Summary Statement of the Asilomar Conference on Recombinant DNA Molecules I. Introduction and General Conclusions This meeting was organized to review scientific progress in research on recombinant DNA molecules and to discuss appropriate ways to deal with the potential biohazards of this work. Impressive scientific achievements have already been made in this field and these techniques have a remarkable potential for furthering our under- standing of fundamentalbiochemicalprocesses in pro- and eukaryotic cells. lutionize the practice of molecular biology. been no practical application of the new techniques, there is every reason to believe that they will have significant practical utility in the future. The use of recombinant DNA methodology promises to revo- While there has as yet Of particular concern to the participants at the meeting was the issue of whether the pause in certain aspects of research in this area, called for by the Committee on Recombinant DNA Molecules of the National Academy of Sciences, U. S. A. in the letter published in July, 1974, should end; and, if so, how the scientific work could be undertaken with minimal risks to workers in laboratories, to the public at large and to the animal and-plant species sharing our eco- systems. The new techniques, which permit combination of genetic information from very different organisms, place us in an area of biology with many unknowns. of research in this field, the evaluation of potential biohazards has Even in the present, more limited conduct proved to be extremely difficult. us to conclude that it would be wise to exercise considerable caution in performing this research. ence agreed that most of the work on construction of recombinant DNA molecules should proceed provided that appropriate safeguards, princi- pally biological and physical barriers adequate to contain the newly It is this ignorance that has compelled Nevertheless, the participants at the Confer- - 2- created organisms, are employed, Moreover, the standards of protection should be greater at the beginning and modified as improvements in the methodology occur and assessments of the risks change. it was agreed that there are certain experiments in which the potential risks are of such a serious nature that they ought not to be dona with presently available containment facilities. problems may arise in the large scale application of this methodology in industry, medicine and agriculture. future research and experience may show that many of the potential bio- hazards are less serious and/or less probable than we now suspect. Furthermore, In the longer term serious But it was also recognized that 11. Principles Guiding the Recommendations and Conclusions Though our assessments of the risks involved with each of the various lines of research on recombinant DNA molecules may differ, few, if any, believe that this methodology is free from any risk. Reasonable principles for dealing with these potential risks are: 1) that containmerit be made an essential consideration in the experi- mental design and, 2) that the effectiveness of the contaiarnent should match, as closely as possible, the estimated risk. Consequently, whatever scale of risks is agreed upon category e it a three, four or multi- ~mkt'tec! tn f !J.I -t.~ fve there should be a commensurate scale of e containment. Estimating the risks will be difficult and at first but this will improve as we acquire additional knowledge; at each stage we shall have to match the potential risk with an appropriate level of containment. would seem to be riskier than equivalent experiments done on a small scale and, therefore, require more stringent containment procedures. The use of cloning vehicles or vectors (plasmids, phages) and bacterial hosts with a restricted capacity to multiply outside of the laboratory would reduce the potential biohazard of a particular experi- ment. levels of containment are matched may vary from time to time parti- cularly as the containment technology is improved. Experiments requiring large scale operations Thus, the ways in which potential biohazards and different The means for -3- asses sing and balancing risks with appropriate levels of Containment will need to be reexamined from the to time. both formal and informal channels of information within and between the nations of the world, the way in which potential biohazards and levels of containment are matched would be consistent. in several ways. The mostt- a~~se it -rant- -y to limiting the spread of the recombinant DNAs, is the use of biological barriers. These barriers are of two types: 1) Fastidious bacterial hosts unable to survive in natural environments, Hopefully, through Containment of potentially biohazardous agents ca.n be achieved h ea c~~krbu h cr\ . .. and 2) non-transmis sible and equally fastidious vectors (plasmids, bacteriophages or other viruses) able tu grow only in specified hosts. Physical containment, exemplified by the use of suitable hoods, or, where applicable, limited access or negative pressure laboratories, provides an additional factor of safety. d , adherence to ~~.rhc&s.~c >crte,bor- (1 s-+rrck .. good microbiological practices which, to a large measure can limit QO d, 4.c re by the escape of organisms from the experimental situation, ea&&&&^ the safety of the operation. Consequently, education and training of all personnel involved in the experiments is essential to the effectiveness of all containment measures. different means of containment will complement one another and documented substantial improvements in the ability to restrict the growth of bacterial hosts and vectors could permit modifications of the complementary physical containment requirements. In practice these Stringent physical containment and rigorous laboratory pro- cedures can reduce but not eliminate the possibility of spreading potentially hazardous agents. "disarmed" hx&emA hosts and vectors for additional safety must rigorously test the effectiveness of these agents before accepting their validity as biological barriers. Therefore, investigators relying upon 111. Specific Recomrnenclztions for Matching Types of Containment with - Tvvzs of Experiments No classification of experiments as ti> risk and no set of Given our containment procedures can anticipate all situations. present uncertainties about the hazards, the parameters proposed here are broadly conceived and meant to provide provisional guide- lines for investigators and agencies concerned with research on recombinant DNAs. for determining whether, in &FIT particular case, special circurn- stances warrant a higher level of containment than is suggested here. d Hkver, each investigator bears a responsibility hi4 A. Types of Containment 1. Ivlinimal Risk: This type of containment is intended for experiments in which the biohazards may be accurately assessed and are expected to be minimal. following the operating procedures recommended for clinical micro- biological laboratories. no drinking, eating or smoking in the laboratory, wearing laboratory coats in the work area, the use of cotton-plugged pipettes or prefer- ably mechanical pipetting devices and prompt disinfection of con- taminated materials ,, Such containment can be achieved by Essential features of such facilities are 2. Low Risk: This level of containment is appropriate for experiments which generate novel biotypes but where the available information indicates that the recombinant DNA cannot alter appreciably the ecological behavior of the recipient species, increase significantly its pathogenicity, or prevent effective treatment of any resulting infections. key features of this containment (in addition to the minimal procedures mentioned above) are a prohibition on mouth pipetting access limited to laboratory personnel, and the use of biological safety cabinets for procedures likely to produce aerosols (e. g. , blending and sonication). conjunction with low risk procedures, safer vectors and hosts should be adopted as they become available. The Though existing vectors may be used in - 5- 3. Moderate Risk: Such containnierit facilities are intended for experiments in which there is a probability of generating an agent with a significant potential for pathogenicity or ecological disruption. The principle features of this level of containment, in addition to those of the two preceding classes, are that transfer operations should be carried out in biological safety cabinets (e. g., laminar flow hoods), gloves should be worn during the handling of infectious materials, vacuum lines must be protected by filters and negative pressure should be maintained in the limited access laboratories. Moreover, sedr experimentskust ?e donm I y wita kctors and hosts that have an ovm Q mo&ersk T\S Gh-6 appreciably capacity to multiply outside of the laboratory. 4. High Risk: This level of containment is int nded for or patho- experiments in which the - e ecological- genicity of the modified organism could be severe and thereby pose a serious biohazarde laboratory personnel or the public. The main features of this facility, which was designed to contain highly infectious microbiological agents, are its isolation from other areas by air locks, a negative pressure environment, a requirement for clothing changes and showers for entering personnel and laboratories fitted with treat- ment systems to inactivate or remove biological agents that may be contaminants in exhaust air, liquid and solid wastes. All persons occupying these areas should wear protective laboratory clothing and shower at each exit from the containment facility. of agents should be confined to biological safety cabinets in which the exhaust air is incinerated or passed through Hepa filters. High risk containment includes, beside the physical and procedural features described above, the use of rigorously tested vectors and hosts whose growth can be confined to the laboratory. poknt.a\ -far di\3rup. % -4 The handling B. Types of Experiments Accurate estimates of the risks associated with different types of exreriments are difficult to obtain because of our ignorance of the probability that the anticipated dangers will manifest themselves. - 6- Nevertheless, experiments involving the construction and propaga- tion of recombinant DNA molecules using DXAs from 1) prokaryotes, bacteriophages sild other -, 2) animal viruses, and 3) eukaryotes have been characterized as minimal, low, moderate and high risks to guide investigators in their choice of the appropriate containment. ments which will need to be revised upward or downward in the light of future experience. p \ qs rc\ 1 d 5 These designations should be viewed as interim assign- The recombinant DNA molecules themselves, as distinct from cells carrying them, may be infectious to bacteria or higher organisms. DNA preparations from these experiments, particularly in large quan- tities, should be chemically inactivated before disposal. 1. +AS rn'\dS Prokaryote s , bacteriophage s and bacterial episepms Where the construction of recombinant DNA molecules and their propagation involves prokaryotic agents that are known to exchange genetic information naturally, the experiments can be performed in minimal risk containment facilities. potential hazard, more stringent containment may be warranted. Where such experiments pose a Experiments involving the creation and propagation of recombinant DNA molecules from DNAs of species that ordinarily do not exchange genetic information, generate novel biotypes. Because such experiments may pose biohazards greater than those associated with the original organisms, they should be performed, at least, in low risk containment facilities. pathogenic organisms, or genetic determinants that may increase the pathogenicity of the recipient species, or if the transferred DNA can confer upon the recipient organisms new metabolic activities not native to these species and thereby modify its relationship with the environment, then moderate or high risk containment should be used. If the experiments involve either Experiments extending the range of resistance of established human pathogens to therapeutically useful antibiotics or disinfectants should be undertaken only under moderate or high risk containment depending upon the virulence of the organism involved. - 7- Om! 't Construction of novel biotypes involving recombinant DNA niolecules containing genes from microorganisms listed as Class I11 etiologic agents by the U.S. Department of Health, Education and Welfare or containing genes which code for toxins lethal to man, pose biohazards of such magnitude that they should not be carried out at the present time. 2. Animal Viruses: Experiments involving linkage of viral genomes or genome segments to prokaryotic vectors and their propa- ith vector-host gation in prokaryotic cells should be performe systems havestricted'growth capabilitid with moderate risk containment , Rigorously purified and characterized seg- ments of non-oncogenic viral genomes or of the demonstrably non- transforming regions of oncogenic viralDNAs canbe attached to presently existing vectors and propagated in moderate risk containment facilities; w&k safer vector-host systems such experiments may be performed in low risk facilities. dtrc~n'-tC~bI ( JU 9."$%- c \e uuk a +-I Lj 4L \I .t\ps 9" Experiments designed to introduce or propagate DNA from non-viral or other low risk agents in only low risk animal &sw DNAs .as vectorsband manipulations should nimal cells should us IeJJ,