INDEX No. Page I - Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 II - Containment **................................... 4 A. Standard practices and training ............ B. Physical containment levels ................ Pl Level (Minimal) ......................... P2 Level (LOW) ............................. i P3 Level (Moderate) ........................ 9 P4 Level (High) ............................ 12 C. Shipment ................................... D. Biological containment levels .............. III - Experimental Guidelines . . . . . . . . . . . . . . . . . . . . . ...* 17 A. Experiments that are not to be perfomed......c....................... 17 B. Containment guidelines for permissible experiments . . . . . . . . . . . . . . . . . . . 19 1. Biological Containment criteria .using I. Coli K-12 host-vectors........ 20 EKl host-vectors ........................ EK2 host-vectors ........................ EK3 host-vectors ............... ..c ...... 32 2. Classification of experiments using the E* Coli K-12 containment systems... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Purified cellular DNAs other than - plasmids, bacteriophages, and other viruses . . . . . . . . . . . . . . . . . . . . . . 35 i No. Page, cc> Plasmids, bacteriophages, and other viruses ........................ 36 (i> Animal Viruses .................. 36 (ii) Plant viruses ................... 37 (iii) Eukaryotic organelle DNAs ....... 37 (iv) Prokaryotic plasmid and phage DNAs ..................... 37 3. Experiments with other prokaryotic host-vectors ........................... 38 4. Experiments with eukaryotic host vectors ........................... 40 Animal host-vector systems ........... 40 CL> Plant host-vector systems ........... 45 cc) Fungal or similar lower eukaryotic host-vector systems ..... 47 IV. - Roles and Responsibilities............ ........... 48 A. Principal investigator ..................... 48 B. Institution ................................ 50 C. NIH Initial Review Group (Study Sections) . . 52 D. NIH Recombinant DNA Molecule Program Advisory Committee ................ 52 E. NIH Staff .................................. 53 V. - Footnotes ....................................... 54 VI. - References ...................................... 56 APPENDICES: A. B. C. D. Statement on the use of Bacillus subtilis in. recombinant moleculewogy......... A-l Polyoma and SV40 Virus...................... B-l Sumnary of Workshop on the Design & Testing of Safer Prokaryotic Vehicles & Bacterial Hosts for Research on Recombinant DNA Molecules.................. C-l Supplementary Information on Physical Containment (Including Detailed Index)..... D-l ii 1 I. Introduction The ptirpose of these guidelines is to recommend safeguards for research on recombinant DNA molecules to the National Institutes of Health and to other institutions that support such research. In this context we define recombinant DNAs as molecules that consist of different segments of DNA which have been joined together in cell-free systems, and which have the capacity to infect and replicate in some host cell, either autonomously or as an integrated part of the host's genome. This is the first attempt to provide a detailed set of guidelines for use by study sections as well as practicing scientists for evaluating research on recombinant DNA molecules. We cannot hope to anticipate all ,possible lines of imaginative research that are possible with this powerful new methodology. Nevertheless, a considerable volume of written and verbal contributions from scientists in a variety of disciplines has been received. In many instances the views presented to us were contradictory. At present, the hazards may be guessed at, speculated about, or voted upon, but they cannot be known absolutely in the absence of firm experimental data--and, unfortunately, the needed data were, more often than not, unavailable. Our problem then has been to construct guidelines that allow the promise of the methodology to be realized while advo- cating the considerable caution that is demanded by what we and others view as Potential hazards. In design,ing these guidelines we have adopted the following principles, which are consistent with the general conclusions that were formulated at the International Conference on Recombinant DNA Molecules held at Asilomar 2 Conference Center, Pacific Grove, California, in February 1975 (3): (i) There are certain experiments for which the assessed potential hazard is so serious that they are not to be attempted at the present time. (ii) The remainder can be undertaken at the present time provided that the experiment is justifiable on the basis that new knowledge or benefits to humankind will accrue that cannot readily be obtained by use of con- ventional methodology and that appropriate safeguards are incorporated into the design and execution of the experiment. In addition to an insistence on the practice of good microbiological techniques, these safeguards consist of providing both physical and biological barriers to the dissemination of the potentially hazardous agents. (iii) The level of containment provided by these barriers is to match the estimated po- tential hazard for each of the different classes of recombinants. For projects in a given class, this level is to be highest at initiation and modified subsequently only if there is a substantiated change in the assessed risk or in the applied methodology. (iv) The guidelines will be subjected to periodic review (at least annually) and modified to reflect improvements in our knowledge of the potential biohazards and of the available safeguards. In constructing these guidelines it has been necessary to define boundary conditions for the different levels. of physical and biological containment and for the classes of experiments to which they apply. Me recognize that these definitions do not take into account existing and anticipated special procedures and information that will allow particular 3 experiments. to be carried out under different conditions than indicated here without sacrifice of safety. Indeed, we urge that individual investi- gators devise simple and more effective containment procedures and that study sections give consideration to such procedures which may allow change in the containment levels recommended here. It is recommended that all publications dealing with recombinant DNA work include a description of the physical and biological containment pro- cedures practiced, to aid and forewarn others who might consider repeating the work.