ment shall not be permitted In the labora- 
tory. 
•(15) Use of the hypodermic needle and 
syringe shall be avoided when alternative 
methods are available. 
*(16) The laboratory shall be kept neat, 
clean, and free of materials not pertinent to 
the research. 
•(17) Experiments of lesser biohazard 
potential can be carried out concurrently In 
carefully demarcated areas of the same 
laboratory. 
b. Containment equipment. ‘Biological 
safety cabinets 1 shall be used to contain 
aerosol-producing equipment such as 
blenders, lyophlllzers, sonlcators, and cen- 
trifuges when used to process organisms con- 
taining recombinant DNA molecules, except 
where equipment design provides for con- 
tainment of the potential aerosol. For 
example, a centrifuge may be operated In the 
open If a sealed head or safety centrifuge 
cups are used. 
c. Special laboratory design. * *An autoclave 
for sterilization of wastes and contaminated 
materials shall be available In the same 
building In which organisms containing 
recombinant DNA molecules are used. 
3. P3 level. — a. Laboratory practices. (1) 
doors shall be kept closed while experiments 
are In progress. 
•(2) Work surfaces shall be decontami- 
nated following the completion of the experi- 
mental activity, and Immediately following 
spills of organisms containing recombinant 
DNA molecules. 
(3) All biological wastes shall be decon- 
taminated before disposal. Other con- 
taminate materials such as glassware, animal 
cages, and laboratory equipment shall be de- 
contaminated before washing, reuse, or 
disposal. 
(4) Mechanical pipetting devices shall be 
used; pipetting by mouth Is prohibited. 
(5) Elating, drinking, smoking, and stor- 
age of food are not permitted In the labora- 
tory area. 
(6) Persons shall wash their hands after 
handling organisms containing recombinant 
DNA molecules and when they leave the 
laboratory. 
(7) Care shall be exercised to m i n i m ize the 
creation of aerosols. For example, manipula- 
tions such as Inserting a hot Inoculating 
loop or needle Into a culture, flaming an 
Inoculation loop or needle so that It splat- 
ters, and forceful ejection of fluids from 
pipettes or syringes shall be avoided. 
(8) Contaminated materials that are to 
be decontaminated at a site away from the 
laboratory shall be placed In a durable leak- 
proof container which Is closed before re- 
moval from the laboratory. 
•(9) Entry Into the laboratory shall be 
through a controlled access area. Only per- 
sons who have been advised of the nature 
of the research being conducted shall enter 
the controlled access area. Only persons re- 
quired on the basis of program or support 
needs shall be authorized to enter the labora- 
tory. Such persons shall be advised of the 
nature of the research being conducted be- 
fore entry, and shall comply with all required 
entry and exit procedures. 
(10) Children under 12 years of age shall 
not enter the laboratory. 
•(11) The universal biohazard sign shall 
be posted on the controlled access area door 
and on all laboratory doors when experiments 
requiring P3 level containment are In prog- 
ress. Freezers and refrigerators used to store 
organisms containing recombinant DNA 
molecules shall also be posted with the uni- 
versal biohazard sign. 
(12) An Insect and rodent control program 
shall be Instituted. 
•(13) Laboratory clothing that protects 
street clothing (l.e., long sleeve solid-front 
or wrap-around gowns, no-button or slip- 
over Jackets, etc.) shall be worn In the lab- 
oratory. Front-button laboratory coats are 
unsuitable. Laboratory clothing shall not be 
worn outside the laboratory and shall be de- 
contaminated before It Is sent to the laundry. 
*(14) Raincoats, overcoats, topcoats, coats, 
hats, caps, and such street out-wear shall 
not be kept In the laboratory. 
*(15) Gloves shall be worn when handling 
materials requiring P3 containment. They 
shall be removed aseptically Immediately fol- 
lowing the handling procedure and decon- 
taminated. 
•(16) Animals and plants not related to 
the experiment shall not be permitted In the 
laboratory. 
* ( 17) Vacuum outlets shall be protected by 
filter and liquid disinfectant traps. 
(18) Use of hypodermic needle and syringe 
shall be avoided when alternative methods 
are available. 
•See sec. II, D. for description of biological containment. 
c. Special Laboratory Design. *(1) The lab- 
oratory shall be separated from areas which 
are open to unrestricted traffic flow within 
the building by a controlled access area. A 
controlled access area Is an anteroom, a 
change room, an air lock or any other double 
door arrangement which separates the lab- 
oratory from areas which are open to un- 
restricted traffic flow. 
• (2) The surfaces of walls, floors, and 
ceilings shall be readily cleanable. Penetra- 
tions through these surfaces shall be sealed 
or capable of being sealed to facilitate space 
decontamination. 
(19) The laboratory shall be kept neat, 
clean, and free of materials not pertinent to 
the research. 
•(20) If experiments Involving other or- 
ganisms which require lower levels of con- 
tainment are to be conducted In the same 
laboratory concurently with experiments re- 
quiring P3 level physical containment, they 
shall be conducted In accordance with all 
P3 level laboratory practices. 
b. Containment Equipment. • ( 1 ) Biologi- 
cal safety cabinets * shall be used for all 
equipment and manipulations that produce 
aerosols (e.g., pipetting, dilutions, transfer 
operations, plating, flaming, grinding, blend- 
ing, drying, sonicating, shaking, centrifug- 
ing etc.) where these procedures Involve 
organisms containing recombinant DNA 
molecules, except where equipment design 
provides for containment of the potential 
aerosol. 
•(2) Laboratory animals held In a P3 area 
shall be housed In partial containment cag- 
ing systems such as Horsfall units, open cages 
placed In ventilated enclosures, solid wall 
and bottom cages covered by filter bonnets, 
or solid wall and bottom cages placed on 
holding racks equipped with ultraviolet ra- 
diation lamps and reflectors. 
Note: Conventional caging systems may 
be used; Provided, That all personnel wear 
appropriate personal protective devices. 
These shall Include at a minimum, wrap 
around gowns, head covers, gloves, shoe cov- 
ers, and respirators. All personnel shall 
shower on exit from areas where these per- 
sonal protective devices are required.) 
•(3) Alternative selection of containment 
equipment. Experimental procedures Involv- 
ing a host-vector system which provides a 
one-step higher level of biological contain- 
ment than that specified In Section IH can 
be conducted In the P3 laboratory using 
containment equipment requirements speci- 
fied for the P2 level of physical containment. 
Experimental procedures Involving a host- 
vector system which provides a one-step 
lower level of biological containment than 
that specified In Section m can be conducted 
In the P3 laboratory using containment 
equipment requirements specified for the P4 
level of physical containment. Alternative 
combinations of containment safeguards are 
shown In Table I on the following page. 
•(3) A foot, elbow, or automatically oper- 
ated hand washing facility shall be pro-* 
vlded near each primary laboratory exit area. 
•(4) Windows In the laboratory shall be 
sealed. 
* (5) Laboratory doors shall be self-closing. 
(6) An autoclave for sterilization of wastes 
and contaminated materials shall be avail- 
able in the same building In which orga- 
nisms containing recombinant DNA colecules 
are used. 
•(7) An exhaust air ventilation system 
shall be provided. This system shall be bal- 
anced so that the direction of air flow Is 
Table I. — Combinations of containment safeguards 
Classification of experiment according Combinations of physical and biological containment 
to guidelines 
Physical 
containment 
Biological* 
containment 
Laboratory 
design 
specified for — 
Laboratory 
practices 
specified for — 
Containment 
equipment 
specified for— 
Biological 
containment 
P3 
HV3 
P3 
P3 
P3 
HV3 
P3 
HV3 
P3 
P3 
P4 
HV2 
P3 
HV2 
P3 
P3 
P3 
HV2 
P3 
HV2 
P3 
P3 
P2 
HV3 
P3 
HV2 
P3 
P3 
P4 
HV1 
P3 
HV1 
P3 
P3 
P3 
HV1 
P3 
HV1 
P3 
P3 
P2 
HV2 
FEDERAL REGISTER, VOL. 42, NO. 187— TUESDAY, SEPTEMBER 27, 197? 
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