Federal Register / Vol. 48, No. 106 / Wednesday, June 1, 1983 / Notices 
24579 
features. Procedures and methods used 
in the testing shall be appropriate for the 
equipment design and for recovery and 
demonstration of the test organism. 
Records of tests and results shall be 
maintained on file. 
Appendix K -III— I. A closed system 
jsed for the propagation and growth of 
/iable organisms containing 
•ecombinant DN.\ molecules shall be 
jermanently identified. This 
dentification shall be used in all records 
eflecting testing, operation, and 
naintenance and in all documentation 
elating to use of this equipment for 
esearch or production activities 
nvolving viable organisms containing 
ecombinant DNA molecules. 
Appendix K— III— J. The universal 
piohazard sign shall be posted on each 
losed system and primary containment 
quipment when used to contain viable 
rganisms containing recombinant DNA 
lolecules. 
Appendix K— III— K. Emergency plans 
equired by Section IV-B-3-f shall 
iclude methods and procedures for 
andling large losses of culture on an 
mergency basis. 
Appendix K-IV. P3-LS Level. 
Appendix K-IV-A. Cultures of viable 
rganisms containing recombinant DNA 
lolecules shall be handled in a closed 
ystem (e.g., closed vessels used for the 
ropagation and growth of cultures) or 
ther primary containment equipment 
:.g., Class III biological safety cabinet 
antaining a centrifuge used to process 
ilture fluids) which is designed to 
revent the escape of viable organisms, 
olumes less than 10 liters may be 
undled outside of a closed system 
'ovided all physical containment 
■quirements specified in Appendix G- 
-C of the Guidelines are met. 
Appendix K-IV-B. Culture fluids 
xcept as allowed in Appendix K-D-3) 
lall not be removed from a closed 
'stem or other primary containment 
juipment unless the viable4>rganisms 
intaining recombinant DNA molecules 
ive been inactivated by a validated 
activation procedure. A validated 
activation procedure is one which has 
:en demonstrated to be effective using 
e organisms that will serve as the host 
r propagating the recombinant DNA 
olecules. 
Appendix K-IV-C. Sample collection 
im a closed system, the addition of 
aterials to a closed system, and the 
insfer of culture fluids from one closed 
stem to another shall be done in a 
anner which prevents the release of 
rosols or contamination of exposed 
rfaces. 
Appendix K-IV-D. ExhaUst gases 
noved from a closed system or other 
imary containment equipment shall be 
treated by filters which have efficiencies 
equivalent to HEPA filters or by other 
equivalent procedures (e.g., incineration) 
to prevent the release of viable 
organisms containing recombinant DNA 
molecules to the environment. 
Appendix K-IV-E. A closed system or 
other primary containment equipment 
that has contained viable organisms 
containing recombinant DNA molecules 
shall not be opened for maintenance or 
other purposes unless it has been 
sterilized by a validated sterilization 
procedure. A validated sterilization 
procedure is one which has been 
demonstrated to be effective using the 
organisms that will serve as the host for 
propagating the recombinant DNA 
molecules. 
Appendix K-IV-F. A closed system 
used for the propagation and growth of 
viable organisms containing 
recombinant DNA molecules shall be 
operated so that the space above the 
culture level will be maintained at a 
pressure as low as possible, consistent 
with equipment design, in order to 
maintain the intergrity of containment 
features. 
Appendix K-IV-G. Rotating seals and 
other mechanical devices directly 
associated with a closed system used to 
contain viable organisms containing 
recombinant DNA molecules shall be 
designed to prevent leakage or shall be 
fully enclosed in ventilated housings 
that are exhausted through filters which 
have efficiencies equivalent to JdEPA 
filters or through other equivalent 
treatment devices. 
Appendix K-IV-H. A closed system 
used for the propagation and growth of 
viable organisms containing 
recombinant DNA molecules and other 
primary containment equipment used to 
contain operations involving viable 
organisms containing recombinant DNA 
molecules shall include monitoring or 
sensing devices that monitor the 
integrity of containment during 
operations. 
Appendix K-IV-I. A closed system 
used for the propagation and growth of 
viable organisms containing 
recombinant DNA molecules shall be 
tested for integrity of the containment 
features using the organisms that will 
serve as the host for propagating the 
recombinant DNA molecules. Testing 
shall be accomplished prior to the 
introduction of viable organisms 
containing recombinant DNA molecules, 
and following modification or 
replacement of essential containment 
features. Procedures and methods used 
in the testing shall be appropriate for the 
equipment design and for recovery and 
demonstration of the test organism. 
Records of tests and results shall be 
maintained on file. 
Appendix K-IV-J. A closed system 
used for the propagation and growth of 
viable organisms containing 
recombinant DNA molecules shall be 
permanently identified: This 
identification shall be used in all records 
reflecting testing, operation and 
maintenance and in all documentation 
relating to the use of this equipment for 
research production activities involving 
viable organisms containing 
recombinant DNA molecules. 
Appendix K-IV-K. The universal 
biohazard sign shall be posted on each 
closed system and primary containment 
equipment when used to contain viable 
organisms containing recombinant DNA 
molecules. 
Appendix K-IV-L. Emergency plans 
required by Section IV-B-3-f shall 
include methods and procedures for 
handling large losses of culture on an 
emergency basis. 
Appendix K-IV-M. Closed systems 
and other primary containment 
equipment used in handling cultures of 
viable organisms containing 
recombinant DNA molecules shall be 
located within a controlled area which 
meets the following requirements: 
Appendix K-IV-M-1. The controlled 
area shall have a separate entry area. 
The entry area shall be a double-doored 
space such as an air lock, anteroom or 
change room that separates the 
controlled area from the balance of the 
facility. 
Appendix K-IV-M-2. The surfaces of 
walls, ceilings, and floors in the 
controlled area shall be such as to 
premit ready cleaning and 
decontamination. 
Appendix K-IV-M-3. Penetrations 
into the controlled area shall be sealed 
to permit liquid or vapor phase space 
decontamination. 
Appendix K-IV-M-4. All utilities and 
service or process piping and wiring 
entering the controlled area shall be 
protected against contamination. 
Appendix K-IV-M-5. Handwashing 
facilities equipped with foot, elbow, or 
automatically operated values shall be 
located at each major work area and 
near each primary exit. 
Appendix K-1V-M-6. A shower 
facility shall be provided. This facility 
shall be located in close proximity to the 
controlled area. 
Appendix K-IV-M-7. The controlled 
area shall be designed to preclude 
release or culture fluids outside the 
controlled area in the event of an 
accidental spill or release from the 
closed systems or other primary 
containment equipment. 
ri5<>] 
