Federal Register / Vol. 46, No. 126 / Wednesday, July 1. 1981 / Notices 
34481 
( /fl) Design Criteria for Viral Oncology 
Research Facilities (1^5). U.S. Department 
of Health. Education and Welfare. Public 
Health Service. National Institutes of Health. 
DHEW Publication No. (NIH) 75-691. 
(77) Kuehne. R. W. (1973). Biological 
Containment Facility for Studying Infectious 
Disease. Appl. Microbiol. 26-239-243. 
(Jg\ Runkle. R. S.. and Phillips (1969). 
Microbial Containment Control Facilities. 
Van Nostrand Reinhold. New York. 
[19] Chaligny, M. A., and D. I. Clinger 
(1969). Contamination Control in 
Aerobiology. In R. L Dimmick and A. B. 
Akers (eds.). An Introduction to Experimental 
Aerobiology. John Wiley & Sons. New York, 
pp. 194-263. 
(/A4) Horsfall. F. L. Jr., and |. H. Baner 
(1940). Individual Isolation of Infected 
Animals in a Single Room. ). Bad. 40. 569- 
saa 
[20] Biological safety cabinets referred to in 
this section are classified as Class 1. Class //. 
or Class III cabinets. A Class / is a ventilated 
cabinet for personnel protection having an 
inward flow of air away from the operator. 
The exhaust air from this cabinet is filtered 
through ■ high-effidency partiodate air 
(HEPA) filter. This cabinet is used in three 
operational modes: (1) with a full-width open 
front. (2) with an installed front dosure panel 
(having four S-Inch diameter openings) 
without gloves, and (3) with an Installed front 
doeure panel equipped with arm-length 
rubber gloves. The face velodty 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 cabinet 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 protection. 
The cabinet exhaust air is filtered through a 
HEPA filter. The face velodty 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 Foundatioa 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 safely cabinets. 
The interior of the cabinet is protected from 
contaminants exterior to the cabinet. The 
cabinet is fitted with arm-length rubber 
gloves and is operated under a negative 
pressure of at least O.S Inches water gauge 
All supply air is filtered through HEPA filters 
Exhaust air is filtered through two HEPA 
filters or one HEPA filter and indnerator 
before being discharged to the outside 
evironmenl. 
[21] Hershfield. V.. H W. Boyer. C. 
Yanofsky. M. A. Lovett, and D R. Helinski 
(1974). Plasmid Co! El as a Molecular 
Vehicle for Cloning and Amplification of 
D\.A Proc. Nat. Acad. Sd. USA 71. 34SS- 
3459. 
[22] Wensink. P. C.. D. |. Finnegan. |. E. 
Donelson. and D. S. Hogness (1974). A System 
for Mapping DSA Sequences in the 
Chromosomes of Drosophila Melanogaster. 
Cell Jl 315-335. 
[23] Tanaka. T.. and B. Weisbium (1975). 
Construction of a Colic in Et-R Factor 
Composite Plasmid In Vitro: Means for 
Amplification of Deoxyribounudeic Acid. |. 
Bacleriol. 121. 354-362. 
(24) Armstrong. K. A., V. Hershfield. and D. 
R. Helinski (1977). Gene Cloning and 
Containment Properties of Plasmid Col El 
and Its Derivatives. Science 196. 172-174. 
(25) Bolivar. F.. R. L Rodriguez. M. C 
Bellach. and H. W. Boyer (1977). Construction 
and Characterization of New Cloning 
Vehicles: I AmpiciUin-Resistant Derivative 
of pMB9. Gene 2. 75-93. 
(26) Cohen, S. N.. A. C. W. Chang. H. Boyer, 
and R. Helling (1973). Construction of 
Biologically Functional Bacterial Plasmids in 
Vitro. Proc. Natl. Acad. Sd. USA 70. 3240- 
3244. 
(27) Bolivar. F., R. L Rodriguez. R. |. 
Greene. M. C. Batlach. H. L Reyneker. H. W 
Boyer. ). H. Crosa, and S. Falkow (1977). 
Construction and Characterization of New 
Cloning Vehicles: II. A Multi-Purpose 
Cloning System. Gene Z 95-113. 
(28) Thomas. M.. |. R. Cameron, and R. W. 
Davis (1974). Viable Molecular Hybirds of 
Bacteriophage Lambda and Eukaryotic DNA. 
Proc. Nat. Acad. Sd. USA 71. 4579-4583. 
(29) Murray. N. E. and K. Murray (1974). 
Manipulation of Restriction Targets in Phage 
Lambda to Form Receptor Chromosomes for 
DN.\ Fragments. 
(30) Rambach. A., and P. Tiollais (1974). 
Bacteriophage Having EcoRJ Endonuclease 
Sites Only in Non-Essential Region of the 
Genome. Proc. Nat. Acad. Sd.. USA 71. 3927- 
3930. 
(31) Blattrer. F R.. B G. Williams. A. E 
Bleche. K. Denniston-Thompson. H. E Faber. 
L A. Furlong. D. |. Gunwald. D. O. Kiefer, D 
D. Moore. ). W. Shumm. E L Sheldon, and O 
Smithies (1977). Charon Phages: Safer 
Derivatives af Bacteriophage Lambda for 
DSA Cloning Science 196. 163-169. 
(32) Donoghue, D. ).. and P. A. Sharp (1977). 
An Improved Lambda Vector. Construction of 
Mode! Recombinants Coding for Kanamycin 
Resistance. Gene 1. 209-227. 
(33) Leder. P.. D. Tiemeier and L Enquist 
(1977). EK2 Derivatives of Bacteriophoge 
Lambda Useful in the Cloning of DNA from 
Higher Organisms: The gl WES System 
Science 196. 175-177. 
(33A) Skalka. A. (1078). Current Status of 
Cohphage EK2 Vectors. Gene 3. 29-35. 
(33B) Szybalski. W., A. Skalka. S. 
Goltetman. A. Campbell, and D. Botslein 
(1978). Standardized Laboratory Tests for 
EK2 Certification. Gene 3. 36-38. 
(.14) We are specifically concerned with the 
remote possibility that potent toxins could be 
produced by acquiring a single gene or cluster 
of genes. See also footnote 2A. 
(.13) Defined as observable under optimal 
laboratory conditions by transformation, 
transduction, phage infection, and/or 
conjugation with transfer of phage, plasmid, 
and/or chromosomal genetic information. 
Note that this definition of exchange may be 
less stringent than that applied to exempt 
organisms under Section l-E-4. 
(J6) As classified in the Third Report of the 
International Gommittee on Taxonomy of 
Viruses: Classification and Nomenclature of 
Viruses. R. E F. Matthews. Ed. Intervirology 
12 (129-296) 1979. (As noted in the Prohibition 
Section, the use of viruses classified |1| as 
Class 4 or 5 is prohibited.) 
[37] The cDNA copy of the viral mRNA 
must be >99% pure; otherwise as for shotgun 
experiments with eukaryotic cellular DNA. 
[37A] For the purpose of these Guidelines, 
viruses of the families Papovaviridae. 
Adenoviridae. and Herpetoviridae (36) 
should be considered as "transforming" 
viruses. While only certain of these viruses 
have been associated with cell 
transformation in vivo or in vitro, it seems 
prudent to consider all members to be 
potentially capable of transformation. In 
addition, those viruses of the family 
Poxviridae that produce proliferative 
responses — i.e., myxoma, rabbit and squirrel 
fibroma, and Yaba viruses-should be 
considered as "transforming." 
(38) >99% pure (i.e., less than 1% of the 
DNA consists of Intact viral genomes); 
otherwise as for whole genomes. 
(39) The viruses have been classified by 
NCI as "moderate-risk oncogenic viruses.” 
See "Laboratory Safety Monograph — A 
Supplement to the NIH Guidelines for 
Recombinant DNA Research" for 
recommendations on handling the viruses 
themselves. 
[40] (Deleted) 
(47) The DNA preparation is defined as 
"purified" if the desired DNA represents at 
least 99% (w/w) of the total DNA in the 
preparation, provided that it was verified by 
more than one procedure. 
(42) The lowering of the containment level 
when this degree of purification has been 
obtained is based on the fact that the total 
number of clones that must be examined to 
obtain the desired clone is markedly reduced. 
Thus, the probability of cloning a harmful 
gene could for example, be reduced by more 
than 10*-fold when a nonrepetitive gene from 
mammals was being sought. Furthereniore. 
the level of purity specified here makes it 
easier to establish that the desired DNA doee 
not contain harmful genes. 
(43) This is not permitted, of course, if it 
falls under any of the Prohibitions of Section 
1-0. Of particular concern here is prohibition 
l-D-5. i.e., "Deliberate transfer of a drug 
resistance trait to micro-organisms that are 
not known to acquire it naturally if such 
acquisition could compromise the use of a 
drug to control disease agents in human or 
veterinary medicine or agriculture." 
(44) Because this work will be done almost 
exclusively in tissue culture cells, which have 
no capacity for propagation outside the 
laboratory, the primary focus for containment 
is the vector. It should be pointed out that 
risk of laboratory-acquired infection as a 
consequence of tissue culture manipulation is 
very low. Given good microbiological 
practices, the most likely mode of escape of 
recombinant D.NAs from a physically 
contained laboratory is carriage by an 
infected human. Thus the vector with an 
inserted DNA segment should have little or 
no ability to replicate or spread in humans. 
For use as a vector in a vertebrate host cell 
system, an animal viral DNA molecule should 
display the following properties; 
(i) It should not consist of the whole 
genome of any agent that is infectious for 
humans or that replicates to a significant 
extent in human cells in tissue culture. If the 
[1691 
