33118 
NOTICES 
yote, the higher the recommended contain- 
ment. at least until the efficiency of expres- 
sion of DNA from higher eukaryotes in pro- 
karyotes can be determined. 
The structure of the classification 
for permissible experiments is based, 
therefore, on the scientific assump- 
tions governing potential risk. It 
should be emphasized that although a 
wide variety of recombinant DNA ex- 
periments have now been performed 
for over 5 years, in hundreds of labora- 
tories throughout the world, no case 
of hazard has been demonstrated. 
Part III of the Guidelines assigns to 
each specified class of experiments a 
level of physical containment and a 
level of biological containment at 
which the experiments shall be per- 
formed. As noted before, there is a 4-5 
log protection in going from PI to P3 
or from P3 to P4. For biological con- 
tainment. there is the criterion for the 
HV2 system that the chances of the 
recombinant DNA escaping, either via 
survival of the organisms or via trans- 
mission of recombinant DNA to other 
organisms, should be less than 10~' (1 
in 100.000.000) under specified condi- 
tions. 
Commentators said that the revi- 
sions did not bring the Guidelines any 
closer to establishing absolute levels of 
hazard. It was brought out. however, 
that In the use of E. coli K-12. a level 
of no risk is neared. Data presented at 
the Falmouth Conference indicate 
that it is essentially impossible for E. 
coli K-12 to be transformed into a 
wild-type pathogen. An E. coli K-12 
containing toxic genes through recom- 
bination could present a risk, tor ex- 
ample, to the laboratory worker who 
ingested It. But it would only be a risk 
to that person. 
Harmful genes will have a very low 
probability of being transfered from E. 
coli to another organism. For exam- 
ple. the plasmids at the HV2 level are 
engineered so that they neither self- 
transfer. nor transfer when another 
plasmid induces conjugation. Current 
work is designed to determine the 
probability of E. coli K-12 as a host 
taking up plasmids from the environ- 
ment that can then receive the recom- 
binant DNA molecules from the engi- 
neered plasmid. Recent data indicate 
that the probability is extremely low. 
Thus, it is clear that this host-vector 
system offers a high degree of safety 
and at present is preferable to any 
other. 
Comments on use of E. coli K-12 
A number of comments were made 
concerning the use of E. coli host- 
vector systems. One commentator 
stressed that because E. coli K-12 is 
currently a "poor" pathogen does not 
mean that one or two genes might not 
convert It to a "good" pathogen. The 
enfeebled nature of E. coli K-12 "is 
presumably the consequence of 
mutation(s) introduced during its labo- 
ratory passage." but different strains 
of K-12 with different histories may 
not all be similarly enfeebled. 
Further, it was claimed that the fail- 
ure to convert K-12 to a pathogen by 
the use of certain plasmids or Salmo- 
nella genes is not definitive. To be de- 
finitive, we must have the detailed 
nature of the mutations in K-12 that 
"prevent the expression of pathogeni- 
city." Also, it was noted that there is 
no way to assess the absolute risk asso- 
ciated with these experiments, and 
that it is important to assess the po- 
tential harm not only to man but to 
plants, animals, and the environment. 
Another commentator urged that 
this section of the Guidelines be sup- 
plemented with evidence from the Fal- 
mouth conference to show that the 
potential risk is minimal. A commenta- 
tor cited the potential risk on the basis 
that "virtually any highly conserved 
physiologically active eukaryotic 
protein * * * or fragment thereof 
could be highly toxic when introduced 
out of context by a bacterium which 
received the appropriate gene in a re- 
combination experiment." This criti- 
cism of E. coli K-12 does not detract 
from the scientific knowledge over the 
past two years of the great safety of 
this system. The evidence is presented 
in detail in the following section. "En- 
vironmental Impact of the Proposed 
Action.” 
Different strains of K-12 with differ- 
ent histories may not all be similarly 
enfeebled, and failure so far to convert 
K-12 to a pathogen does not prove It 
can never happen. However, the safety 
of E. coli K-12 has been clearly shown, 
and there is no need to limit or specify 
particular strains for EK1. After 30 
years of work with many different 
strains, there is still no known patho- 
genic E. coli K-12 strain. Thus, there 
is presumptive evidence that all K-12 
strains are safe. They are well suited 
for laboratory experiments because 
they take up DNA easily, but their cell 
wall makes them unsuited to compete 
in nature with wild-type E. colt 
Still, it is impossible to refute the 
criticism that absolute conclusions as 
to risk have not been reached. There is 
always one more experiment to be per- 
formed that would help in analyzing 
the safety aspects of any potentially 
hazardous research activity. Two years 
ago the Director. NIH. in releasing the 
Guidelines, stated that NIH would 
proceed with recombinant DNA work 
in a deliberately cautious manner 
while simultaneously evaluating all 
the evidence pertaining to the poten- 
tial risks. That statement is reaf- 
firmed. 
General classification 
There was disagreement expressed 
over whether the PRG-RAC were too 
stringent or too lax. Those arguing the 
former position maintain that the 
RAC did not relax the Guidelines 
enough, because all the experimental 
evidence gathered and analyzed in the 
past 2 years Indicates that the initial 
fears concerning the potential hazards 
were severely exaggerated. It was also 
pointed out that recombinant DNA ex- 
periments not allowed under the cur- 
rent NIH Guidelines are proceeding 
with the approval of responsible na- 
tional committees in a number of Eu- 
ropean countries. 
Those concerned that the PRG- 
RAC were too lax point to the inade- 
quacy of experimental data for a 
sound evaluation of the potential 
risks. And they argue that a recombin- 
ant DNA experiment permitted under 
less stringent safety conditions in 
Europe is irrelevant to the establish- 
ment of standards in the United 
States. 
One of the comments at the Decem- 
ber 1977 DAC meeting was that "the 
NIH Guidelines do not adequately 
deal with the use of recombinant DNA 
in plants * * Other commentators 
expressed similar sentiments, includ- 
ing the suggestion that "a subcommit- 
tee be formed to deal with plants and 
plant pathogens and make specific rec- 
ommendations for revision of the 
Guidelines." In response to this, a 
Workshop on Risk Assessment of Agri- 
cultural Pathogens, composed of dis- 
tinguished American plant patholo- 
gists. was held on March 20-21, 1978 
(as announced on March 6 in the Fed- 
eral Register). Sponsored by the U.S. 
Department of Agriculture, the Na- 
tional Science Foundation, and the 
National Institutes of Health, the 
report of the Workshop is Appendix G 
to this document. The report was pre- 
sented to the RAC at its meeting of 
April 27-28. 1978, and was unanimous- 
ly endorsed with certain minor amend- 
ments. These recommendations, with 
certain additional minor amendments, 
have been incorporated into the PRG- 
NIH. 
Two new paragraphs have been in- 
serted at the beginning of Part III of 
the PRG-NTH. The first reminds the 
reader to consult Part I, "where a list- 
ing is given of prohibited experiments 
and experiments exempt from these 
Guidelines." The second is a "general 
flexibility clause.” 
Insertion of the latter passage was 
recommended by the RAC at its April 
27-28 meeting. It recognizes that the 
classification of experiments given in 
Part III will necessarily be imperfect 
as investigators in t the future devise 
ways to conduct recombinant DNA re- 
search wldch are not currently fore- 
seen and therefore not explicitly con- 
sidered in the Guidelines. Also, new 
data may become available showing 
that certain experiments are clearly 
FEDERAL REGISTER, VOL 43, NO. 146 — FRIDAY, JULY 2*. 1978 
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