b 
Dr. Collier pointed out that at this time the specific activity of SLT toxin 
is not known. Dr. Gottesman said arguments on mechanism of entry into the 
animal and treatability of symptoms support a recanmendation to RAC that this 
proposal be recommended to the NIH Pi rector . Dr. Formal said E. coll host- 
vector systems offer an aiditional safety factor since experience has shown 
E. ooli K-12 will not ordinarily colonize the bcwel . 
Dr. Collier moved that the working group approve of Dr. O'Brien's request as 
it appears in the August 19, 1985, Federal Register . Dr. Kbpecko seconded the 
motion . 
Dr. Gill said he would like the record to show the working group vas taking 
this action because it judged that Eh coll host-vector systems carrying the 
SLT gene are no more harmful than Shiga toxin ecpressing strains found in nature. 
By a vote of six in favor, none opposed, and no abstentions, the working group 
recommended RAC approve Dr. O'Brien's request as it appeared m the August 19, 
1RR5, Federal Register . (Drs. Levine and Habig voted in support of the motion.) 
Dr. Newland asked whether 933 E. coll 0157 :H7 would be the standard against 
which activity is measured regardless of '/hat strain was used as the source of 
the SLT gene. Dr. Gill said the strain specified m Appendix F-IV-H of the 
NIH Guidelines would be the standard strain regardless of the source of the SLT 
qene. 
Dr. O'Brien asked the working group to consider substituting in Appendix F-IV-H 
a shigella strain which produces more toxin than Q 33 E. coll 0157 :H7 as the 
standard an a last which to measure toxin production. Shigella dysentenae 1 
strains produce approximately 10 R CDs per mg protein in cell lysate; standard 
strain 933 _Fb_ coli 0157:117 produces 10' CDs per mg protein under optimal 
conditions . 
Dr. Collier said the question is whether there is any reason to believe an 
E. coli K-12 host-vector strain expressing SLT or .Shiga toxin on a high copy 
number plasmid would be any more toxic than Shigella found in nature. 
Dr. Levine pointed out that Eh coli K-12 does not colonize the gut and a K-12 
host- vector system carrying the SLT gene would not be more pathogenic than 
Shigella dysentenae . He thought 10° CDs per mg protein m cell lysates 
would be an appropriate upper limit for permitting a clone to be removed from 
BL3 containment. 
Dr. Gill sard an extra margin of safety is provided by the specification in 
Appendix F-IV-H of 10' CDs per mg protein in cell lysates as the upper limit 
for removal of SLT clones from BL3 containment . He would prefer strain 933 E. 
coli 0157 :H7 which produces 10^ CDs under optimal containment be the standard 
strain for determining whether a particular host-vector strain could be removed 
from RT ,3 containment. If cloning of the Shiga toxin gene on a high copy number 
plasmid in FL_ coli K-12 resulted m a 10 fold increase m toxin production, a 
very high level of toxin, 10^ CDs, would be produced. 
[ 309 ] 
