2.y- 3 Provisional Statement of the Conference Proceedings &[?jyy 4 IdG dW,h b4 I 1. 2 3 the potential biohazards of this work. We have hea about the enormous y scientific progress already achieved in this field, a@ have seen glimpses 5 of the remarkable potential of thesdmethods to accelerate the rate at L which w~-ay++n underst nding of the fundamental processes occurring in eukaryotic cells? Yhe use of recombinant DNA methodology promises Q to revolutionize the practice of molecular biology. While there has as yet q been no practical application of the new techniques, there is every reason f0 to believe that* will have sifinificant impact in the future. (I I2 called for in the July 1974 committee letter, ought not to be left unresolved. 13 They considered whether there were ways in which the scientific work could ,y be undertaken with minimal risks to the workers in laboratories and to Ifsociety at large. It was emphasized that, in the longer term, even more /b difficult problems may arise in the probably large scale applications of this ,*work in industry, medicine and agriculture. Even in the currently more IQ limited area of the conduct of research in this field, the evaluation of potential 20 bining genetic information from very different organisms place us in an 2, area of biology with many unknowns. 22 us to conclude that it would be wise to exercise the utmost caution. This meeting was organized to review scientific progress in the area of recombinant DNA molecules and to discuss appropriate ways to deal with Q I& +h"y The participants at the meeting agreed that the pause in research, biohazards has proved to be extremely difficult. The new techniques com- It is this ignorance that has compelled Never- theless, the work should proceed but with appropriate safeguards. Although future experience may dispel many fears, standards of protection should be rset high at the beginning and each escalation, however small, should be b carefully assessed. Z 2. o lines of research on recombinant DNA molecules may differ, few, if any, Y believe that this m t odology is free %?any risk. Though our assessments of the risks involved with each of the various Reasonable principles for & 30dealing with these isks are to adopt containment as a part of the experimental I strategy and that t 6 e effectiveness of the containment should match the risk. 1 Whatever scale of risks is ultimately devised, we shall need a commensurate Consequently we must seek means for estimating the Vrisks, perliaps subjectively at first but objectively as we acquire additional 6 Experiments requiring large scale operations would seem to be riskier than FJ more stringent containment procedures. Improvements in the methodology, qe. g., a "disarmed" vector or host cell, could permit a reduction in the con- qutainment requirement. Quite possibly the ways in which potential biohazards rand different levels of containment are matched may vary from country to zcountry; 'also, the ways could vary from time to time as the Containment 3 technology is improved. qreaesessment of the balancing of risk against level of containment. scale of containment. knowledge, and then to match that risk@the appropriate degree of containment. the equivalent experiment done on a small scale, and, therefore, require i;: \ J-k 7 Thus, it is essential that there be a continued ./ - 2- Multiple factors define each level in the scale of containment. The most important factors, because they contribute most significantly to limiting the spread of the recombined plasmids, are biological barriers. These barriers are of two types; fastidipus bacterial hosts unable to survive in natural environments and non-transmissable vectors designed to grow only in specified hosts. which, to a large measure, can limit the escape of organisms from the experi- mental situation. Physical containdent, exemplified by the use of suitable hoods, or, where applicable, contained laboratories is an additional factor. Further, education and training of all personnel involved in the experiments is A second factor is adherence to good microbiological practices essential to the effectiveness of all of the above. 3. Specific Recommendations c, These recommendations reflect the principle that a serious evaluation of biohazard potential and the adoption of appropriate biological and physical barriers are integral parts of experiments with recombinant DNA molecules. In this section three levels of containment affording increasing protection are defined. Then, various types of experiments are evaluated for potential bio- hazards and matched with appropriate containment levels. A. Types of Containment The types of containment are an adaptation of the "NCI Safety Standards". 1) Low-This type of containment involves basic good medical microbiological techniques. using mechanical pipettes, no eating in the lab, and that sonication and other procedures which generate large aerosols should be done in biological safety cabinets . The essential factors are wearing lab coats, While existing vectors can be used for this level of containment, as safer vectors become available, their use is recommended. 2) Moderate- Both physical and biological containment enter into this containment s ys tem. The physical containment for moderate risk agents was designed for handling moderate risk oncogenic viruses. transfer operations are to be carried out in biological safety cabinets, gloves are worn in addition to lab coats, vacuum lines are protected by filters, and negative pressure is maintained in limited access laboratories. The main features are that The physical containment procedures are recognized to provide significant but incomplete protection against the accidental spread of biologi- cal agents. The potential hazard of moderate risk agents is such that a strong measure af biological containment is needed to ensure their safe handling. Therefore, experiments with such agents should only use prokaryotic vectors which have been designed with increased safety in mind. Such vectors are currently being designed and created$. anGI--shed$ be-avwhb * S-rl-. -3- 3) High-This type of Containment involves facilities which are isolated from other areas by air locks, clothing changes and shower rooms and which have treatment systems to inactivate or remove biological agents that may be contaminants in exhaust air, liquid and solid wastes. The handling of agents shall be confined to biological safety cabinets and all persons occupying these areas shall wear only laboratory protective clothing and shall shower at each exit from the containment facility. shall be maintained under negative 8ir pressure.. . In addition the containment facility Again, only vectors designed for safety should be used. B. Types of Experime&s The letter published by the Committee on Recombinant DNA Molecules in July 1974 requested that the scientific community join the Committee in deferring two types of experiments. caution regarding a third type of experiment. potential hazards the three original cagegories are maintained, but with some redefinition. The letter also advised In the following assessments of 1 ) Prokaryotes - Potential biohazards of experiments involving genetic exchange among prokaryotic arganisms can in general be accurately asses sed. Experiments involving organisms that normally exchange ' i genetic information involve! no novel biotypes and pose no hazards that 1 cannot be contained by the standard microbiological laboratory techniques ;,appropriate for the handling of these organisms. Experiments involving the introduction of bacterial genes into species in which they have not been found to occur naturally result in novel biotypes and so pose increased potential biohazards. involving genetic determinants affecting pathogenicity for man or other species or antibiotic resistance should be undertaken only under conditions of moderate or high containment; any large scale industrial, commercial, agricultural or other applications should be deferred pending the issuance It Such experiments - Cor+ ius ; of appropriate official guidelines by national'scientific bodies. s,~h ei( 2) Viruses - Experiments employingalow ris fl animal viruses to introduce new genetic material into animal cells can be carried out under moderate physical containment conditions. ,b L segments to prokaryotic vectors and their introduction into prokaryotic cells .I- should be carried out under moderate containment conditions .i* d-d VPE? s. Rigorously purified fragments of the demonstrably Experiments involving linkage of viral genomes or genome non-transforming regions of of non-oncogenic viruses could be at t ached to into E. coli under moderate risk Experiments involving high risk viruses should be carried out under high containment. - 4- F fi6k?qnefs2d, \s: kJuleu i!k+ f&( &t%i4i lhCld&d* 1 L involving the fusion of prokaryotic vectors with DNA from -, lower 3 eukaryotes, plants, invertebrates and cold-blooded vertebrates. Moderate I( risk experiments involve the joining of DNA of warm-blooded vertebrates to -1 fprokaryotic vectors, &?the cop=+- ~w. Experiments with a high risk include the fusion of *eukaryotic or prokaryotic genes to prokaryotic vectors when the resultant gorganism is likely to express a-tuxk-oP-awra11;' 3) Eukaryotic DNA - In the low risk category are most experiments -. J `1 4, Implementation tVI.[C h 3s0, It is clear that in rnanyjcountries steps are already being taken by national bodies to formulate and establish guidelines. are established we urge individual scientists to use the present document as a guide. There are in addition some recommendations which need not wait until that time and which the scientific community could implement directly. Until such guidelines A. Development of Vectors and Hosts. One of the most important and interesting accomplishments of the meeting was the beginning of the design and construction of bacteria and vectors which could radically improve the safety of these procedures by many orders of magnitude. It is certain that in the near future there will become available special variants of X phage, non-transmis sible plasmids and special E. coli which will not only optimize safety but will also bring about considerable technical improvements in the methods themselves. bacterial systems, particularly suitably modified strains of B. subtilis, may also be specially useful for particular purposes. that a suitable vector may be found for simple eukaryotic cells. Other There is also the possibility We think that work which aims to improve the priority. B. Laboratory Procedur s & syzfiqgLa \ L/2.~p~~y(i~~ BLvif- sed kc Before any experime ts of thiskind are initiated, the research staff of the laboratory shall be informed of associated with such experiments. in the containment procedures that are designed to control the hazards, training must include emergency procedures that are to be performed in the event of an accident. surveillance of all be conducted all of the hazards that might be Laboratory workers must also be trained This It is also strongly recommended that appropriate health - `(CUI? 5 periodically to establish a C. Education A continuing reassessment of these problems to take into accountband h4J developing scientific knowledge is essential. This could be achieved by a series of annual courses, workshops and meetings would also serve to train individuals .in the relevant methods. should also be given to the establishment of a newsletter for the rapid dissemination of new information pertinent to the effectiveness of biological containment. which Consideration -5- 5. Proposed Model Containment Review Process A review process should be established which would be able to determine whether a given laboratory given type of experiment. should not lengthen the time required for review of research proposals. specific form of the review procedure in different countries for different scientific and industrial laboratorieg must depend on local circumstances. The following proposal is, therefore, presented as a model. The model is designed for for other situations. has the appropriate containment facilities for a As far-as possible, the biohazard review process The universities in the United States but would have to be modified Each university or ressarch institution should have a committee empowered and trained to grade. the physical containment facilities of its laboratories (e. g., low, moderate or high according to established guidelines). The local committee would provide the laboratory head with a statement certifying the containment rating of the laboratory(subject to periodic reevaluation). When an individual applies to an agency for funds to'support work on recombinant DNA molecules, the certificate of containment rating would be appended. the certified level of containment matched whatever biohazard might result from the proposed work. experiment, the magnitude of proposed growth of bacteria, the type of DNA to be cloned, ` etc., would all enter into the decision. If the reviewing group is satisfied, the grant would be processed for scientific merit in the usual fashion. If a question ariscs `concerning the appropriateness of the certified containment level, the NIH Advisory Committee on Re combinant DNA Molecules or some other body would be asked for an opinion or ruling. The group reviewing the grant would then determine whether The biological barriers incorporated in the This procedure would not guarantee that all experiments would be performed under the required containment conditions but if the investigators have reasonable good will, the system would generate widespread c omplianc e. 6. New Knowledge tl$J This document represents our lses assessment of the potential bio- hazards in the light of current knowledge. will be important to have answers to the following questions. and, if so, can they modify the biohazard potential of these cells? To improve this assessment, it (1) Are eukaryotic genes or viruses expressed in prokaryotic hosts (2) Can free DNA molecules infect animals or plants? (3) Can prokaryote-eukaryote recombinant DNA molecules, either free or encapsulated as phage particles infect animal or plant cells and be expressedthere? free homologous or heterologous DNA? tumors in aniinals? dis semination of cloning vehicles ? (4) `Can mammalian cells in culture be genetically transformed by (5) Can hybrid animal virus DNA or virus-plasmid hybrids cause (6) Can methods be developed to monitor effectively the escape and