Federal Register / Vol. 49, No. 227 / Friday, November 23, 1984 / Notices 
46279 
EKl conditions. The Staphylococcus 
aureus strain used as the donor is to be 
alpha toxin minus. It is suggested that, if 
possible, the donor Staphylococcus 
aureus strain should lack other toxins 
with LD»os in the range of one 
microgram per kilogram body weight 
such as the exfoliative toxin. 
Appendix F-IV-E. Fragments F-l, E-2, 
and F-3 of the diphtheria toxin gene 
(tox) may be cloned in E. coli K-12 
under BLl + EKl containment 
conditions and may be cloned in 
Bacillus subtilis host-vector systems 
under BLl containment conditions. 
Fragment F-l and fragment F-2 both 
contain: (i) some or all of the 
transcriptional control elements of tox; 
(ii) the signal peptide: 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 BLl + EKl conditions. 
Appendix F-IV-G. Genes from Vibrio 
fluvialis, Vibrio mimicus, and non 0- 
1 Vibrio cholerae, specifying virulence 
factors for animals, may be cloned 
under BLl + 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 intact 
structural gene(s) of the Shiga-like toxin 
from E. coli may be cloned in E coli K- 
12 under BL3 -f EKl containment 
conditions. 
E. coli host-vector systems expressing 
the Shiga-like toxin gene product may 
be moved from BL3 + BL2 containment 
conditions provided that: (1) the amount 
of toxin produced by the modified host- 
vector systems be no greater than that 
produced by the positive control strain 
933 E. coli 0157H7, grown and measured 
under optimal conditions: and (2) the 
cloning vehicle is to be an EKl vector 
preferably belonging to the class of 
poorly mobilizable plasmids such as 
pBR322, pBR328, and pBR325. 
Nontoxinogenic fragments of the 
Shiga-like toxin structural gene(s) may 
be moved from BL3 + EKl to BL2 + 
F.Kl containment conditions or such 
nontoxic fragments may be directly 
cloned in E. coli K-12 under BL2 -t- EKl 
conditions provided that the E. coli host- 
vector systems containing the fragments 
do not contain overlapping fragments 
which together would encompass the 
Shiga-like toxin structural gene(s). 
Appendix F-IV-/. 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 BL4 
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 B lA facility. Before any of the 
strains may be removed from the BL4 
facility, data on their safety shall be 
evaluated by the Working Group on 
Toxins and the working group 
recommendation shall be acted upon by 
NIH. 
Appendix F-IV-/. The gene segment 
encoding the A subunit of cholera toxin 
of Vibrio cholerae may be joined to the 
transposons Tn5 and Tn5-131 and the 
A-8ubunit:Tn5-131 hybrid gene cloned in 
E. coli K-12 and V. cholerae under BLl 
containment conditions. 
Appendix G — Physical Containment 
Appendix G-I. Standard Practices and 
Training. The first principle of 
containment is a strict adherence to 
good microbiological practices(7-70). 
Consequently, all personnel directly or 
indirectly involved in experiments on 
recombinant DNAs 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 
organisms 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 PI 
must ensure that everyone in the 
laboratory is familiar with both the 
potential hazards of the work and the 
emergency plan (see Sections IV-B-3-d 
and IV-B-5-e). If a research group is 
working with a known pathogen for 
which there is an effective vaccine, the 
vaccine should be made available to all 
workers. Where serological monitoring 
is clearly appropriate, it shall be 
provided (see Section IV-B-l-f). 
The "Laboratory Safety Monograph" 
and Biosafety in Microbiological and 
Biomedical Laboratories (2) booklets 
describe 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 equipment 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 
BLl, BL2, BL3, and BL4, 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^). 
The National Cancer Institute describes 
three levels for research on oncogenic 
viruses which roughly correspond to our 
BL2, BL3, and BL4 levels(d). 
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 BL3 
and BL4 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 
RAC to other combinations which 
achieve an equivalent level of 
containment (see Section IV-C-l-b-(2)- 
(b)). 
Appendix G-Il-A. Biosafety Level 1 
(BLl) (13). 
Appendix C-II-A-1. Standard 
Microbiological Practices. 
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