Federal Register / Vol. 46. No. 126 / Wednesday. July 1. 1981 / Notices 
34485 
Appeodix C. — Exemptioa* Under l-E-8 
Section l-E-5 (tales that exempt from these 
Guidelines are “Other classes of recombinant 
D.NA molecules, if the Director. NIH. with 
advice of the recombinant DNA Advisor)- 
Comnuttee. after appropriate notice and 
opportunity for public comment Hnds that 
they do itot present a significant risk to 
health or the environment. (See Section fV- 
E-l-b-(lHd) ) Certain classes are exempt as 
of publication of these Rev ised Guidelines." 
The following classes of experiments are 
exempt under Section l-E-5 of the 
Guidelines: 
1. Recombinont DNAs in TiBsue Culture 
Recombinant DNA molecules derived 
entirely from non-viral components (that is, 
no component is derived from a eukaryotic 
virus), that are propagated aiul maintained in 
cells In tissue culture are exempt from these 
Guidelines with the exceptions listed below 
ExceptionB 
Experunenta described in Sections 1-D-l to 
t-D-d as being prohibited 
Expenments involving DNA from Class 3 
organisms (1) or cells known to be infected 
with these agents, or any recombinant D.NA 
molecules nhich increase the virulence and 
host-range of a plant pathogen beyond that 
which occurs by natural genetic exchange 
(See Section IU-O-1.) 
Expenments involving the deliberate 
introduction of genes coding for the 
bios)mthesis of toxins potent for vertebrates. 
(See Appendix G.) 
2. EAfienmentB Involving C coli K-12 hoot 
vector ByttemB 
Expenments which use £ coli K-12 host- 
vector s> stems, with the excepdoo of those 
experiments listed below, are exempt from 
these Guidelines provided that (a) the £ coh 
host shall not contain conjugation proficient 
plasmids or generalized transducing phages, 
and (b) lambda or lambdoid or Ff 
bacteriophages or nuncon)ugative plasmids 
|40| shall be used as vectors. However, 
expenments involving the Insertion into E 
coh K-12 of DNA from prokaryotes that 
exchange genetic Information |3S| with E coli 
may be performed with any £ coli K-12 
vector (e g conjuga live plasmid). When a 
noncon|ugaii\e vector is used the £ coli K- 
12 host may contain con)ugat1ons-pro6cient 
plasmids either autonomous or integrated, or 
generalised transducing phages 
For these exempt experiments. Pi physical 
containment conditions are recommended 
ExceptionB 
Experiments described In Section 1-0-1 to 
l-D-5 as being prohibited. 
Experiments involving DNA from Class 3 
organisms |1| or from cells known to be 
Infected with these agents may be conducted 
at P3 containment. Lower containment levels 
may be specified by NIH. (See Section IV-E- 
l-tM2)-(e).) Experiments In this category 
require prior IBC review and approval. 
Experiments which Increase the virulence 
and host range of a plant pathogen beyond 
that which occurs by natural genetic 
exchange (See Section 111-0-1.) 
Prohibition 1-0-9 concerning large-scale 
experiments (e g., more than 10 liters of 
culture) applies to experiments with £ coU 
K-12 host-vector systems. The Director. NIH 
is responsible for authorizing, under 
procedures specified by the RAC large-scale 
experiments using recombinant DNAs that 
are rigorously characterized and free of 
harmful sequences (3). (See Section lV-E-1- 
b-(3Hd).) 
Experiments involving the deliberate 
cloning of genes coding for the biosynthesis 
of toxins potent for vertebrates (See 
Appendix G.) 
3. Experiments Involving Soccharomyces 
cerevisiae host-vector systems 
Expieriments which use Soccharomyces 
cerevisiae host-vector systems, with the 
exception of experiments listed below, are 
exempt from these Guidelines provided that 
laboratory strains are used. 
For these exempt experiments. Pi physical 
containment conditions are recommended 
Exceptions 
Experiments described in Sections l-D-1 to 
l-D-9 as being prohibited. 
Experiments involving CDC Class 3 
organisms (1) or cells known to be infected 
with these agents, or any recombinant DNA 
molecules wlilch increase the virulence and 
host-range of a plant pathogen beyond that 
which occurs by natural genetic exchange 
(See Section Hl-O-1.) 
Prohibition l-D-9 concerning large-scale 
experiments (e g.. more than 10 liters of 
culture) applies to experiments with S. 
cerevisiae host-vector systems. The Director 
NIR Is responsible for authorizing, under 
procedures specified by RAC. large-scale 
experiments using recombinant DNAs that 
are rigorously characterized and free of 
harmful sequences |3|. (See Section fV-E-1- 
bH3Hd) ) 
Experiments Involving the deliberate 
cloning of genes coding for the biosynthesis 
of toxifu potent for vertebrates (See 
Appendix G.) 
4 Experiments Involving Bacillus subtilis 
host -vector s)s terns. 
Any asporogenic Bacillus subtilis strain 
which does not revert to a sporefoimer with a 
frequency greater than 10' ’can be used for 
cloning D.NA from any nopprohibited source, 
with the exception of those expenments 
listed below Indigenous Bacillus plasmids 
and phages, whose host-range does not 
include Bacillus cereus or Bacillus an throe is. 
may be used as vectors 
For these exempt expenments Pi physical 
containment conditions are recommended. 
Exceptions 
Experiments described in Sections 1-0-1 to 
l-D-9 as being prohibited. 
Experiments involving CDC Class 3 
organisms [1| or cells known to be infected 
with these agents, or any recombinant DNA 
molecules which increase the virulence and 
host-range of a plant pathogen beyond that 
which occurs by natural genetic exchange. 
(See Section Ill-O-I.) 
Prohibition l-D-6 concerning large-scale 
experiments (e g.. more than 10 liters of 
culture) applies to experiments with B. 
subtilis host-vector systems. The Director. 
NIH. is responsible for authorizing, under 
procedures specified by RAC. large-scale 
experiments using recombinant DNAs that 
are rigorously characterized and free of 
harmful sequences (3). (See Section IV-E-1- 
b-(3Hd)) 
Experiments involving the deliberate 
cloning of genes coding for the biosynthesis 
of toxins potent for vertebrates (See 
Appendix G.) 
Appendix D. — HVl and HV2 Host-Vector 
Systems Assigned Containment Levels as 
Specified in the Subsections of Section III-A 
As noted above at the beginning of Section 
lll-A. certain HVl and HV2 host-vector 
systems are assigned containment levels as 
specified in the subsections of Section lU-A. 
liiose so classified as of publication of these 
Revised Guidelines are listed below. 
HVl* — The following specified strains of 
Neurospora crassa which have been modified 
to prevent aerial dispersion; 
(1) ini (inositolless) strains 37102. 37401. 
4631& 64001 and 89601. 
(2) csp-1 strain UCLA37 and csp/2 strains 
FS 590. UCLAlOl (these are conidial 
separation mutants). 
(3) eas strain UCLAlOl (an “easily 
wettable" mutant). 
HVl — The following Slreptomyces species: 
Streptomyces coelicolor. S. lividans. S. 
parvulus. and S. griseus. The following are 
accepted as vector components of certified 
Streptomyces HVl systems: Streptomyces 
plasmids SCP2. SLPl.2. pl|101. actinophage 
phi C31. and their derivatives. 
Appendix E. — Actions Taken Under the 
Guidelines 
As noted In the subsections of Sections (V- 
E-l-b-(1) end IV-E-l-b-{2). the Director, 
NTH. may take certain actions with regard to 
the Guidelines after consideration by the 
RAC. 
Some of the actions taken to date include 
the following- 
1. The following experiment has been 
approved: The cloning in B. subtilis. under P2 
conditions, of DNA derx-ied from 
SaLchorom}xes cerevisiae using EK2 plasmid 
vectors provided that an HVl B. subtilis host 
is used. 
2 Unmodified laboratory strains of 
Neurospora crassa can be used in all 
experiments for which HVl .V. crassa 
systems are approved, provided that only 
DNA from Class 1 agents is used. For agent 
other than Class 1. unmodified laboratory 
strains of N. crassa can be used in all 
experiments for which HVl N. crassa 
systems are approved, prox-ided that these 
are carried out at physical containment one 
level higher than required for HVl. However, 
if P3 containment is specified for HVl N. 
crassa. this level is considered adequate for 
unmodified N. crassa. Care must be exercised 
to prevent aerial dispersal of macroconidia. 
in accordance with good laboratory practice. 
Mutationally modified strains of N. crassa 
specified as HVl in Appendix D can be used 
in all experiments for which HV2 N. crassa 
'These follow the assigned containment levels as 
specified in the subsections of Section III-A with 
one exception. The exception is that experiments 
involving complete genomes of eukaryotic viruses 
will require P3 *■ HVl or P2 -t- HV2 rather than the 
levels given in the subsections of Section lll-A. 
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