Federal Register / Vol. 48, No. 106 / Wednesday, June 1, 1983 / Notices 
24569 
and (iii) fragment A (the center 
responsible for ADP-ribosylation of 
elongation factor 2). Fragment F-3 codes 
for most of the non-toxic fragment B of 
the toxin, and contains no sequences 
coding for any portion of the 
enzymatically-active fragment A moiety. 
Appendix F-IV-F. The gene(s) coding 
for a toxin (designated LT-like) isolated 
from E. coli which is similar to the E. 
coli heat labile enterotoxin (LT) with 
respect to its activities and mode of 
action, but is not neutralized by 
antibodies against cholera enterotoxin 
or against LT from human or porcine E. 
coli strains and sequences homologous 
to the E. coli LT-like toxin gene may be 
cloned under Pi ■+- EKl conditions. 
Appendix F-IV-G. Genes from Vibrio 
fluvialis. Vibrio Mimicus and non 6-1 
Vibrio cholerae, specifying virulence 
factors for animals, may be cloned 
under Pi + EKl conditions. The 
virulence factors to be cloned will be 
selected by testing fluid induction in 
suckling mice and Y-l mouse adrenal 
cells. 
Appendix F-IV-H. The structural gene 
of the Shiga-like toxin from clinically 
isolated strains of E. coli may be safely 
cloned in E. coli K-12 under P4 + EKl 
containment conditions. If the 
investigators wish to proceed with the 
experiments in the NIH P4 facility, a 
prior review will be conducted to advise 
NIH whether the proposal has sufficient 
scientific merit to justify the use of the 
NIH P4 facility. 
Appendix F-IV-I. A hybrid gene in 
which the gene coding for the 
melanocyte stimulating hormone (MSH) 
is joined to a segment of the gene 
encoding diphtheria toxin may be safely 
propagated in E. coli K-12, under P4 
containment, in high containment 
building 550 at the Frederick Cancer 
Research Facility. If the investigators 
wish to proceed with the experiment, a 
prior review will be conducted to advise 
NIH whether the proposal has sufficient 
scientific merit to justify the use of the 
NIH P4 facility. Before any of the strains 
may be removed from the P4 facility, 
data on their safety shall be evaluated 
by the RAC Working Group on Toxins, 
and the Working Group 
recommendation shall be acted upon by 
NIH. 
Appendix F— I V— J. The gene segment 
encoding the A subunit of cholera toxin 
of Vibrio cholerae may be joined to the 
transposons Tn5 and the Tn-131 and the 
A-subunit::Tn5-131 hybrid gene cloned 
in E. coli K-12 and V. cholerae under PI 
containment conditions. 
Appendix C — Physical Containment 
Appendix G-I. Standard Practices 
and Training. The first principle of 
containment is a strict adherence to 
good microbiological practices (1— 10]. 
Consequently, all personnel directly or 
indirectly involved in experiments on 
recombinant DNA's must receive 
adequate instruction. (See Sections IV- 
B-l-e and IV-B-5-d.) This shall, as a 
minimum, include instructions in aseptic 
techniques and in the biology of the 
organism used in the experiments, so 
that the potential biohazards can be 
understood and appreciated. 
Any research group working with 
agents with a known or potential 
biohazard shall have an emergency plan 
which describes the procedures to be 
followed if an accident contaminates 
personnel or the environment. The 
principal investigator must ensure that 
everyone in the laboratory is familiar 
with both the potential hazards of the 
work and the emergency plan. (See 
Section IV-B-3-d and IV-B-5— e.) If a 
research group is working with a known 
pathogen where there is an effective 
vaccine it should be made available to 
all workers. Where serological 
monitoring is clearly appropriate it shall 
be provided. (See Section I V— B — 1— f.) 
The "Laboratory Safety Monograph." 
available from ORDA, describes 
practices, equipment, and facilities in 
detail. 
Appendix G — II. Physical Containment 
Levels. The objective of physical 
containment is to confine organisms 
containing recombinant DNA molecules, 
and thus to reduce the potential for 
exposure of the laboratory worker, 
persons outside of the laboratory, and 
the environment to organisms containing 
recombinant DNA molecules. Physical 
containment is achieved through the use 
of laboratory practices, containment 
equipment, and special laboratory 
design. Emphasis is placed on primary 
means of physical containment which 
are provided by laboratory practices 
and containment. Special laboratory 
design provides a secondary means of 
protection against the accidental release 
of organisms outside the laboratory or to 
the environment. Special laboratory 
design is used primarily in facilities in 
which experiments of moderate to high 
potential hazards are performed. 
Combinations of laboratory practices, 
containment equipment, and special 
laboratory design can be made to 
achieve different levels of physical 
containment. Four levels of physical 
containment, which are designated as 
Pi, P2, P3, and P4, are described. It 
should be emphasized that the 
descriptions and assignments of 
physical containment detailed below are 
based on existing approaches to 
containment of pathogenic organisms. 
For example, the "Classification of 
Etiologic Agents on the Basis of 
Hazard," [2] prepared by the Centers for 
Disease Control, describes four general 
levels which roughly correspond to our 
descriptions for Pi, P2, P3, and P4; and 
the National Cancer Institute describes 
three levels for research on oncogenic 
viruses which roughly correspond to our 
P2, P3, and P4 levels. [3] 
It is recognized that several different 
combinations of laboratory practices, 
containment equipment, and special 
laboratory design may be appropriate 
for containment of specific research 
activities. The Guidelines, therefore, 
allow alternative selections of primary 
containment equipment within facilities 
that have been designed to provide P3 
and P4 levels of physical containment. 
The selection of alternative methods of 
primary containment is dependent, 
however, on the level of biological 
containment provided by the host-vector 
system used in the experiment. 
Consideration will also be given by the 
Director, NIH, with the advice of the 
Recombinant DNA Advisory Committee 
to other combinations which achieve an 
equivalent level of containment. (See 
Section I V— C— 1— b— (2) — (b).) 
Appendix G-III-A. Pi Level. 
Appendix G-II-A-1. Laboratory 
Practices. 
Appendix G-II-A-l-a. Laboratory 
doors shall be kept dosed while 
experiments are in progress. 
Appendix G-II-A-l-b. Work surfaces 
shall be decontaminated daily, and 
immediately following spills of 
organisms containing recombinant DNA 
molecules. 
Appendix G-II-A-l-c. All biological 
wastes shall be decontaminated before 
disposal. Other contaminated materials, 
such as glassware, animal cages, and 
laboratory equipment, shall be 
decontaminated before washing, reuse, 
or disposal. 
Appendix G-II-A-l-d. Mechanical 
pipetting devices shall be used; pipetting 
by mouth is prohibited. 
Appendix G-II-A-l-e. Eating, 
drinking, smoking, and storage of foods 
are not permitted in the laboratory area 
in which recombinant DNA materials 
are handled. 
Appendix G-II-A-l-f. Persons shall 
wash their hands after handling 
organisms containing recombinant DNA 
molecules and when they leave the 
laboratory. 
Appendix G-II-A-1 -g. Care shall be 
taken in the conduct of all procedures to 
minimize the creation of aerosols. 
Appendix G-II-A-l-h. Contaminated 
materials that are to be decontaminated 
at a site away from the laboratory shall 
be placed in a durable leak-proof 
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