1 Sacramento January 28, 1977 Testimony/California State Legis Maxine Singer ature As a result of the lnternat onal Conference on Recombinant DNA at Asilomar, California in February 1975, activities were initiated in every country in the world where scientific capability permits the experiments. These activities were, and are, directed toward providing assurance that potentially hazardous organisms, constructed by the new techniques, will not be inadvertently released. It is important to recognize that these activities are proceeding in the absence of can indeed result from the exper On June 23, 1976, Donald S. of Health, announced publication minimize any potentially hazard0 any demonstration that hazardous organisms ments Fredr of gu s con ckson, Director, National Institutes delines designed to eliminate or equences of recombinant DNA research. The guidelines were subsequently published in the Federal Register (Part II for 7 July 1976). The promulgated guidelines were the result of a year and a half of intensive work by the NIH Recombinant DNA Molecule Program Advisory Committee as well as consideration by the Director of a variety of views expressed by ' individuals and private organizations either in writing or at a public hearing in February 1976. lines as well as the various views expressed by many commentators is available in the Director's Decision Statement, which accompanied publication of the guidelines. A summary of the history of the development of the guile- A Draft Environmental Impact Statement was prepared by the NIH, after determining that publication of the Guidelines was a %ajar federal actiont1 as defined by the National Environmental Policy Act of 1969. The Draft 2 Statement was published in the Federal Register of September 9 1976, and circulated widely. Comments on the Draft Statement were received and exten- sively reviewed by the scientific and administrative staffs of the NIH. And the final Environmental Impact Statement is now being prepared. For the purposes of the guidelines recombinant DNA experiments are defined as those involving 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 reproduce in some host cell, either autonomously or as an integrated part of the host's genome. In the experiments discussed in the guidelines the host cells are generally single living cells, either microorganisms such as bacteria, or animal or plant cells grown as single cells in tissue culture. The guidelines start with a statement of general principles and these are consistent with the conclusions published in the report of the Conference at Asilomar. in the light of currently available information, may be judged to present potential hazards of so serious a nature, should they prove to be hazardous that they should not be attempted at this time. Second, a large group of feasible experiments appear to pose lesser or no potential hazard, and can therefore be performed provided that the information to be obtained, or the practical benefits anticipated, cannot be obtained by conventional methods, and provided that appropriate safeguards for containment of potentially hazardous organisms are incorporated into the design and execution of the experiment. hazardous event, the more stringent should be the safeguards against escape The first principle is that there are certain experiments which, Third, that the more serious the nature of any possible 3 of the potent str i ngent as recombinant. hose generally used to handle Since the estimation of poten speculative, the levels of containment requ organisms should be set high initially, and ally hazardous agents. The safeguards should be at least as the most hazardous parent of the ial hazards is conjectural and red for potentially hazardous modified only when there is substantial relevant information to advise such modifications. And finally that the guidelines are to be reviewed at least annually in order to account for new informat ion. Containment Methods: Three approaches to the problem of containing potentially hazardous organisms form the basis of the recommended safeguards. Each of the three set up barriers to the dissemination of potentially hazardous organ isms worker and the actual containmen practices, rom the laboratory situation, and barriers between the laboratory the organisms. Two of these approaches involve the limitation of physical escape of the organisms, and are referred to as physical . The first such approach is the set of standard microbiological that have been developed over a period of many years, and are widely used for handling pathogenic organisms both in research and clinical laboratories. In the hands of well trained personnel, these procedures have proven to be effective in safeguarding both the worker and the environment from the spread of many pathogenic agents. containment involves the use of special kinds of equipment and facilities - to limit spread of aerosols, - for decontamination and containment of laboratory air and wastes, and for limitating access to laboratories. As with the standard microbiological techniques the type of equipment and facilities are not new, but were developed and have been used for containment The second approach to physical 4 of known pathogenic organisms. judge the efficacy of the various physical barriers in preventing the escape of organ isms. There is documented experience on which to The guidelines go into some detail concerning the practices and facilities required for physical containment: four levels of physical con- tainment are specified. They are termed P1, P2, P3 and P4 in the order of increasing levels of containment. P1, the lowest level, consists of the use of the standard microbiological practices mentioned before. The P2 and the next higher level P3, each require special procedures and facilities (including vertical laminar flow biological safety cabinets and laboratories maintained at lower air pressure than the surrounding building) all designed to limit to increasing extents any possible accidental escape of potentially hazardous organisms. Finally, P4, the maximum level of containment requires sophisticated and isolated facilities designed for maximum containment. Each of the levels, P2, through P4, assumes that the techniques demanded by Pl, the standard microbiological practices, will be followed. Furthermore, for each level, relevant training of personnel is mandatory. The training is to include the nature of the potential hazards, the technical manipulations, and instruction in the biology of the organisms and systems. Spec be used in case of accident, are required and sero appropriate, is to be provided. The third approach to the problem of containi fic emergency plans, to ogical monitoring, where g potential I y hazardous organisms within the laboratory is the use of biological barriers. Biological containment is defined as the use of host cells and vectors with limited ability to survive outside of very special and fastidious laboratory conditions. 5 These conditions are unlikely to be encountered by escaped organisms in natural environments. Biological containment is an integral part of the experimental design, since the host and vector will need to be chosen, in any given experiment, with a view both to the purpose of the experiment and to containment. The guidelines stress that physical and biologic containment procedures are complementary to one another each one serving to control any possible failure in the other. The use of both, in a given experime4ffords much higher levels of containment than either one alone. Therefore, the guidelines always recommend both a particular level of physical containment, and a level of biological containment for any given experiment . The guidelines also recommend that publications describing work on recombinant DNA include a description of the containment procedures used. The guidelines then classify all likely experiments, rank them according to potential hazard and assign the required containment levels. The first class of experiments described in the guidelines are those which are prohibited. very serious hazard, should they in fact be hazardous. The prohibited experiments include the following. These are experiments which are judged to be of potentially (1) Any experiment in which a portion of the recombinant DNA derives from either a highly pathogenic organism - an organism that causes serious disease in man or agriculturally important plants or animals - and classified as such by the Center for Disease Control, of the United States Public Health Service, or derives from cancer causing viruses classified by the National Cancer Institute as moderate risk. Perhaps I should stress here that these 6 viruses cause cancer in animals. There is no virus known to cause cancer in humans. (2) Deliberate formation of recombinants containing the genes for toxins of very high toxicity. or diphtheria toxin, and venoms from insects and snakes. Examples of this class are botulinus toxin (3) Deliberate creation from plant pathogens of recombinant DNAs that are likely to increase either the virulence of the pathogenic material or the range of species susceptible to the disease. (4) Certain of the possible beneficial applications of DNA recombinant research involve the creation of organisms with the ability to carry out useful environmental functions. Release of such organisms into the environ- ment may at some point be required to test their efficacy, and certainly to make use of them. Nevertheless, the guidelines state that deliberate release of any organism containing a recombinant DNA molecule is not to be undertaken at present. (5) Transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally if such acquisition could compromise the use of the drug to control disease agents in human or veterinary medicine or griculture. (6) Finally experiments must be limited in scale to quantities of flu d less than 10 liters with recombinant DNAs known to make harmful products. The guidelines state that the Advisory Committee may make exceptions to this rule for particular experiments deemed to be of direct societal benefit if appropriate equipment is used. The classification of permissible experiments is first made on the basis 7 not known to exchange genetic mater and strictly defined containment is ments it is mandatory to use biolog certified by NIH as unlikely either of the host-vector system that will be used. Recognizing the relation between the host-vector system required by the experiment and the design of suitable biological containment, experiments using the same host-vector system are grouped together. experiments according to the best available estimates of potential risk. In the absence of much needed data, these estimates involve informed judgment in many instances. For example, n3t all recombinant DNA experiments yield novel combinations of UNA .... recombination between the DNAs of organisms known to exchange genetic information in nature do not add uniquely man made cells to She biosphere. In these cases, the guidelines follow the principle that the experiments are to be carried out under conditions generally used to handle the most hazardous parent of the recombinant. When DNAs from species ned, more stringent many such experi- hat have been rigorously defined Tnen the guidelines classify permissible laboratory environments or to transfer the recombined Dl44 to other cells. These agents include certain derivatives of a bacterial species called g. II_ coli Strain K12. Strain K12 has been studied extensively for 30 years and can be readily manipulated for recombinant DNA experiments. This same extensive experience and ease of manipulation permits modification of -- E. coli K12 and its vectors by classical genetic techniques, for the purpose of establishing biological containment. The nature and manner of achieving biological containment with this system is described in the guidelines. -- E. coli K12 appears to be harmless al in nature are recomb required. Moreover, in cally contained agents to propagate outside of 8 i tse cant of a y in sma I extens i ve may be al f, it does not usually either establish itself or multiply signifi- the normal bowel. These facts suggest that accidental injestion number of bacteria by a laboratory worker would not result in spread of the bacterium outside the laboratory. The normal situation ered when people are either taking antibiotics, or have certain abnormal digestive conditions and it is required that such individuals refrain from work for the duration of the abnormal situation. However, while E. coli K12 does not establish itself as a growing strain in normal bowels, it does remain alive during its passage through the tract. Therefore transfer of -- plasmid or bacteriophage vectors containing foreigh DNA from the original E. coli K12 host to bacteria resident in the intestines or bacteria encountered after excretion must be considered. combination used in a recombinant DNA experiment it will be necessary to assess the possibilities for transfer of the recombinant DNA in order to eva uate the degree of biological containment. While we are missing some relevant infor- mation, the available data suggest that the probability of transfer can be quite low, depending on the particular system. e- For any given -- E. coli K12 host-p asmid The use of this bacteria has caused wide concern...and certain facts need to be emphasized. Only one strain of -7 E. coli, called K12, is permitted by the guidelines. Strain K12 is one of a large group of bacterial strains, all of which are called 2. coli because they share certain properties in common. But they do no? all have identical properties. Some -I_ E. coli live normally in the intestines of healthy people and animals: others are pathogens, that is, disease producers. K12, which is rarely found in nature, and does not normally colonize human or animal intestines, is a greatly enfeebled strain 9 whose principle successful ecological niche is in the laboratories of molecular biologists and geneticists. It is not pathogenic. If it were, you and I would not be here worrying about this research, since all the molecular biologists would long since have disappeared. Pathogenicity s a complex phenomenon .... dependent on several properties of the pathogen as well as on the properties of the species being infected. It is very un ikely that alterations in K12 brought about by insertion of recombined DNA will make it into a pathogen .... but it is not impossible. It is this remote possibility with which we are all concerned. We are attempting to protect against an unlikely, uncertain, yet unacceptable event. Thirty years of studey of the genetic chemistry of L- E. coli strain K12 provides confidence that the capacity of these bacteria to escape and spread in the environment can be reduced to immeasurable levels. Thus, should pathogenic organisms arise, it is not likely they would survive to cause disease....nor is it likely that bacteria containing recombined DNA would survive to evolve in unique and fearsome ways. Nevertheless, because of K12s relation to common strains of -__I E. coli, reservations about its use persist. It is important to investigate alternative organisms ... and the Guidelines encourage this but it is not at all certain that useful and safer bacteria exist. Predictions about the existence of rare and fastidious organisms, unable to exchange DNA with bacteria inhabiting man or other living things, are highly speculative. Considering then the properties of 4. coli K-12, as well as those of the certain known plasmid and bacteriophage vector, the guidelines conclude that, using such host-vector systems, recombinant DNAs are unlikely to be spread by the ingestion or dissemination of the few hundred or thousand bacteria, 10 such as might be invo biological practice. a moderate level of b I eve prov They host ved in laboratory accidents, given standard micro- Therefore, these existing systems, are judged to offer ological containment and are defined as EK1, the lowest level of biological containment for experiments with E. coli systems. -- As with physical containment levels, increasing numbers specify increasing levels of biological containment for E. coli systems. The next is called EK2. EK2 host-vector combinations must be demonstrated to de a high level of biological containment by suitable laboratory tests. are obtained by genetic modification of either existing E. coli K12 cells or the relevant vectors or both. More specifically, the guidelines -_I -II state that in order to qualify as EK2 the modified system composed of deri- vatives of -- E. coli K12 combined with a particular vector should not permit survival of the vector in other than specially designed laboratory environ- ments at a frequency greater than 10 (100 million). Various examples of the types of necessary modifications are suggested in the guidelines. -2 in One additional level of contained E. a host-vector systems is defined in the guidelines and is called EK3. the specified containment properties have been demonstrated not only by microbiological and genetic anaylsis but by appropriate tests in animals i nc I ud i ng humans or pr i mates and other re I evant env i ronments. EK3 systems are EK2 systems for which EK2 and EK3 host vector systems must be recommended for approval by the decombinant DNA Program Advisory Committee and then certified by the Director, NIH before use. system must be submitted for consideration by the Committee. several EK2 systems have been approved and certified. No EK3 systems have Detailed data on the relevant properties of the Thus far, 11 been submitted for certification as yet. Having ranked experiments according to potential hazard, and defined the several levels of physical containment and biological containment the guidelines then assign to each type of experiment both a required physical containment level, that is a P level, and a required biological containment level, that is an EK level and the particular combination of the two reflects the severity of for the E. coli DNA. In genera relatedness of -- the estimated potential hazard. The Guidelines are organized, systems, according to the source and nature of the foreign it is assumed that potential hazard increases with increasing he foreign DNA to man. Experiments in which the foreign DNA is highly purified, and does not contain harmful genes are assigned lower containment requirements than those in which the foreigh DNA is an unpurified mixture of fragments - the so-called shotgun experiments. Experiments involving such mixtures of DNA fragments are assumed to be of relatively higher potential hazard because of the greater likelihood of dangerous and unknown genes being introduced into a recipient cell compared to experiments with a single, highly purified fragment. In addition to experiments using E. coli K12, the Guidelines specify containment conditions for certain other host-vector systems. Many recom- binant DNA experiments will involve the use of systems in which the host cells are derived from animals or plants but are grown as single cells in the laboratory. Given the current state of technology, DNAs from animal viruses are most likely to be used as vectors in the near future. The cel themselves are fragile and fastidious and there is little or no chance tha a living cell could escape from a laboratory in the way that an -- E. coli S 12 and whole human be and the reports of the early Salk pol inoculated with it the recipients and SV40 cause classified as Because SV40 i cell might. Therefore containment considerations focus on the viruses since they might escape a laboratory in a viable form. There are two animal viruses whose DNAs are, now, technically useful as vectors; polyoma and simian virus 40 (SV40). In their respective normal hosts, mouse for polyoma, rhesus monkeys for SV40, neither virus causes a known disease. Polyoma does not infect human cells grown as single cells in the lab and also does not appear to infect humans, since humans exposed to polyoma do not produce antibodies. SV40 does infect both human cells grown as single cells in the laboratory, ngs, as evidenced by the active production of antibodies isolation of SV40 from humans. This virus contaminated o vaccines and millions of people were inadvertently in the middle 1950s. To date, there is no indication that of the vaccine suffered any related difficulty. Both polyoma tumor formation in newborn small laboratory mammals. They are ow risk oncogenic viruses by the National Cancer Institute. fects human beings, and also because SV40 and related viruses have been isolated in connection with several human disease states, the proposed guidelines assume that polyoma inherently affords a higher level of biological containment: therefore more stringent physical containment is required for SV40 than for pol yma. The guidelines require that the viral DNA used for recombination with a foreign DNA must itself be defective - in a manner analogous to the EK2 systems. Physical containment conditions are specified, and these depend, as before, on the nature of the llforeignll DNA fragment to be spliced onto the viral DNA. levels only. Experiments with polyoma and SV40 are restricted to P3 and P4 The Guidelines also contain recommendations for experiments plant cells will serve as hosts for recombinant DNA. The cells m single plant cells grown under laboratory parts, or small whole plants. This is in guidelines address the question of recomb organisms. Directions are given for modif defining the roles and assuring compliance wi described, are primari responsibilities of individua h required containment levels y directed at grantees of the n which ght be conditions, or seedlings, plant fact the only instance where the nant DNA experiments with whole cation of the specifications for P1, P2, and P3 physical containment in order to provide conditions appropriate for work with plants. Implementation of the Guidelines. The guidelines contain a large section s and institutions in The procedures, as Hat ional Institutes of Health. Similar procedures are in force for work carried within the NIH laboratories themselves, and for work carried out under contract arrangements with the NIH. The principle investigator is required to assess any potential biohazards, to institute appropriate safeguards and procedures, to minimize effects of possible accidents by planning, to train and inform all personnel, to report any serious or extended illness of a worker or any accidents, and s. Thus, the primary o the guidelines is in out experiments with recombinant DNA, the investigator must include an estimate of the potential biohazards as well as a statement as to the containment procedures that will be used. The application must include certification as to the existence and availability of appropriate facilities, procedures, and training. The all of these must be carried out on a continuing bas responsibility for conducting experiments according his hands. Further, in applying for grants to carry 14 guidelines indicate that institutions in which recombinant DNA experiments are carried out must establish biohazard committees which can serve to exam equipment and facilities and certify their compliance with the requirements Such committees will also serve as a source of advice and reference on physical containment facilities, on properties of biological containment, and on training of personnel. According to the proposed guidelines review of the certification and scientific in it i ated, Guidelines the invest document. ne of the investigators judgment concerning the extent of potential hazard and the required containment would be by NIH study sections, during the normal review of the application. After approval, but before work is tment to abide by the understanding. Both nstitution must sign this the investigator must formalize his comm by submitting to the NIH a memorandum of gator and a responsible official of his Failure, on the part o an investigator, to comply with the provisions of the guidelines, may resu t in cancellation of his research grant by the NIH. The NIH has no statutory regulatory authority and is thus limited in the sanctions it can use. Application of the guidelines to work not supported by the National Institutes of Health. Several agencies of the U.S. government other than the National Institutes of Health provide support for biological and medical research and are currently, or may in the future, sponsor recombinant DNA experiments. All of these agencies have now adopted the NIH Guidelines and will apply them to all research under their sponsorship. These include the National Science Foundation, the Energy Research and Development Adminis- tration, the Department of Defense, the Department of Agriculture, and 15 the National Aeronautics, Space Administration. At present there are no mechanisms except voluntary ones for extending the provisions of the guidelines to work supported by private funds - either for research or commercial purposes or to work supported by state and local funds. Both the executive and legislative branches of the federal government as well as concerned private individuals and organizations have recognized the urgent need to establish such a mechanism. A federal Interagency committee, chaired by the Director of NIH, and formed at the request of the President is at work. Both research and regulatory agenc whether ex i st i ng to extend contro as to how this m es are involved. The Committee's charge was to determine powers within the Federal regulatory agencies, are sufficient to the non-federal sector....-to formulate recommendations iy best be done, and to recommend legislation should that be deemed necessary. The Committee has not yet completed its work, but I can report to you on its progress to date. A careful examination of existing regulatory authorities within the EPA, the OSHA, the Center for Disease Control, and the FDA has been made. It is probable that none of the existing authorities can properly cover the particular requirements of -the recombinant DNA situa- tion. The NIH itself has no regulatory authority. Thus the Interagency Committee may conclude that new legislation will be required. In that instance an appropriate legislative framework will need to be devised. The various concerned F deral agencies may be expected to consult with their particular constituenc es for advice on ways to proceed - the Department of Commerce with interested Agriculture with agricultural interests and the NlH with scientific community. ndustry - the Department of Labor with Unions, the Department of 16 A high level of interest in the Congress is stimulating the Interagency Committee to prompt resolution of this problem: we may therefore expect action to extend the applicability of the guidelines - or some modification of them - to all privately or locally sponsored work with recombinant DNA in this country.