Federal Register / VoL 50. No. 226 l Friday, November 22, 1985 [ Notices 
48345 
requested by Dr. O’Brien under much 
less restrictive conditions. He though 
U.S. investigators are unfairly shackled 
by differences in rules on the ctoning of 
toxin genes. 
“Dr. Gottesman said there are two 
approaches to dealing with differences 
in rides between countries: (1) The 
working group may suggest the NTH 
Guidelines be modified; and (2) the 
investigator may request more generic 
approvals. 
"Dr. Gottesman suggested the working 
group proceed by voting on those 
portions of request which were 
published in the August 19. 1965, Federal 
Register and subsequently propose and 
vote on other actions the working group 
deems reasonable. 
"Dr. Gottesman asked whether the 
SLT structural gene is carried or» a 
phage. Dr. O'Brien replied that high 
toxin production in bacteria usually 
appears to be associated with the 
presence of SLT converting phages such 
as 933J and H18A/J but not always. A 
low level of toxin production can 
consistently b« detected in the absence 
of phage, and may be associated with a 
chromosomal gene. Dr. O'Brien 
hypothesized that the phage gene may 
be a variant of the chromosomal gene. 
She suggested other variants of the gene 
might be found in other bacterial 
species. She added that the Shiga toxin 
gene appears to be a chromosomal gene 
in ShSgeUa dysenteriae 1 since the 
phage cannot be induced. 
"Dr. Gottesman asked what is known 
about the host range of the SLT 
converting phages. Dr. John Newland of 
USUHS said the phages can infect other 
E. coli strains; other bacteria) families 
have not been studied to determine 
whether these phages wiU infect 
members of these families. 
"Dr. Gottesman asked how much 
toxin was produced when the SLT gene 
under its own regulation on a multicopy 
plasmid was introduced into the E. coli 
host-vector system. Dr. O’Brien replied 
that a seven fold increase in toxin 
production compared to the standard 
strain 933 E coli 0157:H7 occurs when 
the SLT gene under its own regulation is 
introduced into the E coli host-vector 
system. 
"Dr. O'Brien said the issue of 
increased SLT production by the E coli 
host-vector system should be kept in 
perspective. Under optimal laboratory 
conditions. Shigella dysenteriae 1 strain 
00 R produces 10 s cytotoxic toxin doses 
per milligram [mg) protein . . 4 the 
standard strain 933 E coli 0157:117 
produces 10 7 cytotoxic doses (CDs), per 
mg protein under optimal conditions. 
The E. coli host-vector strain carrying 
the SLT gene on a multicopy plasmid 
produces 7x10’ CDs per mg protein 
under optimal conditions. This level of 
toxin production is still less than that 
produced by the Shigella dysenteriae 1 
strain. She added that Shigella flexneri 
produces approximately 10 4 CDs per mg 
protein under optimal conditions. The 
Vibrio cholerae vaccine strain produces 
10 4 CDs per mg protein under optimal 
conditions. 
"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 recommendation to RAC that 
this proposal be recommended to the 
NLH Director. Dr. Formal said E coli 
host-vector systems offer an additional 
safety factor since experience has 
shown E coli K-l 2 will not ordinarily 
colonize the bowel. 
"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. Gill said he would like the record 
to show the working group was taking 
this action because it Judged that E coli 
host-vector systems carrying the SLT 
gene no more harmful than Shiga toxin 
expressing 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 in the August 19, 1965, Federal 
Register. . . . 
"Dr. Newland asked whether 833 E 
coli 0157:H7 would be the standard 
against which activity ws measured 
regardless of what strain was used as 
the source of the SLT gene. Dr. Gill said 
the strain specified in Appendix F-IV-H 
of the N1H Guidelines would be 
standard strain regardless of the source 
of the SLT gene. 
"Dr. O'Brien asked the working group 
to consider substituting in Appendix F- 
IV-H a shigella strain which produces 
more toxin than 933 E coli 0157:H7 as 
the standard against which to measure 
toxin production. Shigella dysenteriae 1 
strains produce approximately 10* CDs 
mg protein in cell lysate; standard strain 
933 E. coli 0157; H7 produces 10 1 CDs 
per mg protein under optimal conditions. 
"Dr. Collier said the question is 
whether there is any reason to believe 
an Ecoli1C~\Z 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 E 
coli K-12 does not colonize the gut and a 
K-l 2 host -vector system carrying the 
SLT gene would not be more pathogenic 
than Shigella dysenteriae. He thought 
10 s CDs per mg protein in cell lysates 
would be an appropriate upper limit for 
permitting a clone to be removed from. 
BL3 containment. 
"Dr. GiU said an extra margin of 
safety is provided by the specification h» 
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 coh 
0157dl7 which produces 10 7 CDs under 
optimal containment be the standard 
strain for determining whether a 
particular host- vector strain could be 
removed from BL3 containment. M 
cloning of the Shiga toxin gene on a high 
copy number plasmid in E. coK K-12 
resulted in a TO fold increase in toxin 
production, a very high level of toxin, 
10* CDs, would be produced. 
"Dr. Kopeckosaid it was important to 
clone the Shiga gene from Shigella 
dysenteriae 1 in order to understand 
how toxin gene expression is controlled 
since Shigella dysenteriae 1 , an 
epidemic strain, produces more toxin 
than other bacteria. He thought 10*CDs 
per mg protein fs equivalent to the 
highest level of Shiga toxm expression 
found £» nature; therefore, the working 
group should recommend E. coli K-T2 
host-vector systems expressing 10* CDs 
per mg protein be permitted to be moved 
from BL3 to lower containment. K-12 
strains producing more than 10*CDs per 
mg protein will not be permitted to leave 
BL3 containment under the proposed 
language. 
“Dr. Gottesman said the issue is 
whether E. coli K-12 producing high 
levels of Shiga toxin would be 
hazardous to animals. 
' "Dr. Habig said he did not see why E 
coli K-12 host-vector systems carrying 
the Shiga toxin gene would pose a 
greater danger than Shigella dysenteriae 
1. Dr. Kopecko pointed out that clinical 
laboratories routinely work with 
Shigella dysenteriae 1 at BL2 conditions. 
He did not think E coli K-12 host-vector 
systems would be more dangerous than 
Shigella dysenteriae 1 . Dr. Gottesman 
said she feh comfortable with permitting 
£■. coli host-vector systems expressing 
10* CDs of SLT to be used at BL2 
containment since E coli K— 12 does r>of 
colonize the gut. 
"Dr. Levine suggested a number such 
as M 8 CDs be chosen as the upper limit 
of toxin a host-vector system might 
express if it is to be removed from BL3 
to a lower containment level. 
"Dr. Levine said E call K-12 is not an 
invasive pathogenic organism. Several 
factors are necessary for pathogenicity, 
and even for Shigella more i* required 
than simply expression of the toxin 
gene. An attenuated Shiga bacillus 
[ 527 ] 
