3 
at the P4 level of containment; and they could proceed under the previous 
permission which appears in the Guidelines (48 FR 24569) under Appendix F-IV-H. 
In a letter dated April 4, 1984 , Drs. O'Brien and Holmes have now asked RAC to 
address several specific issues. These issues deal with containment conditions 
for cloning the intact Shiga toxin structural gene(s) and for cloning portions 
of the structural gene(s) (Attachment II). 
Dr. Gottesman felt the Working Group on Toxins should consider two items: 
(1) the working group should offer to RAC a recommendation on Dr. O'Brien's 
present request (Attachment II); and (2) the working group should discuss the 
status of Appendix F, Containment Conditions for Cloning Genes Coding for the 
Biosynthesis of Molecules Toxic for Vertebrates . 
Dr. Gottesman then described the genesis of Appendix F of the NIH Guidelines 
for Research Involving Recombinant DNA Molecules; in Appendix F, experiments 
involving the cloning of toxin genes are classified by the pharmacological 
potency of the toxin. 
Dr. Formal asked Dr. Gottesman to explain how the fact that the gene to be 
cloned in EL_ coli is isolated from EL_ coli will effect the evaluation. 
Dr. Gottesman explained that proposals involving the cloning of genes encoding 
toxins are covered by Section III-A of the Guidelines. Section III-A overrides 
Section III-D which specifies the exemption for self cloning (Section III-D-2). 
Thus, these types of proposals must be reviewed by RAC and approved by NIH. 
The fact that a gene isolated from EL_ coli would be cloned in EL_ ooli would be 
a consideration in that review. 
Dr. Gottesman suggested the working group begin the evaluation of Dr. O'Brien's 
request by discussing the first item of the request. That item reads as follows 
"1. We request that the containment conditions required for cloning of the 
intact structural gene(s). for Shiga- like toxin of EL_ coli into EL_ coli 
K-12 be reduced from P4 + EK1 to P3 + EK1." 
Dr. Levine said he had participated in formulating Appendix F. In his opinion, 
cloning of the Shiga toxin gene was placed under Section F-l which requires 
NIH review and approval because at that time the working group did not possess 
sufficient data to evaluate pertinent questions. Dr. Levine thought pertinent 
data were now available. He cited the data generated through feeding experi- 
ments with 140 human volunteers. These volunteers were fed Shiga toxin-produ- 
cing Shigella which lacked invasive characteristics. No disease symptoms 
were observed in 139 individuals; in one individual, the strain reverted to an 
invasive form and the volunteer developed shigellosis. He also cited the 
evidence generated by Branham, Dack, and Riggs (Attachment III) which shows that 
large amounts of Shiga toxin instilled directly into monkey intestinal pouches 
has no effect. Dr. Levine said the containment specified for cloning Shiga 
toxin in E. coli K-12 should be lowered on the basis of these data. 
[412] 
