33114 
NOTICES 
siderations of safety and risk associat- 
ed with the use of E. coli K-12 will, of 
course, apply. 
Mouth pipetting at the PI level 
Both the 1976 guidelines and the 
PRG-RAC prohibit mouth pipetting 
at the P2. P3. and P4 levels. For the 
PI level, however, they state. "Al- 
though pipetting by mouth is permit- 
ted. it is preferable that mechanical 
pipetting devices be used.” A number 
of commentators have urged that 
mouth pipetting be prohibited at the 
PI level of physical containment. The 
decision accompanying the PRG-NIH 
discusses in detail the arguments of 
various groups on both sides of the 
Issue. In resolving this, the Director, 
NIH. has decided to adopt the conser- 
vative position and ban mouth pipet- 
ting. Accordingly, language has been 
Inserted in the PRG-NIH saying that 
at the PI level "mechanical pipetting 
devices shall be used; pipetting by 
mouth is prohibited." Since mouth pi- 
petting had already been banned at 
the P2-P4 levels, this means that 
mouth pipetting would now be banned 
for all experiments covered by these 
guidelines. 
Proposed Action: Environmental 
Impact Assessment 
Physical containment 
The PRG-NIH Include the following 
specific changes at each P level, aimed 
at clarifying and strengthening physi- 
cal containment requirements and 
thereby reducing the probability of re- 
combinant DNA molecules being acci- 
dentally released Into the environ- 
ment. 
PI Level The 1976 guidelines estab- 
lish as a principle of containment the 
adherence to good microbiological 
practices. At the PI level, however, 
certain practices are not required but 
merely encouraged. The PRG-NIH 
now make adherence to certain of 
these practices mandatory at all four 
physical containment levels. 
A major change in the PRG-NIH is 
the banning of mouth pipetting at the 
PI level, meaning that mouth pipet- 
ting is now banned for all experiments 
covered by the guidelines. Since the 
only plausible way E. coli K-12 could 
gain entry Into laboratory workers is 
by oral ingestion, this ban greatly re- 
duces the possibility that any organ- 
isms containing recombinant DNA will 
escape and thus minimizes the risk of 
environmental Impact. 
P2 Level The PRG-NIH have ex- 
tended the prohibitions against eating, 
drinking, smoking, and storage of 
foods from just the work area to the 
entire P2 laboratory. This change was 
made to achieve consistency with rec- 
ommended practices in laboratories 
where research with low-risk human 
pathogens is conducted. 
The PRG-NIH call for posting of 
the universal biohazard sign on stor- 
age freezers and refrigerators. This ad- 
ditional requirement was specified to 
facilitate safe storage of organisms 
containing recombinant DNA mole- 
cules when research with them is not 
in progress. 
P3 Level In the PRG-NIH. a con- 
trolled access area is defined as one 
that separates the P3 laboratory from 
areas open to unrestricted traffic flow. 
Access procedures for this area are 
specified, clarifying the juxtaposition 
of the P3 laboratory and uncontrolled 
areas. 
The PRG-NIH require the installa- 
tion of foot, elbow, or automatically 
operated facilities for washing hands 
in laboratories in which P3-level work 
is conducted. This additional require- 
ment was made to eliminate the possi- 
bility of contaminating faucets 
through contact. The requirement is 
consistent with practices common to 
Infectious disease laboratories. 
The PRG-NIH would permit recircu- 
lation of untreated air within individu- 
al rooms of the P3 laboratory. Such 
recirculation can conserve energy 
without compromising safety. Refer- 
ence to recirculation of treated air to 
other areas of the building has been 
eliminated, because this approach to 
energy conservation Is generally not 
practical. 
Some commentators pointed out 
that the PRG-RAC did not require an 
autoclave in the P3 laboratory itself 
but only within the building. It has 
been suggested that the autoclave 
should be as close as possible to the 
controlled area of the P3 laboratory. 
The language of the 1976 guidelines, 
stating a preference for the autoclave 
to be within the controlled laboratory 
area, is therefore retained in the 
PRG-NIH. A requirement, however, 
that the autoclave be within the con- 
trolled area would Increase costs, and 
In the view of NIH would not add mea- 
surably to safety. 
P4 Level In the PRG-NIH the mini- 
mal age for entry Into a P4 laboratory 
has been raised to 18 years, the com- 
monly accepted legal age. 
The PRG-NIH specify containment 
equipment appropriate for the isola- 
tion of experimental anirrals. This was 
added because the lack of guidance in 
the current guidelines has led to con- 
fusion in their application to animal 
experimentation. 
The PGR-NIH provide flexibility in 
selecting combinations of physical and 
biological containment to be used for a 
given experiment. All possible combi- 
nations available for selection achieve 
P4-level safety objectives. This ap- 
proach was patterned after the guid- 
ance provided in the NIH/EMBO 
r«*i ort. 
The PRG-NIH specify design crite- 
ria for a suit area where personnel 
wear positive-pressure suits that are 
ventilated by life-support systems. 
This provides an option to the class III 
cabinet-system requirement at the P4 
level of physical containment. This 
option has been used successfuly in re- 
search with extremely hazardous 
human pathogens. Without compro- 
mising safety, it provides an opportu- 
nity to conduct research procedures 
that cannot be confined to convention- 
al class III cabinet systems. 
Shipmenl In the PRG-NIH. the la- 
beling requirements for the shipment 
of etiologic agents apply to all organ- 
isms containing recombinant DNA 
molecules, rather than to Just those 
containing molecules derived from an 
etiologic agent listed in 42 CFR 77.25. 
This change was made In order to 
Insure that the Center for Disease 
Control. U.S. Public Health Service, 
would be notified in the event of acci- 
dental breakage during shipment. The 
PRG-NIH also specify that agents re- 
quiring P4 physical containment must 
be shipped by registered mail or an 
equivalent system that provides for 
sending notifications to the shipper 
immediately upon delivery. This 
change would Impose on the shipment 
of organisms requiring P4 contain- 
ment the same standards used for 
shipment of high-risk human patho- 
gens. 
Biological containment 
A number of alternate systems tor 
biological containment are being de- 
veloped by NIH grantees, including 
ones using Bacillus subtilis and Sac- 
charomyces cerevisiae. To assure ade- 
quate safety control, a new section of 
certification of host-vector systems 
has been added to the guidelines. HV1 
systems other than E. coli K-12 are re- 
viewed by an expert working group, 
then by the RAC, which makes appro- 
priate recommendatons to the Direc- 
tor. NIH. Descriptions of the organ- 
ism. Its biology, and the characteristics 
of the particular strains to be used are 
considered. The same standards of 
safety and risk associated with the use 
of E. coli K-12 will apply to any new 
host-vector systems to be certified in 
the future. 
HV2 Level For the HV2 level of con- 
tainment. the RAC, on June 23. 1977. 
unanimously approved a document en- 
titled “Instructions to Investigators 
Concerning Data To Be Submitted on 
Host-Plasmid Systems Proposed for 
EK2 Certification." Although not offi- 
cially part of the PRG-RAC. this doc- 
ument sets forth criteria that any pu- 
tative EK2 host-vector systems must 
meet before recommendation by the 
RAC for certification (see appendix H 
of the October 1977 EIS). The commit- 
tee applied these criteria in reviewing 
new systems (pBR322 and pBR313 in 
FEDERAL REGISTER, VOL. 43, NO. 146— FRIDAY. JULY 28, 1978 
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