Federal Register / Vol. 59, No. 127 / Tuesday, July 5, 1994 / Notices 
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I Appendix F-II-B. Cloning of genes for 
j the biosynthesis of molecules toxic for 
: vertebrates that have an LD 50 of >1 
'! microgram per kilogram and <100 
j microgram per kilogram body weight 
' may proceed under BLl + EKl 
I containment conditions (e.g., 
[ Staphylococcus aureus alpha toxin, 
' Staphylococcus aureus beta toxin, ricin, 
! Pseudomonas aeruginosa exotoxin A, 
Bordet eJla pertussis toxin, the lethal 
I factor of Baciilus anthracis, the 
‘ Pasteurella pestis murine toxins, the 
I oxygen-labile hemolysins such as 
I streptolysin O, and certain neurotoxins 
present in snake venoms and other 
venoms). 
Appendix F-II-C. Some enterotoxins 
I are substantially more toxic when 
I administered enterally than 
i parenterally. The following enterotoxins 
j shall be subject to BLl + EKl 
containment conditions; cholera toxin, 
the heat labile toxins of Escherichia coli, 
Klebsiella, and other related proteins 
that may be identified by neutralization 
with an antiserum monospecific for 
cholera toxin, and the heat stable toxins 
^ of Escherichia coli and of Yersinia 
enterocolitica. 
Appendix F-llI. Cloning of Toxic 
Molecule Genes in Organisms Other 
Than Escherichia coli K-12 
Requests involving the cloning of 
genes coding for molecules toxic for 
vertebrates at an LDjo of <100 
nanograms per kilogram body weight in 
host-vector systems other than 
j Escherichia coli K-12 will be evaluated 
by NIH/ORDA in consultation with ad 
hoc toxin experts (see Sections III-B -1 
I and IV-C-l-b-{2}-{e)). 
Appendix F-IV. Specific Approvals 
I An updated list of experiments 
■ involving the deliberate formation of 
I recombinant DNA containing genes 
coding for toxins lethal for vertebrates at 
j an LD50 of <100 nanograms per kilogram 
I body weight is available from the Office 
I of Recombinant DNA Activities, 
National Institutes of Health, Building 
I 31, Room 4B11, Bethesda, Maryland 
20892, (301) 496-9838. 
j Appendix G. Physical Containment 
I Appendix G specifies physical 
containment for standard laboratory 
I experiments and defines Biosafety Level 
' 1 through Biosafety Level 4. For large 
I scale (over 10 liters) research or 
production. Appendix K supersedes 
I Appendix G. Appendix K defines Good 
Large Scale Practice through Biosafety 
Level 3 — Large Scale. For certain work 
with plants. Appendix P supersedes 
Appendix G. Appendix P defines 
Biosafety Levels 1 through 4— Plants. 
For certain work with animals. 
Appendix Q supersedes Appendix G. 
Appendix Q defines Biosafety Levels 1 
through 4 — Ani.mals. 
Appendix G-I. Standard Practices and 
Training 
The first principls of conlainnieril is 
strict adherence to good microbiological 
practices (see Appendices G-III-A 
through G-III-J). Consequently, ail 
personnel directly or indirectly 
involved in experiments using 
recombinant DNA shall receive 
adequate instruction (see Sections IV- 
B-l-e and IV-B—4-d). At a minimum, 
these instructions include training 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 that are known or potential 
biohazards shall have an emergency 
plan that describes the procedures to be 
followed if an accident contaminates 
personnel or the environment. The 
Principal Investigator shall ensure that 
everyone in the laboratory is familiar 
with both the potential hazards of the 
work and the emergency plan (see 
Sections rV-B-4-d and IV-B 4 - 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. Serological 
monitoring, when clearly appropriate, 
will be provided (see Section IV-B- 1 - 
The Laboratory Safety Monograph 
(see Appendix G-III-0) and Biosafety in 
Microbiological and Biomedical 
Laboratories (see Appendix G-III-B) 
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 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 hazard are performed. 
Combinations of laboratory practices, 
containment equipment, and special 
labo-f-atory design can be made to 
achieve different levels of physical 
containment. Four levels of physical 
containment, which are designated as 
DLl, BL 2 , 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 
(see Appendix G-III-B). The National 
Cancer Institute describes three levels 
for research on oncogenic viruses w'hich 
roughly correspond to our BL2, BL3, 
and BL4 levels (see Appendix G-lIl-C). 
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 NIH 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 w'ill be 
given by the NIH Director, with the 
advice of the RAC to other combinations 
which achieve an equivalent level of 
containment (see Section IV-C-l-b-( 2 )- 
(c)). 
Appendix G-II-A. Biosafety Level 1 
(BLl) (see Appendix G-III-M) 
Appendix G-II-A-1. Standard 
Microbiological Practices (BLl). 
Appendix G-lI-A-l-a. Access to the 
laboratory is limited or restricted at the 
discretion of the Principal Investigator 
when experiments are in progress. 
Appendix G-II-A-l-b. Work surfaces 
are decontaminated once a day and after 
any spill of viable material. 
Appendix G-II-A-1 -c. All 
contaminated liquid or solid wastes are 
decontaminated before disposal. 
Apf>endix G-U-A-l-d. Mechanical 
pipetting devices are used; mouth 
pipetting is prohibited. 
Appendix G-II-A-l-e. Eating, 
drinking, smoking, and applying 
cosmetics are not permitted in the work 
area. Food may be stored in cabinets or 
refrigerators designated and used for 
this purpose only. 
Appendix G-II-A- 1 -f. Persons wash 
their hands: (i) After they handle 
rhaterials involving organisms 
containing recombinant DNA molecule:- 
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