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33180 
Federal Register / Vol. 56. No. 130 / Thursday. July 18. 1991 / Notices 
1 1 closed system and primary containment 
I equipment when used to contain viable 
' organisms containing recombinant DNA 
molecules. . 
; Appendix K-TVMC Emergency plans 
jrequired by Secbon shall • 
I indude methods and procedures for 
Jhandling large losses' of culture on an 
iemergency basis. . - 
\Appendix K-V BL3-LS Level, * • ■*-' ' 
1 ; Appendix K-V-A. Cultures ofjvjable 1 
jorganisms' contaming recomhlnaht bNA 
{molec^es ^^_be handed in a^osed 
! 'system (e.g., dosed ve^ds for &e’,_ 
; propagation arid grow^ of-cdture^ dr ' 
other primary bontai^rat.e^jpmra^^ ^ 
(e.g.. Class in biolo^^ safety cabinet 
i containing a centii^e used to process ' 
, culture fluids] which is designed to 
prevent the escape of viable organisbis. 
: Ivolumes less than 10 liten may be 
jhandled outside of a closed system 
I provided all physical containment 
F iquirements specified in appendix G— 
-C of the Guidelines are met 
Appendix K-V-B. Culture fluids 
I (except as allowed in appendix K-V-C] 
ishaU not be removed from a closed 
jsystem or other primary containment 
j pquipment unless the viable organisms 
1 containing recombinant DNA molecules 
'have been inactivated by a validated 
inactivation procedure. A validated 
' inactivation procedure is one which baa 
been demonstrated to be effective using 
, tlie organisms that will serve as the host 
; lor propagating the recombinant DNA • 
; imolecales. 
; Appendix K-V-C Sample collection 
horn a closed system, the addition of 
materials to a closed system, and the 
' transfer of culture fluids from one closed 
system to another shall be done in a 
ca.nner which prevents the release of 
aerosols or contamination of exposed 
. surfaces. 
Appendix K-V-D. Exhaust gases 
!, removed from a closed system or other 
'primary 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 
erganisms containing recombinant DNA 
Dolecules to the environment 
Appendix K-V-E. A closed system or 
)lhcr primary containment equipment 
hat has contained viable organisms 
xsntaining recombinant DNA molecules 
ihall not be opened for maintenance or 
ither purposes unless It has been 
ilerilized by a validated sterilization 
irocedure. A validated sterilization 
•rocedure is one which has been 
lemonstrated to be effective using the 
irganisms that will serve as the host for 
Topagating the recombinant DNA 
3 jlecules. 
Appendix K-V-F. A closed system 
used for the propagation and growth bf- 
viable organisms contaming 
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 integrity of containment ' 
features. . ■ -rvv; • -■ 
Appendix. K-V-G. Rotating seeds and 
other mechanical devices directly 'J-'i'. 
, associated with a closed system nsed to 
... contain viable organisms containing - - - ' - 
..recombinant DNA molecules shall.be .. . 
■ designed .to prevemt leakage or shall be - 
fully enclosed in ventilated housings ■ 
that are exhausted through filters which ^ 
have effidendes equivsdent to HEPA 
filters or through other d}uivalent 
treatment devices. ‘ 
Appendix K-V-^ A closed system, 
used for the propagation wd growth of 
viable orgainisms containing 
recombinant DNA molecules and other 
primary containment equipment used to 
contain operations involving viable 
organisms containing recombinant DNA 
molecules shall indude monitoring or ' 
sensing devices that moniter the 
integrity of containment during 
operatiSns. 
Appendix K-V-L A dosed system 
used for the propagation and growth of 
viable organisms containing 
recombinant DNA molecules shall be 
tested for integrity of the contaimheht 
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 contaimnent 
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 end results shall be 
maintained on file. 
Appendix K-V-J. A dosed 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 
resear^ production activities hu'olving 
viable organisms containing 
recombinant DNA molecdes. 
Appendix K-V-K. The imiversal 
biohiizard sign shall be posted on each 
closed system and primary containment 
equipment when used to contain viable 
organisms containing recombinant DNA 
molecules. 
Appendix K-V-L. Emergency plans 
required by section IV-B-3-f sh^ ... 
Indudc methods and procedures for . 
• handling large losses of culture on an 
emergency basis. - . .. 
Appendix K-V-M. Closed systems and 
other primary containment equipment 
used in handling cultures of viable ' . 
organisms rantaining recombinant DNA . 
molecules shall be located wlthin a .- ■ 
controlled area which meets the ■>.• •. . 
•ToUowingTequifements: .vr-V'.-.-v. «■. .. >■ 
• • ' AjJpendix K-V-M-1- The controlled 
'area sh^'have a -separate ditiy area.' 
The eritry area shall be a double-door^ 
space such as an air lock^ anterboi^ br 
diange room that separates' the ' ‘ r’ l;' . ; ' 
controlled area £rpm the balance, of the 
facility. ' ' 
Appendix K-V-M-2. The sn^aces of ' 
walls, ceilings, and floors In the 
controlled area shall be such os to 
permit ready cleaning and - . . 
decontamination. , 
Appendix K-V-M-3. Penetrations into 
the controlled area shall be sealed to 
permit liquid or vapor phase space 
decontamination. 
Appeiidix K-V-^-4i AH utilities and 
service or process piping and wiring 
entering the coritrqlled area shall be . 
pixitected against contamiriation. 
Appendix K-V-M-& Hand-y/ashing 
facihties equipped with foot, elbow, or 
automatically operated valves shall be 
located at each major work area and 
near each primary exit ' 
Appendix K-V-M-6, A shower facility 
shall be provided. This facility shall be 
located in close proximity to the 
controlled area. 
Appendix K-V-M-7, The controlled 
area shall be designed to preclude 
release of culture fluids outside the 
controlled area in the event of an 
accidental spill or release from the 
closed systems or other primary 
contaimnent equipment 
Appendix K-V-M-8. The controlled 
area shall have a ventilation system that 
is capable of controlling air movement - 
The movement of air shall be fiom areas 
of lower contamination potential to 
areas of higher contamination potential 
If the ventilation system provides 
positive pressure supply air, the system 
shall operate in a manner that prevents 
the reversal of the direction of air 
movement or shall be equipped with an 
alarm that would be actuated in the 
event that reversal in the direction of air 
movement were to occur. The exhaust 
air from the controlled area shall not be 
recirculated to other areas of the ' - • . 
facility. The exhaust air from the 
controlled area may not be discharged 
to the outdoors without being HEPA 
filtered, subjected to thermal oxidation. 
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Recombinant DNA Research, Volume 14 
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