APPENDIX D— 20 
Welfare, the Assistant Secretary for 
Health, Department of Health, Educa- 
tion, and Welfare, and the Director, Na- 
tional Institutes of Health, on a progi’am 
for the evaluation of potential biological 
and ecological hazards of recombinant 
DNAs (molecules resulting from differ- 
ent segments of DNA that have been 
joined together in cell-free systems, and 
which have the capacity to infect and 
replicate in some host cell, either au- 
tonomously or as an integrated part of 
their host’s genome) , on the development 
of procedures which are designed to pre- 
vent the spread of such molecules within 
human and other populations, and on 
guidelines to be followed by investigators 
working with potentially hazardous re- 
combinants. 
The NIH Recombinant DNA Molecule 
Program Advisory Committee has re- 
sponsibility for: (i) Revising and updat- 
ing guidelines to be followed by investi- 
gators working with DNA recombinants, 
(il) for the time being, receiving infor- 
mation on purported EK2 and EK3 sys- 
tems and evaluating and certifying that 
host-vector systems meet EK2 or EK3 
criteria, (iii) resolving questions con- 
cerning potential biohazard and ade- 
quacy of containment capability if NIH 
staff or NIH Initial Review Group so 
request, and (iv) reviewing and approv- 
ing large scale experiments with re- 
combinant DNAs known to make harm- 
ful products (e.g., more than 10 liters of 
culture) . 
E. NIH Staff. NIH Staff has responsi- 
bility for: (i) Assuring that no NIH 
grants or contracts are awarded for DNA 
recombinant research tmless they (a) 
conform to these guidelines, (b) have 
been properly reviewed and recom- 
mended for approval, and (c) include a 
properly executed Memorandum of Un- 
derstanding and Agreement, (ii) review- 
ing and responding to questions or 
problems or reports submitted by institu- 
tional biohazards committees or princi- 
pal Investigators, and disseminating 
findings, as appropriate, (ill) receiving 
and reviewing applications for approval 
to lower containment levels when a 
doned DNA recombinant derived from a 
shotgun experiment has been rigorously 
characterized and there is sufficient evi- 
dence that it is free of harmful genes, 
(iv) referring items covered under (11) 
and (ill) above to the NIH Recombinant 
DNA Molecule Program Advisory Com- 
mittee, as deemed necessary, and (v) 
performing site inspections of all P4 
physical containment facilities, engaged 
In DNA recombinant research, and of 
other facilities as deemed necessary. 
V. FOOTNOTES 
1 Biological Safety Cabinets referred to In 
this section are classified as Class I, Class II 
or Class III cabinets. A Class I cabinet is a 
ventilated cabinet for personnel protection 
having an inward flow of air away from the 
operator. The exhaust air from this cabinet 
Is Altered through a high efllcienoy or high 
eflaclency particulate air (HEPA) filter before 
being discharged to the outside atmosphere. 
This cabinet Is used In three operational 
modes; (1) with an 8 Inch high full width 
open front, (2) with an Installed front clos- 
ure panel (having fotu: eight Inch dlametm 
openings) without gloves, and (3) with an 
in.stalled front closure panel equipped with 
arm length rubber gloves. The face velocity 
of the inward flow of air through the full 
width open front Is 75 feet per minute or 
greater. A Class II cabinet is a ventilated cab- 
inet for personnel and product protection 
having an open front with Inward air flow 
for personnel protection, and HEPA filtered 
mass recirculated air flow for product pro- 
tection. The cabinet exhaust air Is filtered 
through a HEPA filter. The face velocity of 
the inward flow of air through the full width 
open front Is 75 feet per minute or greater. 
Design and performance specifications for 
Class II cabinets have been adopted by the 
National Sanitation Foundation, Ann Arbor, 
Michigan. A Class III cabinet is a closed front 
ventilated cabinet of gas tight construction 
which provides the highest level of personnel 
protection of all Biohazard Safety Cabinets. 
The interior of the cabinet is protected from 
contaminants exterior to the cabinet. The 
cabinet is fitted with aim length rubber 
gloves and is operated under a negative pres- 
sure of at least 0.5 Inches water gauge. All 
supply air filtered through HEPA filters. Ex- 
haust air is filtered through HEPA filters or 
incinerated before being discharged to the 
outside environment. 
‘ Defined as observable under optimal lab- 
oratory conditions by transformation, trans- 
duction, phage infection and/or conjugation 
with transfer of phage, plasmid and/or chro- 
mosomal genetic information. 
^ The bacteria which constitute Class 2 of 
ref. 5 (‘‘Agents of ordinary potential haz- 
ard * • *.”) represent a broad spectrum of 
etlologlc agents which possess different levels 
of virulence and degrees of communicability. 
We think it appropriate for our specific pur- 
pose to further subdivide the agents of Class 
2 into those which we believe to be of rel- 
atively low pathogenicity and those which are 
moderately pathogenic. ‘The several specific 
examples given may suffice to Illustrate the 
principle, 
‘ The terms "characterized” and "free of 
harmful genes” are unavoidably vague. But, 
in this instance, before containment condi- 
tions lower than the ones used to clone the 
DNA can be adopted, the investigator must 
obtain approval from the National Institutes 
of Health. Such approval would be contin- 
gent upon data concerning: (a) the absence 
of potentially harmful genes (e.g., sequences 
contained in indigenous tumor viruses or 
which code for toxic substances), (b) the 
relation between the recovered and desired 
segment (e.g., hybridization and restriction 
endonuclease fragmentation analysis where 
applicable), and (c) maintenance of the bio- 
logical properties of the vector. 
^ A DNA preparation is defined as enriched 
if the desired' DNA represents at least 99% 
(w/w) of the total DNA in the preparation. 
TTie reason fqr lowering the containment 
level when this degree of enrichment has 
been obtained is based on the fact that the 
total number of clones that must be exam- 
ined to obtain the desired clone is markedly 
reduced. TTius, the probability of cloning a 
harmful gene could, for example, be reduced 
by more that l(F-fold when a rionrepetltlve 
gene from mammals was being sought. Fur- 
thermore, the level of purity specified here 
makes it easier to establish that the desired 
DNA does not contain harmful genes. 
* ‘The DNA preparation is defined as puri- 
fied if the desired DNA represents at least 
99% (w/w) of the total DNA in the prepara- 
tion, provided that it was verified by more 
than one procedure. 
‘'In special circumstances, in consultation 
with the NIH Office of Recombinant DNA 
Activities, an area biohazards committee may 
be formed, composed of members firom the 
institution and/or other organlzatlone be- 
yond its own staff, as an alternative when ad- 
ditional expertise outside the institution is 
needed for the Indicated reviews. 
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