7,. kc THURSDAY, SEPTEMBER 9, 1976 I `, /I PART 111: DEPARTMENT OF HEALTH, National Institutes of Health O RECOMBINA RESEARCH GUIDELINES Draft Environmental impact Statement 3s 126 NOTICES DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE National Institutes of Health RECOMBINANT DNA RESEARCH GUIDELINES Draft Environmental Impact Statement On Wednesday, June 23, 1976. the Director of the National Institutes of Health, with the concurrence of the Sec- retary of Health, Education. and Welfare and the Assistant Secretary for Health, issued Guidelines that will govern the conduct of NIH-supported research on recombinant DNA molecules. The decision by the NIH Director to release the Guidelines was reached after extensive scientific and public airing of the issues. The issues were discussed at public meetings of the Recombinant DNA Molecule Program Advisory Committee (Recombinant Advisory Committee) and the Advisory Committee to the NIH Director. The Recombinant Advisory Committee debated three different ver- sions of the Guidelines during this period, and made detailed recommendations to the NIH Director on how this line of re- search could proceed effectively with maximum protection of workers and the environment against possible hazards. The Advisory Committee to the NIH Director, augmented with consultants representing law, ethics, consumer af- fairs, and the environment, was asked to advise on whether the proposed Guide- lines balanced responsibility to protect the public with the potent.ial benefits through the pursuit of new knowledge. The many points of view expressed at an open meeting of the Committee on Feb- ruary 9 and 10, 1976. and in subsequent correspondence, were taken into con- sideration in the Direct,or's decision. A number of public commentators urged NIH to consider preparing an en- vironmental impact statement on re- combinant DNA research activity. They evoked the possibility that organisms containing recombinant DNA molecules might escape and affect the environment in potentia.lly harmful ways. It should be noted that the development of the guide- lines was in large part tantamount to conducting an environmental impact assessment. For example, the objectives of recombinant DNA research were con- sidered and the potential hazards and risks analyzed. Possible alternative ap- proaches to the objectives were thor- or~ghly explored, to maximize safety and minimi7c potential risks. And an elab- orate review st.ructure to ensure safet!, has been created. present draft environmental impact statement in accordance with the Na- tional Environmental Policy Act of 1969. Notice of the availability of this docu- ment appeared in the FEDERAL REGISTER of September 2. In order to extend the opportunity for public comment and consideration, the present draft environmental impact statement is offered for general comment. Please address any comments on this draft statement to the Director, National Institutes of Health. 9000 Rockville Pike, Bethesda, Maryland 20014. All comments should be submitted by October 18, 1976. Additional copies of this draft are available from Dr. Rudolf G. Wanner, Associate Director for Environmental Health and Safety, Building 12A, Room 4051, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20_014. Dated: August 26,1976. DONALD S. FREDRICKSON, Director, National Institutes of Health. DRAFT ENVIRONMENTAL IMPACT STATEMENT GUIDELINES FOR RESEARCH INVOLVING RE- COMBINANT DNA MOLECULES NATIONAL INSTITUTES OF HEALTH BETHESDA, MARYLAKD August 19, 1976 GUIDELINES FOR RESEARCH INVOLVING RECOMBINANT DNA MOLECULES National Institutes of Health, Public Health Service, DHEW, Bethesda, Maryland (X) Draft ( ) Final Environmental Impact Statement. Name of Action (X) Administrative Action. Additioml Information Additional information on the proposed abtion, including technical documents psrti- nent to this statement may be obtained from: Dr. Donald S. Fredrickson, Director. Na- tional Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20014, Tele- phone: (301) 496-2433. A copy of the "Guidelines for Research Involring Recombinant DNA Molecules" is attached. (Appendix D) "he Guidelines are premised on physi- cal and bioiogical containment to pre- vent the release or propagation of DNI\ recombinants outside the laboratory. Deliberate release of organisms into the environment is prohibited. The stipulated physical and biological containment en- sures that this research will proceed with a high degree of safety and precaution. The Department. in issuing tlus draft, is requesting comments on the accuracy of the factual information (including the absence of relevant material) and projections con- tained therein. Comments shall be submitted by October 18. 1976, the Council on Environ- mental Qnality weekly notice in the FZDER.AL REGISTER. Address comments to Dr. Donald S Fredrickson. COrcTFEiTS I. Foreword. II. Authority. 111. Objective of the NIH Action IV. Background. With a view to promoting public un- derstanding of its issuance of the Guide- lines, NIH conducted an environmental impact assessment and prepared the A. Description of the recon?bi!xnt DNI experimental process. B. Events leading to the development of guidelines. C. Description of issl.les raised by re- combinant DNA research. ( ) Legi9ative 2. Expected benefits of DN.4 recom- binant rffiearcil. 3. Long-range implicatloxs. 4. Possible deliberate misuse V. Description of the proposed action VI. Description of alternatives. A. No action. B. NIH prohibition of fm~ding of al! experiments u-it,h reconll,lllaI~t DNA. C. Development of different guideline; D. No guidelines but NIH consideration of each proposed project on an in- diridual basis before fUnding E. General Federal regulation of all such research VII, Environmental impact of the guidelines. A. Impact of i%aance of NIH gnide- lines. 1. Impact on the safety of labora- tory personnel and on the spread of possibly hazartious agents by infected laboratory personnel. 2. Impact on the environmental spread of possibly haznrdoas agent- Y. 3. C&impact. 4. Secondary impacts. B. Impact of experiments conduc:ed under the guidelines. 1. Possible undesirable impacted 2. Beneficial impacts of DNA recom- binant research. *PPENDICES A. Glwsarp. B. Suggested references for add!tional reading. C. Documents describing the impie- mentation of the guidelines. D. "Recombinant DNA Research" con- taining "Decision of the Director, National Institutes of Health to Release Guidelines for Research on Recombinant DNA Molecules" and "Guidelines for Research In- volving Recombinant DNA Mole- cules" as published in the FF.DER.AL REGISTER, Part II, July 7. 1976. FOREWORD Recent developments In molecular genetics, particularly in the last 4 years. open avenues to science that were previ- ously inaccessible. In the "recombinant DNA" experiments considered here. genes-deoxyribonucleic acid (DNA j molecules-from virtually any living organism can be transferred to cells of certain completely unrelated organisms. For example, the genes from one species of bacteria have been transferred to bacteria of another species. And genes from toads and from fruit flies have been introduced into the bacterium Escherichia coli. If the recipient bacterium is then allowed to m@tipl:;. it will propagate these newly acquired genes as part of its own genetic complement. It appears likely that any kind of gene from any kind of or::anism could be introduced it?to E. coli and certain other organisms. This ability to join together genetic material from two different sources and to propagate these hybrid elements in bacterial and animal cells has resulted in a profound and qualitative cl;ange il: the field of genetics. Non`, for the first time. there is a methodology for cro~sin:: very large evolutionary boundaries. and for maying genes betv;een organi,cms that are believed to hai-e previously had litrle genetic contact. FEDERAL REGISTER, VOL. 41, NO. I76--THURSDAY, SEPTEMBER 9, 1976 NOTICES 38427 The promise of recombinant DNA research for better understanding and improved treatment of human disease is great. There is also a possible risk that microorganisms with foreign genes might cause disease or alter the environment should they escape from the laboratory and infect human beings, animals. or plants. However, in the absence of fur- ther experimental data neither the bene- fits nor the risks can be precisely identi- fied or assessed. On June 23, 1976, the Director of the National Institutes of Health released Guidelines governing the conduct of NIH-supported research on recombinant DNA molecules (See Appendix D) . Pro- mulgation of these Guidelines followed 2 years of intensive discussion and debate within the scientific community and NIH itself, with public participation, concern- ing the possible hazards of such research and the best means for averting them, al- though the wssible hazards remain spe&ative. The Guidelines prohibit cer- tain kinds of recombinant DNA experi- ments and, for those experiments that are permitted, they specify safety pre- cautions and conditions designed to pro- tect the health of laboratory workers, the general nublic. and the environment should tde putative hazards prove real. The issuance of Guidelines establish- lng conditions and precautions with re- sect to such exneriments is viewed bv NIH as a Fed&al action that may significantly affect the quality of the &nan environment, and NIH Director Dr. Donald S. Fredrickson ordered the preparation of this statement pursuant to the National Environmental Policy Act. Although NEPA assumes that such Federal actions will not be taken until the NEPA procedures are completed, the Director of NIH concluded that the pub- lic interest required immediate issuance of the Guidelines, rather than deferral for the months that would be required for completion of the NEPA process. This was because the escape of potentially hazardous organisms was more likely in the absence of NIH action. Further, prompt issuance of the Guidelines was believed necessary in order to promote their acceptance by scientists in the United States and abroad who do not come under the purview of NIH. Issuance of and compliance with the Guidelines is, in itself, expected to de- crease the chance of any detrimental environmental impact. However, since there has been little actual experience to date with recombinant DNA experiments, the indicated confidence in the Guide- lines rests essentially upon the judgment of scientists. Their confidence is based on two premises. First, it is believed that the containment measures specified in the Guidelines make the escape of poten- tially harmful recombinant organisms into the environment highly improbable. Second, it is believed that, even if an experiment performed in accordance with the Guidelines does result in acci- dental release of recombinant organisms, adverse effects will either not occur or not be serious. In the absence of an adequate base of data derived from either experiments or experience, it must be recognized that future events may not conform to these judgments. There is some statistical probability that recombinant organisms will find their way into the environment either from experiments under NIH auspices or from-the activities of others. It is not difficult to construct scenarios in which injury could result. Although the possibility of significa.nt environmental consequences is entirely speculative, the chance of an event that could cause severe injury, however low the probabil- ity, must be treated as an environmental impact. The NIH Guidelines, in addition to en- suring the safety of NIH-supported re- searchers, the general public and the environment, are serving as a model for other laboratories throughout the world, thereby promoting environmental pro- tection beyond that achievable through other actions available to the Federal Government. And the experiments them- selves may be expected ultimately to lead to an increase of knowledge and the ad- vancement of medicine and other sciences. Although the action in auestion-that is, issuance of the Guidelines-has al- ready been taken, the Director of NIH believes that the NEPA review will fur- ther enlighten the public and focus at- tention on the important issu,es involved, in the Interest of gaining the under- standing and views of the broadest DOS- sible segment of the American people. In issuing the Guidelines, the NIH Director pointed out that they will be subject to continuous review and modification in the light of changing circumstances. Constructive modification could result from information received during the NEPA process. II. AUTHORITY The Federal action discussed in this document is taken under the authority of Title III of the Public Health Service Act--General Powers and Duties of Public Health Service; Part A-Re- search and Investigation: sections 301 and 307 (42 U.S.C. 241 and 2421). III. OBJECTIVE OF THE NLH ACTION The objective of the proposed action- release of the NIH Guidelines-is the protection of laboratory workers, the general public, and the environment from infection by possibly hazardous agents that may result from re- combinant DNA research. The Guide- lines are meant to ensure that experi- ments involvinK recombinant DNA molecules and which are supported by NIH, are carried out under conditions and safeguards that minimize the possi- bility of the harmful exposure of any human being or other component of the environment to these possibly hazardous agents. It is NIH policy that all work sup- ported by NIH, either in its own labora- tories or through grants or contracts to various organizations, must be carried out according to the Guidelines. As pa.rt of this objective, the Guidelines describe procedures that will be used to ensure implementation. A further objective of establishing the Guidelines is to in- fluence, to the extent possible, other Federal, non-Federal, and foreign or- ganizations in their efforts to assure that recombinant DNA exneriments will be carried out with minimal risk to labora- tory workers, the general public, and the' environment. IV. BXKGRO~ND A. DESCRIPTION OF THE RECOMBINANT DNA EXPERIMENTAL PROCESS All living things, from subcellular particles to higher organisms, require specific information for their reproduc- tion and functions. The basic source of this information is deoxyribonucleic acid (DNA), which is the principal substance of the genes, the units of heredity (1). Each cell of an organism is composed of various organized structures, several of which cbntain DNA. Figure IV-l il- lustrates a typical cell. DNA plays two roles: (1) Provides in- formation for the reproduction, growth, and functions of the cell. and (2) Dre- serves and directs replication of thg in- formation and transfers it to the off- spring. These two roles of DNA are com- mon to animals, plants, single-cell or- ganisms, and mnny viruses. The DNA cjf cells is mainly found in organized strut- tures called chromosomes. Intracellular DNA also occu:`.s c::t;;ide Of the Cll?O~lO.~Gi~l~S as separately rep- licating molecu!cs. S:~ch DNA4 molecules include the plasmids, found in bact.erin; the DNA of chloroplasts, common to green plants; nr.d the DNA of mito- chondria, the e:iergy-producing units of the cells of complex organisms. These DNA% while not st,rictls part of the in- herent genetic make-up of a cell, help define the cell's functional capability. Another type of DNA con:mo:?ly fou::d in cells is the DNA of infecting vYusec. In the past 30 years the structure of the DNA molecule has been studied in- FEDERAL REGISTER, vol. 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976 3S428 tensively, and it can now be described in much detail. The molecule may be com- pared to a very long, but twisted step- ladder with thousands to millions of rungs (shown in Figure IV-2). The sides of the ladder are formed of sugar mole- cules (deoxyribose) attached end to end through phosphate groups. At right angles to each sugar molecule is one of four possible bases-adenine, guanine, thymine, and cytosine. The precise se- quence of these bases, the rungs of the ladder, codes the information content. The "reading" of the code contained in the sequence of bases results in the for- mation of proteins which in turn permit the essential functions of the cell. A gene is a portion of the DNA mole- cule which codes for the manufacture of a single protein. In higher organisms, much of the DNA may not serve as genes in this sense, but may regulate the activity of nearby genes. It is possible to break open cells and isolate DNA, free of other cellular constituents. NOTICES and allowed to multiply. The resulting population of identical cells is called a "clone." In some experiments the DNA will be extracted from the cells for study: in others, the properties of the cells themselves will be investigated. In the experiments discussed in the Guidelines, the host cells are generally single-cell microorganisms such as bac- teria, or animal or plant cells that were originally obtained from living tissue but are grown as single cells under special laboratory conditions. The process of producing recombinant DNA molecules and introducing them into cells is illustrat.ed in Figure IV-3. FIGURE IV-2 In recombinant DNA experiments, DNA is first isolated from two different cell types. Each DNA is then broken into segments. Each segment may contain one or more genes, or it may contain a por- tion of the DNA that lacks functional genes. The breaking is accomplished by means of bacterial enzymes (restriction endonucleases), which cut the DNA in such a way that the chemical structure at the ends of the segments permits in- terchangeable rejoining when the two different DNAs are mixed. In this way single `DNA molecules containing per- tions of the t,wo different DNAs are con- structed. The DNA recombined in these experiments can be derived from widely divergent sources. The DNA from one of the sources serves as a carrier, or vector, for the insertion of the recombined DNA into a cell, or host. The vector ma.y be DNA from a virus or a plasmid, usually derived from the same species as w-ill serve as the host of the recombinant DNA. From a growth cuhure of the host cells, those containing the DNA frag- ment of particular interest are selected wu FIGURE IV-3 The cell represented at the upper left con- tains chromosomal DNA and several sep- arately replicating DNA molecules. The non- chromosomal DNA molecules can be isolated from the cell and manipulated to serve as vectors (carriers) for DNA from a foreign cell. Most DNA molecules used as vectors are circular. They can be cleaved, as shown, by enzymes (restriction endonucleasea) to yield linear molecules with rejoinable ends. At the upper right is another cell, repre- sented here as a rectangle. It serves as the source of the foreign DNA to be inserted in the vector. This DNA can also be cleaved by enzymes. The rectangular cell could be de- rived from any living species, and the foreign DNA might contain chromosomal or non- chromosomal DNA, or both. In the next steps, the foreign DNA frag- ment is mixed and combined with the vector DNA, and the recombinant DNA 1s reinserted into a host cell. In most experiments this host cell will be of the same species as the source of the vector. The recipient cells are then placed under conditions where they grow and multiply by division. Each new cell will contain recombinant DNA B. EVENTS LEADING TO DEVELOPMENT OF GUIDELINES On June 23, 1976, the Director, NIH, released "National Institutes of Health Guidelines for Research Involving Re- combinant DNA Molecules" (see Appen- dix D) This action was approved by the Secretary of Health, Education, and Wel- fare and the Assistant Secretary for Health. The Guidelines established care- fully controlled conditions for the con- duct of experiments involving the inser- tion of recombinatn genes into orga- nisms, such as bacteria. The chronology leading to the present Guidelines and the decision to release them are out- lined below. It was some of the scientists engaged in recombinant DNA research who called for a moratorium on certain kinds of ex- periments in order to assess the risks and devise appropriate guidelines. The capability to perform DNA recombina- tions, and the potential hazards, had be- come apparent at the Gordon Research Conference on Nucleic Acids in July 1973. Those in attendance voted to send an open letter to Dr. Philip Handler, Presi- dent of the National o Academy of Sciences, and to Dr. John R. Hogness. President of the Institute of Medicine, NAS. The letter, appearing in "Science" (2) , suggested that the Academy "estab- lish a study committee to consider this problem and to recommend specific ac- tions or guidelines, should that seem appropriate." In response, NAS formed a committee, and its members published another letter in "Science" in July of 1974 (3). Under the title "Potential Biohazards of Re- combinant DNA Molecules," the letter proposed : First, and most important, that until the potential hazards of such recombinant DNA molecules have been better evaluated or until adequate methods are developed for prevent- ing their spread, scientists throughout the world join with the member of this com- mittee in voluntarily deferring * o o [cer- tain] experiments * o *. Second, plans to link fragments of ani- mal DNAs to bacterial plasmid DNA or bacteriophage DNA should be carefully weighed + o o . Third, the Director of the National In- stitutes of Health is requested to give lm- mediate consideration to establishing an ad- visory committee charged with (i) oversee- ing an experimental program to evaluate the potential biological and ecological haz- ards of the above types of recombinant DNA molecules; (ii) developing procedures which will minimize the spread of such molecules within human and other populations: and (111) devising guidelines to be followed by in- vestigators working with potentially hazard- ous recombinant DNA molecules. Fourth, an international meeting of ln- valved scientists from all over the world should be convened early in the coming year to review scientific progress in this area and to further discuss appropriate ways to deal with the potential biohazards of recom- binant DNA molecules. On October 7, 19'74, the NM Recom- binant DNA Molecule Program Advisors Committee (hereafter "Recombinant Advisory Committee") was established to advise the Secretary of HEW, the As- sist.ant Secretary for Health, and the Di- rector of NIH" concerning a program for developing procedures which will minimize the spread of such molecules within human and other populations, and for devising guidelines to be followed by investigators working with potentially hazardous recombinants." The international meeting proposed in the "Science" article (2) was held in February 1975 at the Asilomar Confer- ence Center, Pacific Grove, California. It was sponsored by the National Academy of Sciences and supported by the Na- tional Institutes of Health and the Na- tional Science Foundation. One hundred and fifty people attended, including 52 foreign scientists from 15 countries, 16 representatives of the press, and 4 attorneys. The conference reviewed progress in research on recombinant DNA molecules and discussed ways to deal with the po- tential biohazards of the work. Partic- ipants felt that experiments on con- FEDERAL REGISTER, VOL. 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976 NOTICES combinant DNA Molecules," which were referred to the Director, NIH, for a final decision in December 1975. The Director of the National Institutes of Health called a special meeting of the Advisory Committee to the Director to review these proposed guidelines. The meeting was held at NIH, Bethesda, on February 9-10, 1976. The Advisory Com- mittee is charged to advise the Director, NIH, on matters relating to the broad setting-scientific, technological, and socioeconomic-in which the continuing development of the biomedical sciences, education for the health professions, and biomedical communications must take place, and to advise on their implica- tions for NIH policy, program develop- ment, resource allocation, and admin- istration. The members of the committee are knowledgeable in the fields of basic and clinical biomedical sciences, the so- cial sciences, physica sciences, research, education. and communications. In addi- tion to &rent members of the commit- tee, the Director, NIH, invited a number of former committee members as well as other scientific and public representa- tives to participate in the special Feb- ruary session. 1 38.429 struction of recombinant DNA mole- cules should proceed: Provided, that ap- propriate containment is utilized. The conference made recommendations for matching levels of containment with levels of possible hazard for various types of experiments. Certain experiments were judged to pose such serious poten- tial dangers that the conference recom- mended against their being conducted at the present time. A revort on the conference was sub- mitted-to the Assembly of Life Sciences, National Research Council, NAS, and approved by its Executive Committee on May 20, 1975. A summary statement of the report (4) was published in "Science, Nature," and the "Proceedings of the National Academy of Sciences." The re- port noted that "in many countries steps are already being taken by national bodies to formulate codes of practice for the conduct of experiments with known or potential biohazards. Until these are established, we urge individual scientists to use the proposa,ls in this document as a guide." The NIH Recombinant Advisors Com- mittee held its first meeting in San Fran- cisco immediately after the Asilomar conference. It proposed that NIH use the recommendations of the Asilomar con- ference as guidelines for research until the committee had an opportunity to elaborate more snecific tidelines. and that NIH establis`h a newsletter %r in- formal distribution of information. NIH accepted these recommendations. At the second meeting, held on May 12-13, 1975, in Bethesda, Maryland, the committee received a report on biohaz- ard-containment facilities in the United States and reviewed a proposed NIH contract program for the construction and testing of microorganisms that would have very limited ability to survive in natural environments and would thereby limit any possible hazards. A subcom- mittee chaired by Dr. David Hogness was appointed to draft guidelines for research involving recombinant, DNA molecules, to be discussed at the next meeting. The NIH committee, beginning with the draft guidelines prepared by the Hog- ness subcommittee, prepared proposed guidelines for research with recombinant DNA molecules at its third meeting, held on July 18-19, 1975, in Woods IIde, Massachusetts. FolIowing this meeting, many Ietters were received which were critical of the guidelines. The majority of critics felt that they were too lax, others that they were too strict. The committee reviewed all letters, and a new subcommittee, chaired by Dr. Elizabeth Kutter, was ap- pointed to revise the guidelines. A fourth committee meeting m-as held on December 4-5, 1975, in La Jolla, Cali- fornia. For t,his meeting a "variorilm edi- tion" had been vrevared. comanrine line- for-line the Hog&s, Woods Hole, and Kutter guidelines. The committee re- viewed these, voting item-by-item for their preference among the three varia- tions and, in many cases, adding new material. The result was the "Proposed Guidelines for Research Involving Re- The purpose of the meeting was to seek the committee's advice on the guidelines proposed by the Recombinant Advisory Committee. The Advisory Committee to the Director was asked whether, in their judgment, the guidelines balanced scientific responsibility t0 the public with scientific freedom to pursue new knowl- edge. Public responsibility weighs heavily in this genetic research area. The scientific com&mity must have the public's con- fidence that the goals of this profoundly important research accord respect to im- portant ethical, legal, and social values of our society. A key element in achiev- ing and maintaining this public trust is for the scientific community to ensure an openness and candor in its proceedings. Representatives of the jnternational press were invited to the Asilomar con- ference, and the proceedings received ex- tensive coverage. The meetings of the Di- rector's Advisory Committee and the Recombinant Advisory Committee have also reflected the intent of science to be an open community in considering the conduct of recombinant DNA experi- merits. Notification of all the meetings was published in the FEDERAL REGISTER and all the meetinps were attended and reported by representatives of the press. At the Director's Advisory Committee meeting, there was ample opportunity for comment and an airing of the issues, not only by the committee members but by public witnesses as well. All major points of view were broac?ly represented. The guidelines n-ere reviewed in light of the comments and suggestions made by particivants at that meeting. as well as the written comments received after- ward. As part of that review the Recom- binant Advisory Committ,ee was asked to consider at its meeting of April l-2, 1976, a number of selected issues raised by the commentators. Those issues and the response of the Recombinant Ad- visory Committee were tallen into ac- count in arriving at the final decision on the Guidelines. The history of the events and discus- sions leading to the development of the Guidelines are described in greater de- tail in the "Decision of the Director, NIH," published as a preamble to the Guidelines in the FEDERAL REGISTER,~~~~ II, July 7, 1976 (See Appendix D). C. DESCRIPTIONOFISSIJESRAISPD BP RECOMBINANTDNARESEARCH 1. Possible hazardous situations. The stable insertion of DNA derived from a different species into a cell or virus (and therefore the progeny thereof) may change certain properties of the host. The changes may be advantageous, detri- mental, or neutral with regard to (a) the survival of the recipient species, (b) other forms of life that come in contact with the recipient and cc) aspects of the nonliving environment. Current knowl- edge does not permit accurate assess- ment of whether such changes will be advantageous, detrimental or neutral, and to what degree, when considering a particular recombinant DNA experiment. At present it is only possible to speculate on ways in which the presence of recom- binant DNA in a cell or virus could bring about these effects. It should be empha- sized that there is no known instance in which a hazardous agent has been created by recombinant DNA technology. The following discussion is speculative and consider ways in which hazardous agents might be produced. a. The effect of foreian DNA on the survival of-recipient sp&ies (host cells or viruses). The effect of foreign DNA on the surviva1 of recivient svecies is im- portant to the discussion of possible haz- ards of recombinant DNA experiments because although a recipient species may acauire a notential for harmful effects as a result bf the foreign DNA, the possi- bility that the harmful effect will occur will depend on the survival of the recipi- ent and its ability to multiply. If acqui- sition of foreign DNA increases the prob- ability of survival and multiplication the possibility of harmful effects will in- crease. Similarly, if acquisition of for- eign DNA decreases the probability of survival or multiplication, the possibility of harmful effects will decrease. It is important to recognize, in evaluating the potential for harmful effects, that sig- nificant infections of animals and plants by bacteria or viruses may require con- tact with either a large or small number of the infectious cgent. depending on the agent. There are various i:ldir ?tie% that bar- teria and Mruses containin~ir!iertedfc\r- eian DNA are less liire!v to survive and multiply than are the oricinnl organisms. Natural evolution rc;;ilts in the survival of well-balanced and eiiiOcient organisms. Essential functions Rre carefullv co::- trolled, and can be switched on &d off as needed. It is unlikely that uncon- trolled, nonessential properties such as might be introduced by foreign genes would result in ang advanbage to the survival and multiplication of an cther- FEDERAL REGISTER, VOL. 41, NO. I76-THURSDAY, SEPTEMBER 9, 1976 38430 NOTiCES desirable elects. The case in which bac- terial cells are used as carriers of foreign DNA is discussed first. A foreign protein, specified by the foreign DNA, might act after being liberated from the micro- orga.nism, or it could funct,ion within the microorganism and alter, secondarily, normal microbial cell function in such a way that the cell is rendered harmful to other living things. Either means depends on the expression of the foreign genes; that is, the information in the foreign genes must be used by the recipient bac- terium to produce a foreign protein. Examples of protein that might prove harmful to other organisms are hor- mones, enzymes and toxins. The weight of present evidence sug- gests that foreign DNA from bacteria of one species, when inserted into bacteria of another species, may be expressed in the recipient. For example, if the donor of the foreign DNA produces a toxic sub- stance, then the recipient call may pro- duce such a substance if the gene for the toxic substance is present in the re- combinant. The recipient may or may not be more hazardous than the original donor organism, depending on the Gela- tive ability of the two organisms to grow and infect an animal or plant species at risk. wise well-balanced organisms. It is more likely that the new properties accom- panying insertion of foreign genes will confer some relative disability to the recipient organisms. Therefore it is likely that bacterial cells containing inserted foreign DNA will multiply more slowly than the same cells without foreign DNA Thus, in a natural competitive environ- ment, bacteria containing recombinant DNA would generally be expected to dis- appear. The rate of disappearance will depend on the relative rate of growth compared to other, competing bacteria. The following calculation demonstrates this point. Assume that a new organism constitutes 90 percent of a population, but grows 10 per- cent less rapidly than its natural counter- part. The new organism will drop from a con- centmtion of 90 percent to a concentration of 0.0001 percent (1 part in l,OOO,OOO) in 207 generations. If the generation time of the natural organism is one hour, this amounts to about 8% days. One example of a situation in which the capability of recipient bacterial host cells to survive may be significantly in- creased as the result of the nresence Of a foreign DNA is the case of resistance to antibiotics and drugs. It is well known that such resistance is often genetically determined and genes specifying resist- ance have been described. Furthermore it is well known that such genes may be transferred, by natural DNA recombina- tion, from one species of microorganism to another. Such natural events are in fact responsible for the rapid and wide spread of resistance to clinically im- portant drugs that has been observed during the last 20 Years. The ability of recipient bacterial host cells to survive and multiply might also be enhanced by acquisiton and expres- sion of a foreign gene conferring the ability to metabolize particular nu- trients. In an environmental niche con- taming the metabolite, such a recombi- nant might compete succesfillly against organisms native to the niche. This could result in destruction of an environ- mental componenGthat is, the metabo- lite. Also, if the native organisms were performing beneficial functions, those functions could be lost upon the success- ful establishment of the recombinant in the niche. b. The eflect of bacteria and viruses containing recombined DNA m other forms of life. The analysis leading to the Guidelines centered on the possibility of deleterious effects, since the concern was the health and safety of living orga- nisms, including humans, and the en- vironment. Agents constructed by re- combinant DNA technology could prove hazardous to other forms of life by be- coming pathogenic (disease-producing) or toxigenic (toxin-producing), or by be- coming more pathogenic or toxigenic than the original agent. There are two basic mechanisms by which a recipient microorganism might be altered with regard to its pathe- genicity or toxicity as a result of a reei- dent recombinant : (1) The recombinant DNA may resWt in formation of a protein that has un- The evidence available at present is in- sufficient to predict whether or not for- eign genes derived from a comnlex orga- nism(animals, plants, yeasts, and fungi) will be expressed in a bacterium in any particular instance. It may be that spe- ciflc manimrlations will be reouired to permit bacteria to express information of a foreign DNA efficiently. Faithful ex- pression of a gene requires ac&rate func- tioning of the complex bacterial machin- ery involved in protein synthesis. At each step, speciilc signals originating in the fore@ gene must be recognized by the bacterial machinery. Evolutionar; diver- gence has resulted in different signals in bacteria and complex organisms. Attempts to translate animal virus and animal cell genes into portein, using cell- free systems containing the protein- synthesizing machinery isolated from bacteria such as E. coli yield some pro- tein-like products. The protein products characterized to date were not faithful products of the information in the genes. In a few cases, intact bacteria contain- ing recombined genes from complex or- ganisms have been tested for evidence of expression of the inserted gene. By and large, accurate expression of the genes has not yet been demonstrated, although it may occur at a low frequency. In some instances, a new protein has been found, replacing one encoded by a bacterial gene. This result is expected if a bacterial gene is interrupted by insertion of the new DNA sequence within it. and does not necessarily indicate expression of the foreign gene. DNA fragments from yeast have been inserted into a strain of the bacterium E. coli which c nnot manu- facture the amino acid I! lstidine (5). (Histidine is a comnonent of most oro- teins and therefore is required for-the growth of all organisms.) After insertion, some cells no longer required histldine; thus, the presence of the yeast DNA over- came the requiremenl for histidine. This is the first suggestion that a foreign gene from an organism more comulex than bacteria may actually function in a bacterial cell. (Although yeast is a single- cell organism, it contains an organized nucleus like cells of higher organisms.) However, the detailed mechanism ex- plaining this observation is unknown. Analogous issues must be considered for the case in which animal viruses are the carriers of foreign DNA. Many viruses are simply described as DNA molecules enclosed and protected by coats of pro- tein molecules. The protein coat protect.6 the DNA from environmental effects, thus increasing the ability of the viral DNA to infect a cell. If viral DNAs are re- combined with foreign DNAs in such a way that necessary viral genes remain intact, then the recombinant DNA may in turn be able to produce, and be packagpd in, the coat of the virus. Inadvertent dis- persal of such a viral particle outside of the laboratory might then result in entry of the recombinant DNA into cells of living organisms. The foreign genes mav be expressed, resulting in ormation of a protein foreign to the infected cell, or the uncontrolled synthesis of a normal protein. The likelihood of expression of the foreign genes will probably depend on the degree of relatedness between its source and the infected organism as well as its location in the viral DNA used as vector. Currently, few if any relevant ex- perimental data are available so that estimates of the probability of expression are, in these instances, impossible. (2) The recombined DNA mau itself cause pathogenic or toxic ebects. Foreign DNA inserted in a bacterial gene, might so alter the microbial cell's properties that it becomes harmful to other orga- nisms. This might happen, for example, through a change in the growth rate and comnetitive advantage of the recipient microbial cell, resulting in increased virulence of a mildly pathogenic bacteria. In general, one would expect the inserted DNA to result in a reduced growth rate and a selective disadvantage to the oiga- nism, as discussed in "a" above. Similar issues arise where animal viruses serve as carriers of foreign DNA. It is also necessary to consider situa- tions in which DNA molecules themselves may escape from the laboratory or from the exoerimental host cell and enter cells of living organisms with which they come in contact. Although free DNA molecules are themselves relatively fragile (and the probability that they would survive, in a significant form or for a significant time, in air, water, or any other medium, is considered remote), they can be pro- tected in nature in a variety of ways and zTlea.sed either into, or close to, a living When a cell or virus dies, or comes close to or invades the tissue of another living organism, the recombinant DNA may effectively enter a new cell. A haz- ardous situation similar to that described above might ensue if foreign proteins were manufactured in this "secondary" recipient. The recombinant DNA might survive as an independent eellula.r cem- ponent, or it could recombine by natural FEDERAL REGISTER, VOL. 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976 NOTICES process with the DNA of the secondary recipient. Various possible deleterious consequences of such a recombination may be considered. If the secondary recipient is another microorganism, the same considerations described in IV-C-l-a apply. If the sec- ondary recipient is one of the cells of an animal or plant, different considerations apply. The latter include alterations of normal cellular control mechanisms, syn- thesis of a foreign protein (such as a hor- mane), a.nd insertion of genes involved in cancer production (if, for example, the foreign DNA were derived from a cancer- producing virus). It should be Pointed out that the hke- lihood of causing inheritable changes in the offspring of complex organisms by such a mechanism is extremely low in animals because of the protection afforded germ-line cells (eggs and sperm) by their location. Thus, the pas- sibility that recombined foreign DNA would reach germ line cells at a time in the life of such cells when secondary re- combination can occur is extremely re- mote. With one-celled oraanisms, plants, or simple multicellular -organisms, the probability of causing heritable change by secondary recombination may be higher. What is the probability of secondary recombination between prokaryotes and eukarvotes in nature? It is generally held that &combination in nature is-more likely if similar or identical sequences of bases, (rungs in the DNA ladder) occur in the two recombining DNA.?. The greater the degree of similar sequences, the more likely is recombination. In gen- eral. the more closelv two snecies are re- lated, the more likely it s that similar sequences will be found in their DNA?,. Thus, DNA from primates has more DNA sequences in common with human DNA than does DNA from mice, or fish, or plants. Recombination may also occur between DNAs not sharing sequences but at lower frequencies. It is Possible that the capacity for interspe&s recombination. between dis- tantly related species exists in nature. For example, bacteria in animal intes- tines are constantly exposed to fragments of animal DNA released from deadlntes- tlnal cells. Significant recombination re- quires the uptake of intact segments of animal DNA and their subsequent incor- poration into the'bacterial DNA. The frequency of such events is unknown. There are very few available data per- mitting assessment of the reverse proc- es&-namely, the incorporation of bac- terial DNA into the cells, or DNA, of more complex organisms. Although there are reports of experiments in which bac- terial DNA was inserted into animal and plant species and production of the bacterial protein followed, the process is very inefficient and many investigators have been unable to repeat these experi. ments (6-S). There are certain well-documented in- stances in which the DNAs of different living things become more or less per- manently recombined in nature. These instances Involve recombination between the DNAS of nonchromosomal genes, such as those of viruses or plasmids. or re- combination between the DNAs of viruses or plasmids and chromosomal genes. The former instance, for example, is the mechanism behind the rapid spread of resistance to ant.ibiotics among different bacterial species (9, 10). This spread ac- companied the prevalent use of antibi- otics in medicine and agriculture. Some viral DNAs recombine into and Persist in chromosomal DNA of cells of recep- tive organisms (11, 12) . Some viral DNAs acquire, in stable form, DNA sequences derived from their host cells (13, 14). There is also strong evidence for re- combination of the DNA form of RNA tumor virus genes with chromosomal genes (15-17). 2. Expected benefits of DNA recombi- nant research. Benefits may be divided into two broad categories: An increased understanding of basic biological proc- esses, and practical applications for med- icine, agriculture, and industry. At this time the practical applications are, of course, speculative. It is impor- tant to stress that the most significant results of this work, as with any truly innovative endeavor, are likely to arise in unexpected ways and will almost cer- tainly not follow a predictable path. a. increased understanding of basic biological processes. There are many im- portant fundamental biomedical ques- tions that can be answered or approached by DNA recombinant research. In order to advance against diseases in inherit- ance. we need to understand the struc- ture `of genes and how they work. The DNA recombinant methodology provides a simale and inexepensive way to PrePare large- quantities of specific genetic- in- formation in pure form. This should per- mit elucidation of the organization and function of the genetic information in higher organisms. For example, current estimates of the fraction of this informa- tion that codes for proteins are simply educated guesses. There are almost no clues about the function of the portions of DNA that do not code for proteins, although these DNA sequences are sus- pected of being involved in the regula- tion of gene expression. The existing state of ignorance is largely attributable to our previous in- abilitv to isolate discrete segments of the DNA"in a form that permits detailed molecular analysis. Recombinant DNA methodoloPlv remove this barrier. Pur- thermore, &cillary techniques 6ave been developed whereby pure DNA segments that contain particular sequences of in- terest can be identified and selected. Of particular interest is the isolation of pure DNA segments that contain the genes for the variable and constant portions of the immunoglobin proteins. The analyses of such segments obtained from both germline and somatic cells should be of inestimable value in determining the mechanism of immunologic diversity. A major problem in understanding the mechanism by which certain viruses cause cancer is how and where the in- fecting or endoaenous viral senomes are Meg&ted into- the cell's chromosome. This bears on the question of how the expresdon of the integrated viral genes ::p.:::1 affects cellular ?egu!~?:ion. thus leading to the abnormal grovvvth characteristics of cancer cells. With the recombinant DNA techniques for isolation and purifi- cation of specif?c genes, this research problem is reduced to manageable pro- portions. It, is possible t.o isolate the de- sired DNA segment in pure form. Large quantities can be obtained for detailed study by simply ext.ract.ing a culture of the bacteria carrying the viral DN,4 seg- ment in a plasmid. b. Potential practical applicatiom for medicine. aariculture and industru. Cer- tain of ihe potential applications will only be realized if the reproduction of the recombined foreign DNA in a recipient host cell is followed by expression of the genetic information contained in the DNA in the form of synthesis of pro- teins. Since the efEcient translation of eukaryote genes in bacterial (prokary- ate) hosts has get to be proved, these po- tential applications are speculative at this time. ApplicaUons that depend on the expression of foreign prokaryotic genes in prokaryotic recipient cells are presently more cerbain. (1) Synthesis of medically important proteins and other substances. It has been suggested that genes coding for medically important substances be at- tached to bacterial vectors, and that the bacteria then be used to produce large uuantities of the desired material. A nymber of costly and/or rare substances would be prime candidates for such syn- thesis : Human insulin ia future shortage of cur- rently used animal insulin appears to be likely) ; Human growth hormone (preeenbly avaiI- able only from human cadavers and in short SUPPlY) i Clothing factor VIII rfor xeatme:lt of hemophilia). Specific antibodies and antigens (for pre- venting and treating infectiow, allergic, and autoimmune dise-lse, ar.d per!:aps even ca!l- cer) ; Certain enzymes, such as Ebrinolysin and urokinase (promismg agents In the trent- ment of embolism) and lysosomal enzJ`mes. (2) Endouiment of plants with new synthesis capabilities. Whole plants may be generated from a single cell, and thus insertion of recombinant DNA into such cells might make it possible to endow plant species with the capability of- Improved photosgntte:ic f%eEion of car- bon dioxide; Nitrogen fixation by presently inept species (thereby reducing the need for costly chem- ical fertilizers that cause PollUtiOn-e.g., eu- trophication) : producing a higher Qvallty or q2iantiry of food protein. (3) Some industrial applications. A number of industrial processes utilize microorganisms containing enzymes (which are proteins) to produce impor- tant chemicals (e.g., steroid hormones or other drugs, vitamins) or foodstuffs (e.g.. cheese). Such processes could be im- proved through innovations effected by DNA recombinant research. COmPletelY new biosynthetic reactions may thereby become available, Permitting the synthe- sis of large amounts of complex and FEDERAL REGISTER, VOL. 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976 ::5&32 valuable compounds with ease and at low cost. Some highly speculative applications relate to the area of energy production and neutralization of pollutants-e.g., as in oil spills. Genetic modification through DNA recombination might it possible to devise microorganisms tailor-made for such important purposes. 3. Long-range implications. The exper- imental situations treated in the Guide- lines are those that appear feasible either currently or in the near future. The ex- periments primarily involve insertion of recombined DNA into bacteria or into single cells derived from more complex organisms and maintained under special laboratory conditions. It is only in the case of plants that the Guidelines cover exueriments involving insertion of DNA &to cells capable of d&eloping into com- plex, multicellular organisms. The Guide- iin& and the discussions leading to their development have focused on problems of safety. It is possible that techniques similar to or derived from current recombinant DNA methodology may, in the future, be applicable to the deliberate modifica- tion of complex animals, including hu- mans. Such modification might have as its aim correction of an inherited defect in an individual, or alteration of herit- able characteristics in the offspring of individuals of a given species. The latter type of alteration has been succe&ull~ achieved in agriculture for centuries, by classical breeding techniques. It may be that recombinant DNA methods, should they develop in appropriate ways, may offer new opportunities for specificity and accuracy &`I- animal breeding. The deliberate application of such methods for the correction of individual genetic defects or the alteration of herit- able characteristics in man raises con+ plex and diftlcult problems. In addition to philosophical, moral, and ethical ques- tions of concern to individuals, serious societal issues are involved. Broad dis- cussion of these problems in a variety of forums will be required to inform both private and public decision-making. 4. Possible deliberate misuse. In the event that recombinant DNA technology can yield hazardous agents. such agents migl& be considered fir deliberate-per- petration of harm to animals (including humans), plants or the environment. The possibilities include biological warfare or sabotage. Because it is not known whether recombinant DNA technology can yield such agents, discussion of these problems such as theft by saboteurs is hypothetical and difficult. With regard tdbiolotical warfare. a July 3. 19%let- ter to cr. David Baitimore- frbm James L. Malone. General Counsel of the United States A&s Control and Disarmament Agency says, "you raise the question as to whether the Biological Weapons Conven- tion prohibits production of recombinant DNA molecules for purposes of construct- ing biological weapons. In our opinion the answer is in the affirmative. The use of recombinant DNA molecules for such purposes clearly falls within the scope of the Convention's provisions." NOTICES (1) Handler, Philip, (ea.) (1970). Biology and the Future of Man. Oxford University Press. New York, N.Y. (2) Singer, M. F. and D. Soil (1973). Guide- lines for DNA Hybrid Molecuies. Science 182:1114. (3). Berg, P., D. Baltimore, H. W. Bayer, S. N. Cohen. R. W. Davis. D. S. Hoaness, D. Nathans, R. 6. Roblin, J. ti. Watson, 5. Weiss- man, and N. D. Zinder (1974). PotentiaZ Bio- hazards of Recombinant DNA Molecules. Science 185:303. (4) Berg, P.; D. Baltimore, S. Brenner, R. 0. Roblin, and M. F. Singer (19'75). Summary Statement of the Asilomar Conference on Recombinant DNA Molecules. Science 188:991; Nature 225:442; Proc. Nat'l. Acad. Sci. 72:1981. (5) Struhl, K., J. R. Cameron, and R. W. Davis (1976) Functional Expression of Eukaryotic DNA in Esoherichia Colt. Proc. Nat'l. Acad. Sci. U.S.A. 73:1471-1475. (6) Goebel, W. and W. Schiess (1975). The Fate of a Bacterial Plasmid in Mammalian Cells. Mol. Gen. Genet. 138:213-223. (7) Merril, C. R., M. R. Geier and J. C. Petricciani (1971). Bacterial Virus Gene Ex- pression in Human Cells. Nature 233 : 398-400. (8) Doy, C. H., P. M. Gresshoff and B. G. Rolfe (1973). Biological and Molecular Evi- dence for the Transgenesis of Genes from Bacteria to Plant Cells. Proc. Nat'l. Acad. Sci. U.S.A. 70:3: 23-726. (9) Novick. R. P. and S. I. Morse (1967). In Viva Transmisston of Drug Resistance Factors Between Strains of Staphylococcus Aureus. J. Exp. Med. 125:45-59. (10) Anderson, J. D., W. A. Gillespie and M. H. Richmond (1974). Chemotherapy and Antibiotic Resistance Transfer Between En- terobacteria in the Human Gastrointestinal Tract. J. Med. Microbial. 6:461473. (11) Hays, W. (1968). Genetics of Bacteria and Their Viruses. John Wiley and Sons, Inc., Second Edition, New York, N.Y. (12) Tooze, J. (1973). Molecular Biology of Tumour Viruses. Cold Spring Harbor Lab- oratory, Cold Spring Harbor, N.Y. (13) Lavl. S.. and E. Winocour (1972). Acquisition of Sequence Homologous to Host Deoxyribonucleic Acid by Closed Circular Simian Virus 40 Deoxyribonucleic Acid. J. of Virology 9:309-316. (14) Brbckman, W., T. N. H. Lee and D. Nathans (1973). The Evolution of New Species of Viral DNA During Serial Passage of Simian Virus 40 at High Multiplicity. Virology 543384-397. (15) Gillespie, D.. W. C. Saxinger and R. C. Gallo (1976). Information Transfer in CeZls Infected by RNA Tumor Viruses and Exten- sion to Human Neoplasia. Prog. NUC. AC. Res. and Mol. Biol. 15:1-108. (16) Markham, P. D. &d M. A. Baluda (1973). Integrated State of Oncornauirus DNA in Normal Chicken Cells and in CeZ& Transformed by Atian Myeloblastosis Vfsw. J. Viral. 12:721. (17) Hill, M. and H. Hillova (1972). Virus Recovery in Chicken CeZZs Tested with Rous Sarcoma Cell DNA. Nature New Biology 237:35. V. DESCRIPTION OF THE PROPOSED ACTION The Director, Nat&iii&l Institutes of Health, has issued Guidelines that will govern the conduct of NIH-supported re- search on recombinant DNA molecules. The Guidelines will apply to all MH- supported research on such molecules- that is, molecules which are made by combining segments of DNA from differ- ent organisms in a cell free-system and which can be inserted into some living cell, there to replicate. The objective of the Guidelines is the protection of the laboratory worker, the general public, and the environment from infection by possibly hazardous agents that may re- sult from this research. The complete text of the Guidelines is found in the FEDERAL REGISTER, Part II, for Wednes- day, July 7, 1976. As an integral part of this Draft Environmental Impact State- ment the Guidelines are found in Appen- dix D. The mechanisms by which the MH will implement the application of the Guide- lines are outlined in the Guidelines them- selves and are specified in greater detail in Appendix C. Noncompliance with the Guidelines will result in termination of funding of research grants and contracts. The Guidelines describe (1) safeguards that protect the laboratory worker, the general public, and the environment, (2) the criteria for assessing the possible dangers from experiments involving re- combinant DNA molecules, (3) the cri- teria for matching the assessed possible dangers of individual experiments with the appropriate safeguards, and (4) the roles and responsibilities of principal in- vestigators, their institutions, and MH for ensuring the implementation of the requirements specified in these Guide- lines. The emphasis on protection of lab- oratory workers from infection reflects the fact that laboratory workers are the persons at the greatest risk of infection and that the most likely route of escape of possibly hazardous agents from the laboratory is the laboratory worker. The physical safeguards have been grouped into four levels providing in- creasing capability for containment. The four 1eveIs approximate those rec- ommended by the Center for Disease Control for the control of known in- fectious agents that have been deter- mined to be of (1) no or minimal. (2) ordinary, (3) special, or (4) extreme hazard to man and other living things. These correspond to the terms Minimal, Low, Moderate, and High risk, respec- tively, as used in the NIH Guidelines. The safeguards include u;Sual and spe- cial microbiologicA safety practices, priniary physical barriers that isolate the experiment from the laboratory worker, and facility installations that either markedly reduce or eliminate the potential for accidental dissemination of recombinant DNA mol&les to the en- vironment. The four levels. designated Pl to P4, provide increasing protection against contact with or accidental re- lease of microorganisms containing re- combinant DNA molecules, Additional safeguards are provided by the use of host cells and vectors with demonstrably limited ability to survive in other than specially designed labora- tory environments. This concept is called "biological containment" in the Gtide- lines. In the case of bacterial host cells and vectors, this means that particu- lar strains of cells and vectors with genetically determined and fastidious survival requirements must be used. For those experiments fudgti to be of Poten- tially moderate or high risk, the propcr- ties of the bacterial strains to be used FEDERAL REGISTER, VOL. 41, NO. 176THURSDAY. SEPTEMBER 9, 1976 must be certified by the NIH Recom- binant Advisory Committee Prior to in- itiation of experiments. In the case Of a vector derived from an animal virus, the virus itself must be a low risk agent (CM: or National Cancer Institute), and a strain of the virus that is defective in infection must serve as the source of the vector DNA. The selection of containment (safe- guard) levels is dependent on the assessed possible dangers of the experi- ment. The Guidelines provide standards for evaluating the conceivable dangers of particular experiments involving re- combinant DNA molecules. In the ab- sence of evidence of any hazard actually occurring, these standards are based on relevant current knowledge. Permis- sible experiments are placed into four classes of increasing possible danger which correspond to the four levels of in- creasing containment capability (safe- guards). Certain experiments, judged to have the potential for extreme hazard, should they prove dangerous, are pro- hibited. The possibility for danger depends on- (1) The biohazard associated with the DNA or the cell or microorganism that serves as the DNA source (e.g., genes for toxin pro- duction), (2) The degree to which the DNA seg- ment has been purified away from other genes and shown ta be free of harmful char- 6cteristics, (3) The biohazard associated with the vec- tor that 6erve6 to transmit the 6ource DNA to a recipient host cell, (4) The ability of the vector to survive in natural environment6 or habltsts. (6) The kinds and number of different organism6 that are susceptible to infection by the recipient or vector, (6) The biohazard of the recipient host cell that serves to replicate the recom- binant DNA molecule. (7) The abilfty of the recipient cell to 6urvlve in natural environments or habitats, (8) The ability of the recipient 6611 to transmit the recombinant DNA molecule to other cells capable of surviving in natural environments or habitats. (9) The potential of the recipient cell to obtain the source DNA by natural means, and (10) The evolutlonary relatedness of the DRA source to humans. - The Guidelines prohibit a number of types of experiments, including those in which an organism contributing DNA i itself a biohazard of greater than low risk 8s determined by conventional methods of risk assessment (low risk cor- responds to class 2 agents as defined by the Center for Disease Control). The host cells and vectors are required to be of no or minimal risk. The potential dangers are considered to increase as the organism providing the source DNA ap- proaches humans phylogenetically. `I'bus, source DNA from primate cells is considered to have greater potential dangers than source DNA from lower eukaryotes. In general, greater possible dangers are assigned to recombinants than are present In the most hazardous component used to construct the DNA. The risk-assessment standards are specified in detail for one prokaryote host-vector system employing a variant of 1. coli called strain K12, which is, by itself, of no or minimal risk. Eukaryote host-vector systems using defective viral vectors are also described. The descrip- tions of these systems provide principles by which the potential dangers of recom- binant DNA experiments with other host-vector systems can be assessed. The Guidelines also establish an ad- ministrative framework for assigning the responsibility for ensuring safety in rec- combinant DNA research supported by NIH. This responsibility is shared among the principal investigators, their institu- tions, and NIH. The principal investiga- tors have the primary responsibility for hazard assessment and for implemen- tation of appropriate safeguards. The in- stitutions are responsible for ensuring that the principal investigators have the capabilities for meeting the requirement6 stipulated in the Guidelines. NIH is re- sponsible for securing an independent as- sessment of the potential dangers of this research and for ensuring that no re- search is supported unless it conforms to the requirements stipulated in the Guide- lines. The Guidelines require that the insti- tutions establish biohazard committees to carry out the institutional responsibility, and stipulate the qualmcations and ex- pertise of the committee membership. NIEI responsibilities are detailed in the Guidelines and are divided among (1) NIH Initial Review Groups, (2) the NIH Recombinant DNA Molecule Program Advisory Committee, and (3) the NIH staff. Physica containment requirements The safeguards in the Guidelines re- quire the use of procedures and physical containment systems to protect labora- tory workers and the environment from exposure to potentially harmful orga- nisms. The requirements include pro- cedures and equipment in which work is to be done and special laboratory room and building features, as well as appro- priate training of workers. The systems are grouped into four levels of contain- mentP1, P2, P3, and P4-each provid- ing a level of containment greater than the one preceding it. The level of con- tainment that must be provided by a lab- oratory in which an experiment is to be done is based on an assessment of the degree of hazard involved. The following description of the physi- cal containment levels is presented to outline these requirements. A complete description may be found in the Guide- lines (Appendix B) . Pi Level (Minimal). A laboratory suit- able for experiments involving recom- binant DNA molecules requiring physica. containment at the Pl level is shown in Figure V-l. Such a laboratory passesses no special engineering design features. Work in this laboratory is generally con- ducted on open bench tops. Special con- tainment equipment is neither required nor generally available. The laboratory is not separated from the general traffic Patterns of the building, and public ac- eess is permitted. Control of biohazards is provided by standard microbiological practices. P2 Level (LouJ). A laboratory suitable for experiments involving recombinant DNA molecules requiring physical con- tainment at the P2 level (see Figure V-2) is similar in construction and design to the Pl laboratory. The P2 laboratory must have access to an autoclave within the building, and it may have a biological safety cabinet. Work that does not pro- duce a considerable aerosol is conducted on the open bench. However, when exces- sive aerosols may be produced, low-risk experiments must be conducted in special cabinets (biological safety cabinets) that provide physical barriers against possible release of organisms. Although this laboratory is not separated from the general traffic patterns of the building, access to it is limited when experiment requiring Pa-level physical containment. arc being conducted. FIC~RE V--l RGURE v--2 PS Level (Moderate). As shown in Frg- ure V-3, a laboratory suitable for experi- ments involving recombinant DNA mole- cules requiring physical containment at the P3 level has special engineering design features and physical contain- ment equipment. The laboratory is sepa- rated from areas that are open to the general public. Separation is generally achieved by controlled access corridor6 and air locks, locker rooms, or other dou- ble-doored facilities not available for use by the general public. Access to the labo- ratory is controlled. Biological safetg cabinets are available within the con- trolled laboratory area. An autoclave shall be available within the building and preferably within the controlled labo- ratory area. Environmental protection i6 provided by waste sterilization tech- niques. The surfaces of walls, floors, FEDERAL REGISTER, VOL. 41, NO. ?76-THURSDAY, SEPTEMBER 9, 1976 bench tops, and ceilings are easily clean- able to facilitate housekeeping and space decontamination The laboratory ventl- l&ion system is balanced to provide for an inflow of supply air from the access corridor into the laboratory. No work in open vessels ls conducted on the open bench: all such procedures are confined to biological safety cabinets. P4 Level (High). As shown ln Figure V-4, experiments involving recombinant DNA molecules requiring physical con- tainment at the P4 level shall be con- fined to work areas ln a maximum-secu- rity facility of the type designed to con- tain mieroorganlsms that are extremely hazardous to man or may cause serious epidemic disease, The facility is either 8 separate bulldlng or a controlled interior area completely isolated from all other areas of a buildllg. Access to the facility is under strict control. Class ID hiolog- ical safety cabinets are available. Fxcrrxx V4 A P4 facilit,y has engineering fea- tures, shown in Figure V-5, designed to prevent the escape of 7 icroorganlsms to the environment (l-4 . The special features in a P4 facility include: Monolithic walls, floors, and ceilings in which all penetrations such ax for air ducts. electrical conduits, snd utility pipes are sealed to ensure the physical isolation of the work area and to facilitate houaekeep- lug and space decontamination. Air locks through which supplies and materials can be brought safely into the facility. Contiguous clothing change and shower rooms through which personnel enter Into and exit from the facility. Double-door autcclaves to sterilfie and safely remove wastes and other materials from the faculty. . A biowesti treatment system to steriliee liquid effluenta lf facility drains are In- stalled. k --iw- a .._ . w-m "*g$ FIGUFS v-8 A separate ventilation system that maln- tains negative air pressures and directional airflow within tha facilitv. A treatment system to decontaminate ex- haust air before It Is dlsperaed to the at- mosphere. A c+ral vacuum utility system Ia not enmuraged: lf one b lnstall~ each branch line leading to a laboratory shall be protected by 6 high-efficiency par- ticulate air filter. %CFERENCES 1. Design Criteria for Viral Oncology Re- oearch Facuftfes. UA Department OL Health, Education and Welfam, Publie Health Service, National Institutes of Health: DHEW Publication No. (NIB) `76- 891, 1976. 2. Kuehne. R. W. (1973). B<~bgfcd Con- tainment Factlfty @r Studying In~eCtiour Df.%?u.ve. Appl. Mlcrobiol. 26.239-145. 8. Runkle. R. 8. snd G. B. Phillips (1060). b?imobial Containment Control Facilities. Van Nostrand Reinhold, New York. 4. Cbatigny, M. A. and D. L Clinger (1969). Contamination control in Aerobiology. In. R. L Dimmick and A. B. Akem (e&a.). An Introduction to Experlmental Aerobiology. John Wiley L Sons, New York, pp, 194- 263. VI. DESCRIPTION OF ALTERNATIVES The following general classes of action have been considered ae alternatives to, or in addition to, the proposed action. The impact of each is described briefly, and reference is made to other portions of this document which have a more complete discussion of the particular lm- pact in question. A. NO ACTION This alternative would perpetuate the situation existing prior to June 23, 1976. At that time the only restrictions on recombinant DNA research stemmed from voluntary compliance of f&e re- search community with the guidelines developed at the International Confer- ence on Recombinant DNA Molecules, held at Asilomar, California, in Febru- ary of 1975. which were published in scientific journals. The Asilomar guide- lines differ in substance from the NIH Guidelines, and are considerably less stringent and less detailed in their re- quirements for containment of poten- tially hazardous organisms. For example, experiments that may be carried out with minimal containment according to the specific language of the Asilomar guide- lines (e.g., the construction of an E. coli plasmid containing the noncancer-pro- duclng DNA segment of SV40) require P3 or P4 according to the NIH Gulde- lines. In addition, while the Asflomar guidelines recommend that certain ex- periments be deferred, the list of experi- ments to be deferred is expanded h the NIH Guidelines. Furthermore, disregard of the Asilomar guidelines carries no sanctions on investigators, and it could be expected that the currently high level of voluntary compliance would be eroded with time. The "no action" alternative would greatly increase the probability that pos- sibly hazardous organisms would be re- leased into the environment. In addi- tion, public concern would be increased in the absence of any Federal action It is concluded that the "no action" alter- native would not afford adequate pro- tection of laboratory workers, the gen- eral public, and the environment from the possible hazards described in sec- tion IV-C-l. The alternative of "no action" would essentially remove from the conduct of research the restrictions inherent in the NIH Guidelines. Experiments concerning basic biological processes, and the devel- opment of technology applicable to medi- cal, agricultural, and industrial prob- lems, would proceed at a faster rate. Moreover, the immediate cost of con- ducting research would be markedly de- creased with the "no action" alternative. since the need for costly physical con- tainment would be less. B. NIH PROHIBITION OF FUNDING OF ALL EXPERIMENTS WITH RECOMBINANT DNA NIH could refuse to fund ani any re- combinant DNA experiments. This would not necessarily result in the cessation of such research, since it may still be sup- ported by non-NIH funds both in this country and abroad. Therefore a reduc- tion of risks but not elimination of risks might be achieved by total NIH prohibi- tion. Because the NIH funds a large pro- portion of the total biomedical research effort, a signiilcant delay might be ex- pected in the achievement of the goals and missions of programs designed to elucidate basic biological processes and, in turn, the mechanisms underlying vari- ous disease states. It is widely antici- pated that a variety of research-im- pacting on health and other areas of hu- man concern-will beneflt from recom- binant DNA technology (see Section IV-c-2 ! . American scientists have played a leading role in bringing the potential hazards of recombinant DNA research to the attention of scientists, governments, and international organizations. As a re- sult, there is an effort to adopt safety procedures for the conduct of this re- seearch in many countries. Although na- tions differ in their perceptions of the need to adopt safety measures, and of what the exact measures should be, the NIH Guidelines are being used as a model. NIH. prohibition of the work would undermine American leadership in the establishment of worldwide stand- ards for safety. Finally, prohibition would be likely to have important impacts on American science, both in research and in develop- ment of technology. The leadership of RDERAL REGISTER, VOL. 41, NO. 176THURSDAY, SEPTEMBER 9, 1976 NOTICkS 38435 tie United States in biological research would be threatened. Further, historiqal precedents indicate that measures which interfere with free inquiry in one area of interest, often inhibit the vitalit,y of other aspects of society. C. DEVELOPMENT OF DIFFERENT GUIDELINES Each of the stipulations in the NIH Guidelines was made after assessment of the pcesible hazards associated with Par- ticular experiments. The available data, however. were limited. and different con- clusions could have been reached. Some issues addressed in the preparation of the Guidelines which could have led to different speciflcatlons are as follows: 1. Levels of physical containment. For certain experiments in which the poten- tial risk is controversial, the physical containment level could have been high- er or lower. Examples of controversial issues are the recommendations with re- spect to containment levels for recombi- nant experiments involving bacterial cells and DNA derived from cold-blooded animals, and for -experiments involving the use of DNA from animal viruses. 2. Establishment of a few national P3 facilities openly available to all investf- gators, with the requirement that all ex- periments requiring P3 containment be conducted therein. In effect, this will be the situation with respect to P4 facilities under the Guidelines. There are several advantages to working in regiona. cen- t43-S: a. It m-ould he le!;a expensive io construct and staff s few such regional ce~?ters than many 6uch facilities. b. ~!rraining would be centralized. E. PB facilities would be more unifornllv accessible to qualified investigator6 from a variety of institutions. d. There would be greater assurance that the facilities meet the specified require- ments. e. Banks of cells cont.aining recombinant BNA could be maintained, with a view to decreasing the number of times the actual recombination process would be performed (such banks can also be maint.ained in the absence of centralized P3 facilities). 1. The sliks could be placed away from populat,ion centers. The disadvantages of establishing re- gional centers include: a. Long-range planning would be neces- SarY. b. Scheduling would be a problem. e.. The Investigator's independence would be diminished. d. Competition for access might favor es- tiblished investigators or established ideas. e. The nature of the urocess, which might require only brief acce& of p3 facilities & a given day but over 8 lengthy period of time. f. Acce.~ problems might unnecessarily dis- fxxxrage valuable research. 2. AU permissible recombinant DNA experiments be conducted in P4 facili- ties. This alternative implies no distinc- tion among experiments. It does not rec- ognfze that certain recombinant DNA experiments are widely agreed to pose little, if any, possible hazard. It is equl- valent t.e a total prohibition on much recombinant DNA research because af the limited number of P4 facilities that a,re available a.nd the high cost of con- struction. Because of access problems, interesting and important research of low or moderate possible hazard would be discouraged. 4. Exaeriments vrohibited at this time Certain types of `experiments are pro- hibited by the Guidelines. Their selec- tion was a matter of judgment, and de- pended on the assessment of the seri- ousness of the uossible hazard. Alteina- tive assessments would result in either an expansion or a contraction of the list of prohibited experiments and consequent decrease or increase in the Possible risks. Some of the controversial recom- mendations are- a. The prohibition of experiments in- volving more than 10 liters of culture fluid containing recombinant DNAs known to make harmful products with- out the express approval of the NIH Re- combinant Advisory Committee. Contro- versy over this recommendation relates to the fact that some investigators and laboratories contend that larger volumes of culture fluid can be safety contained by special procedures and facilities. The recommendation places responsibility for evaluating the containment on the NIH Recombinant Advisory Committee. b. Sanction of the use of the bacterium Escherichia coli as a recipient for recom- binant DNA molecules. This organism has been studied extensively and is well suited to recombinant DNA research. It has been argued, however, that E. COli should not be used at the present time. This is because many E. coli strains are intimately associated with humans and other living things, and because they readily exchange DNA (genes) with cer- tain other bacteria in nature. Theoretically, the most desirable bac- terial recipient of recombinant DNA would be a species uniquely adapted to carefully controlled laboratory environ- ments and unable to survive or transmit DNA to other organisms in any natural environment. This means that the bac- teria should be unable to survive in nor- mal ecological niches, either in the lab- oratory or neighboring areas. It should be unable to colonize or survive in or on other living things, or in soil or water. In addition, these properties should not be significantly altered by the insertion into the bacterium of the recombined DNA. The bacteria must also be able to be manipulated for successful execution of the proposed experiment. No bacteria is known to meet all these requirements. The guidelines permit the use of various forms of a aarticular strain _-- -~ .~~~~.~ of E. coli called K12. ?I'he forms are called EKl, EK2 and EK3 in the Guide- lines where they are discussed in detail.) Some of these forms already exist, others need ta be constructed. Although related to other E. coli strains that do not in any way meet the definition of the ideal or- ganism, these permissible strains of E coli partially fulfill many of the criteria in the definition of the ideal strain. At present, no other bacterial species is known to approximate the definition as closely as E. coli K-12 and its derivatives. In the future, other bacteria, closer to the ideal, may become known, or the properties of already known species may be shown to approach the ideal more closely than E. coli strain K12 and its de- rivatives, 85 defined in the Guidelines. c, Sanction of the use of Simian Virus 40 (SV 40) as a carrier of a foreign DNA fragment. It has been argued that SV40 should not be permitted, since it is known to cause cancer in laboratory am- mals, There is little evidence that SV40 results in disease in humans. However, SV40 infects humans, and demonstrable antibodies to SV40 indicate that h&c- tion has occurred in some members of the general population. Some of the infection may have resulted from the inadvertent inoculation of millions of individuals dur- ing the initial mass program of immunl- zation aganist polio virus before SV40 was identiiied as a contaminant in the VW- tine. The antibodies may have been formed against SV40-like viruses known to exist naturally in humans (1). It is possible that a recombined DNA carried by SV40 could infect humans and sig- nifirantly affect their health (2). The Guidelines restrict the use of SV40 DNA to DNA from strains of the virus that. are I-- defective in the infection process. In addition, stringent physical containment is required. d. Sanction of experiments involv*iW the transfer of uncharacterized mfxtures of DNA segments deri,ved from wam- blooded animals into bacteria. Such fiv- periments are believed to present a greater possible risk than others because they involve a conglomeration of un- defined genes that might include DNA ca.pable of causing disease. e. Sanction of the use of oncogenic uirmes. It has been argued that tir* . . ..- int~roduction into E. cob of the whole DNA or any purified segment of the DNA _- .-- of any virus oncogenic ln any species should not be permitted. II. n;o guidelines but NIH consideration oj eucil proposed project on an individual oasis before funding. With this alterna- tive, mclividual investigators requesting NIH funds for projects involving recom- binant DNA research would bring plans for proposed experiments to an NIH comrl~itt.ee t,hat would, without the us* of _- -_ foirnal guidelines, recommend suitable ronminnaent measures. Depending on the criteria used by the committee, this might result in lower or higher containment levels than a.re currently imposed by the Guidelines. The advantages of such a procedure would include constant -reI eraluation of potential hazards and con: taimnent measures, and up-to-date in- formation for investigators. The dis- advantages include the enormous time and resources required for review, given the size of the biological research enter- prise in the United States, the problem of finding knowledgeable individuals to serve on such a committee--essentially a full-time occupation-the opportunny for arhil,rary decisions, and the bypasx- uig of local input in asscisment. of hazar&. It should be pointed out that under the present NIH Guidelines, loc:ll institu- tional biohazards committees must con- sider proposed research projects on an IndivldunJs basis and may im5)ose more FEDERAL REGISTER, VOL 41. NO. 176THURSDAY, SEPTEMBER 9, is'76 , 3&m -:NQF##ES stringent safeguards than those required bv the Guidelines. The judgnients of the i&estigator and hts lo&l c&mittee will be reevaluated by the NIH Study Section reviewing the scientific merit of the .proposal. E. GENERAL FEDERAL REGULATION OF ALL SUCH RESEARCH The NIH Guidelines control only re- combinant DNA research supported by NIH. Nevertheless, NIH has assumed a real responsibility to work toward the promulgation of safety measures for all such research. Nationally, NIH has con- ducted and is continuing to conduct meetings' with representatives of other Federal agencies and of private industry. In the case of the Federal Government, consideration is being given to the im- wsition of the Guidelines either bs indl- iidual agency adoption or through an Executive Order. Non-Federa. groups have indicated that they will voluntarily comply with reasonable gujdelines de- signed to be applicable to their specific needs. From the international standpoint, the NIH has been in communication with relevant national bodies, the World Health Organization, the European Mo- lecular Biology Organization, and the In- ternational Council of Scientific Unions, among others, to encourage the widest possible application of the Guidelines. A variety of administrative mecha- nisms could be employed to regulate re- combinant DNA research. Relevant agencies are the Center for Disease Con- trol (CDC). including the National In- stitute fbr. Occupational Safety and Health (NIOSH), or the Occupational Safetv and Health Administration. De- partment of Labor (OSHA) . For ex$mple NIH could petition OSHA ti enforce and monitor such research through its stand-= ard procedures. If OSHA concurred, the adopted guidelines could be extended to all facilities under OSHA's responsibility. Legislation could be passed to impose procedures and specify containment for recombinant DNA experiments. Specific guidelines, as well 85 appropriate en- forcement mechanisms and penalties, could be established as statute. The ad- vantages of this approach would include uniformity in coverage and process. The disadvantages include the need for estab- lishment of a new administrative mech- anism and consequent costs. the long time aeneiallv reauired for enactment of legislation, and the relative inflexibility of law. Flexibility is desirable because presently recommended containment pro- cedures will surely require timely revision as knowledge and experience are accu- mulated. A body like the National Commission on the Protection of Human Subjects of Biomedical and Behavioral Research could be legislatively established. It should be noted that a bill (S. 2515) cur- rently under consideration in the Con- gress would assign responsibility for con- sideration of recombinant DNA experi- ments to a permanent President's Com- mission for the Protection of Human Sub- jects of Biomedical and Behavioral l3e- search. A real concern would be the in- ability of a group with such a broad man- date to deal effectively with the highly specialized subject of recombinant DNA research. REI`EREiW'ES (I) Millarkey. M. F.. J. F. Hruska and,K. K. Takemoto (1974). Comparison of Human Papooa Viruses With Simian Virus 40. J. Virol. 13 : 1014-1019. (2) Shah, K. and N. Nathanson (1975). Human Eqosure to SV40: Review and Cm- ment. A resource document for the meeting on Recombinant DNA molecules. Asilomar Conference Center, February 24-26, 19'75. VII. ENVIRONMENTAL IMPACT 0F THE GUIDELINES A. IMP.\CT OF ISSi'ANrE OF NIH GUIDELINES The primary impact of issuance of the Guidelines is to provide a mechanism for the protection of the laboratory worker, the general public, and the environment from the possible hazards that might re- sult from recombinant DNA molecule re- search. These hazards are purely specu- lative at present; the speculations may prove to be wrong. Nevertheless the Guidelines take cognizance of the possi- bility of dangers to the laboratorg work- er, other persons, and the environment posed by the emergency research tech- nology involving recombinant DNA mole- cules, and call for a number of measures aimed at reducing or eliminating human and environmental exposure to materials containing recombinant DNA molecules, in case they should prove hazardous. The Guidelines govern only work supported by the NIH, including NM supported re- search at various institutions (grants and contracts) and research carried out with- in NIH intramural laboratories. With regard to the anticipated but speculative benefits of recombinant DNA research, adherence to the Guidelines may postpone their realization. Certain experiments are prohibited; many per- missible experiments will be delayed pending availability of suitable contain- ment facilities and certification of ap- propriate hosts and vectors. 1. Impact on the safety of laboratory wrsonnel and m the swead of vossiblv jurzatdous agents by injected iadorator$ perso?meZ. The ND3 Guidelines are di- re&l. concerned with reducing and elim- inating exposures of 1aborat.G~ person- nel and all other persons to host cells and microorganisms containing recombinant DNA molecules. Because laboratory per- sonnel would be the chief source of in- fection of other people, protection of personnel is of primary importance. Lack of knowledge about the real risks of such molecules makes it immble to deter- mine either the nature of the hazards or the extent to which laboratory personnel are endangered by exposures to the ma- terials. Nevertheless present understand- lng of biology permits a ranking of the possible risks that may be associated with a given experiment. Four levels of possible risk have been established: minimal, low, moderate, and high. PiotecUon of personnel from min- lmrd risk materials is provided by or- dinary microbiological techniques. SincS these procedures are generally per- formed on the open bench, exposures may occur. The avoidance of harmful effects depends more on the exceedingly low potential of these materials to cause a harmful infection than of the elimina- tion of potential exposures. Potential harmful effects would require exposure to large numbers ol organisms, e.g., due to accidental ingestion by poor pipetting techniques or self-inoculation by needIe and syringe). Such exposures should be prevented-by adherence to practices rec- ommended for this risk level. The safety of personnel handling ma- terials of minimal risk in the prescribed manner is supported by the absence of any documented laboratory-acquired bacterial or viral infections involving known human etiologic agents that are customarily handled in the same fash- ion-i.e., CDC class 1 agents (see Glos- sary ) , The protection of personnel from po- tential dangers associated with-low- and moderate-risk materials is provided by a greater reliance on physical barriers sep- arating the laboratory personnel from the experimental process as well as on safe microbiological practices. Acci- dental exposure by ingestion would be prevented by the adherence to the re- quired use of mechanical pipetting for low- and moderate-risk materials. Po- tential exposure to low-risk materials through aerosols is reduced by the re- quirement that all processes that pro- duce significant aerosols are to be con- fined to biolonical safety cabinets. Po- tentlal exposure to moderate-risk ma- terials through aerosols is further re- duced by the requirement to contain all processes that produce any aerosol. The use of Class I and Class II biological safety ca.binets that comply with the standards specified in the Guidelines can reduce the potential exposure by a factor of 10,000 (1). Potential exposures of laboratory personnel not involved in these experiments are further controlled by the specified laboratory access pro- cedures. These measures do not provide absolute protection from exposures, and the required primary barriers can be compromised by lack of attention to technique, poor placement of equipment, and human error. Experience demon- strates that the use of these measures reduces but does not prevent the poten- tial for laboratory-acquired infections with relatively infectious agents such as class 2 and class 3 agents. The nature of the harmful effects from exposures to low- and moderate-risk re- combinant DNA materials cannot be de- termined. However, the ability for these materials to cause disease or injury, should they be hazardous can be esti- mated by comparison of their infectivity with that of known class 2 and class 3 agents. The requirement that recipient bacterial cells be class 1 asrents (no or minimal risk) and that animal virus iec- tars be similarly low risk agents (in the absence of recombined DNA) reduces the likelihood that they will have the infec- tious properties of class 2 or 3 agent3 upon insertion of foreign DNA FEDERAL REGISTER, VOL. 41, NO. 176--THURSDAY, SEPTEMBER 9, 1976 Recombinant DNA experiments as- sessed to have high-risk potential require special precautions designed to prevent exposures, as specified in the Guidelines. All such experimental procedures are re- auired to be surrounded by absolute Prl- mary barriers that are gas-tight. These are barriers that physically isolate the experimental process from the laboratory worker. Research is conducted within these barriers through attached gloves. Mate&& are not removed from the bar- riers until they have been sterilized or put into hermetically sealed containers, which are then surface sterilized. Experience with class 3 and 4 human etiologic agents demonstrates that the absolute primary barriers can be oper- ated without exposure of the operators under standardized procedures, employ- ing stable, well trained and well-disci- plined personnel (2) . This conclusion is based on those data in reference 2 that refer to the experience of recent years; the earlier experience is less relevant be- cause of important recent developments in the design and availability of contain- ment equipment. The procedures for combining segments of DNA and insert- ing them into recipient cells can bc standardized, and the Guidelines require that research personnel be well trained and proficient in the necessarv onera- tional practices. Inspection and certifica- tion of all high-risk research facilities by NIH personnel provide additional assur- ances that these requirements will be met. Thus. uotentialls harmful effects from research- with high risk recombinant DNA molecules should be extremely un- likely given strict adherence to the NIH Guidelines. Insofar as research sponsored by NIH 1s concerned, potentially harmful effects from experiments judged to present the possibility of very severe hazard should bc prevented completely since those ex- periments are prohibited. 2. Impact on the environme&al spread of possibly hazardous agents. The NTH guidelines a,re directly concerned with preventing the release of cells and micro- organisms containing recombinant DNA m&cules, or the release of recombinant DNA molecules themselves, into the en- vironment, thus preventing potential ex- posures of humans, other animals and plant communities. The Guidelines require decontamina- tion of all liquid and solid wastes gen- erated by low-, moderate-, or high-risk experiments. As the potential risk of these materials increases (low -+ high). further measures are required to in- crease the certainty of containment. The Guidelines recommend the decontamina- tion of no- or minimal-risk materials before their disposal to the environment. This is standard microbiological practice. The Guidelines prohibit the release of contaminated air under ordinary condi- tions. Procedures involving low- and moderate-risk materials that may pro- duce aerosols are confined to primary barriers. Contaminants In the exhaust air from these barriers are removed by filtration. The potential for accidental release of recombinant DNA materials into the atmosphere, however, increases with de- creasinsz containment reauirements (moderate --) minimal). Harmful sec- ondary effects from such a&dental re- lease of minimal-, low-, or moderate-risk materials are exceedingly remote. An analysis of 36 reported laboratory-ac- quired micro-epidemics in the period 1925-19'75 involving over 1,000 infections with class 2, class 3, and class 4 human etiologic agents demonstrated no infec- tions among persons who were never in the laboratory building or who were not associated in some way with the labora- tory (2). Almost all of these outbreaks occurred in the absence of genuine efforts to control contaminated air, liquid wastes, refuse, and laundry. Any potential release of high-risk ma- terials to the environment should be pre- vented by adherence to the NIH Guide- lines. All high-risk materials are required to be isolated in physically contained, ab- solute primary barriers. All effluents from these barriers are sterilized. The bar- riers themselves are located In maxi- mum-security facilities, which are pro- vided with additional barriers to prevent any accidental release. Air locks, nega- tive air pressure, clothes-change rooms, filtration and incineration of all air ex- hausted from the facility, and the sec- ondary sterilization of all liquid and solid wastes, provide additional protection to the environment. The NIH Guidelines also define re- quirements for protecting the environ- ment from potential dangers that may be associated with the shipment of recom- binant DNA materials. Federal packag- ing standards appropriate for the ship- ment of class 4 human etiologic agents are required for the shipment of all re- combinant materials. 3. Cost impact. The direct cost impact of the NIH guidelines is the cost of com- plying with their provisions. The costs will vary according tc the level of poten- tial risk of the research. There are no special facility requirements for work with minimal- and low-risk recombinant DNA materials (Pl and P2). There are equipment requirements for work involv- ing low-risk recombinant DNA materials that will involve little cost impact. Low- risk research requires a biological safety cabinet for procedures that may produce significant aerosols and an autoclave for sterilizing waste materials. These items of equipment, however, are generally available within the existing facilities where such research is being conducted. The cost impact of the NIH guidelines on minimal- and low-risk research is there- fore not significant. Special equipment and facility require- ments are specified for moderate-risk re- combinant DNA research (P3). All work at this level of potential risk is to be conducted within biological safety cabi- nets (Class I or II). This requirement will necessitate the acquisition of many additional cabinets, the number being dependent on the scope of the research effort. It is estimated that one eabinet will be required for every three persons involved in the research. The cost. of earth cabinet is approximately $5,000. Directional air flow, single-pass ven- tilation, and provisions for ensuring re- stricted access are facility recmirementc specified for moderate risk (P3) recom- binant DNA research. While many new facilities (those constructed in the last decade) have been constructed with this capability, few older facilities can provide this capability without extensive renova- tion. Creating adequate access control by construction of architectural barriers (e.g., air locks, double-door alcove, etc.! is not expensive. However, th cost of ren- ovat.ion of air-handling systems to pro- vide for single-pass, directional air flow may prevent some institutions from con- ducting moderate-risk research. It has been estimated that installation of air- handling systems that comply with the NIH Guidelines would cost approximate- ly $200 per square foot of space serviced by the system. The NIH Guidelines require that high- risk tP4) research involving recombinant DNA materials be conducted only in class III biological safety cabinets (glove boxesi that are installed in maximum security facilities. Fewer than 30 facil- ities within the United States have the potential for meeting the requirements specifieti in the Guidelines for such facil- ities. A smaller number may actually be available for this research. It is esti- mated that approximately $75G,OOO would be required to construct and equip a maximum-security facility having two IO-font by 20-foot laboratory modules with class III cabinetry. This great cost is due to sophisticated mechanical sup- port s:;stcms (e.g., negative pressure, ex- haust sir filtration, air waste treatment plant) and architectural barrier 1e.g.. clothes-change rooms, air locks. waste- staging are& and `monohthic walls floors, and ceilings), The cost of class III csbinetry installed is' approximately $3000 per linear foot. In addition, the c.abinei;ry line and the facilny each re- quire a double-door autoclave, costing a minimum of $15;000 and $65,000 re.;pec- tively. 4. Scc~~cia-ry impacts. There are three secondary impacts which further pro- vide for environmental protection-i.e., reduce the potential risk to the environ- ment from recombinant DNA research' a. Limited maximum-security contaiti- me& capability. The small number of facilities available to support high-risk research greatly restricts the number of such experiments that can be conducted The reduction in the number of emri- mentti minimizes the probability of acci- dental exposure of laboratory workers and subsequent seconda.ry environmcmal impa.ctr. b. Safety uwarencss. The safe perform- a.nce of biomedical research is dependent on an awareness of the risks and the safegutirds required to control the risks. Issuance of the NIH Guidelines should strengthen safety performance in gen- eral by providing safety information and increasing the awareness of the labora- tory worker to the potential hazards as- socia tw3 with biomedical research. FEDERAL REGISTER, VOL. 41, NO. I76--THURSDAY, SEPTEMBER 9. )?76 c. Early recognition of potential iLo:- ards. The Guidelines require that the principal investigator notify NIH of any serious or extended illness or accident that may result in serious exposure to man or to the environment. This moni- toring procedure will provide an early warning of possible unforeseen hazard. For example, if a laboratory infection from exposure to a recombinant DNA molecule is confirmed, indicating a real hazard, an increase in safeguards or ces- sation of emeriments can be required to minimize the hazard to other lnvestiga- tom. conducing similar studies. This up- grading willalsoreduce anypotentin! for environmental effects. 9. IMPACT OF EXPERIMENTS CONDUCTED UNDER THE GUIDELINES 1. Possible undesirable impact--a aspersion of potentianr haaardous agents. The hypothetical mechanisms by which insertion of foreign genes into cells or viruses might result ln the for- mation of hazardous agents are de- scribed in Scct.ion IV-C!. There is, as stated before, no known instance In which a hazardous agent has been created by recombinant DNA technology. Current knowledge permits no more than specn- latlon that such agents may be produced and an equaliy speculative assessment of the nature and extent of hazards that may follow upon a particuh recombinant DNA experiment. This is the underlying reason that the thrust of the Guidelines is to mlnimize contact of organisms con- taining recombinant DNA with other or- ganisms or the environment. Therefore the following analysis of possible un- desirable impacts due to dispersion of notentialls hazardous agents emnhasizes the likelihood of sign&ant dispersion rather than the nature of the hazard lt- self. The analysis given does not appiy in detail to all the- possible situations, but can serve as a model for analyzing different situations. In order that any potential hazard be realized, it is necessary that each of a number of sequential events occur. Each event in the sequence is possible only if the earlier events have occurred. The or- ganism mu&+ (a) contain foreign genes, (b) Escape from the eXperlnle?>I;ii -.`~a- uon, (c) Survive after eecape. -(d) Become established in an environment permitting Its growth and multlpllcation, (e) Contact other living organisme In a signfI%ant manner, including contact by a sufllcient number of organisms to ensure sur- vival and growth and to cause infection. (Note that the environment in (d) map be a living organism itseli). \ In those cases where the detrimental effect results from the formation of a harmful protein, the organism contain- ing the recombinant DNA must- (r) Contain a gene for a potentially harm- rui protein, (g) Be able to express the foreign gene- that is. synthesize the foreign stein, (h) Synthesize the protein in sufacteti quantity to be deleterious to the infected organfsm. NOTICES In those cases where the foreign DNA itself may be the cause of undesirable effects, another set of events must be considered. In the case where the foreign DNA increases the pathogenicity of the inltlal host cell or virus, the inserted DNA must- (i) Impart a selectiv+ advsntage for growth to the carrier of the recombinant DNA as compared with the original cell or virus, (j) Alter the metabolism of the carrier so that it becomes disease producing. -I& the case where the foreign DNA causes undesirable effects by virtue of its transfer out of the original recipient and reinsertion into cells of another species, the DNA must- (k) Leave the original reciplcni wit.hout being destroyed, (1) ~urvlve transfer to another cell, (m) Become associated wlth the other cell ln a stable manner. either aa an independ- ent element or by natural recomblnatioa For example, in a hypothetical experi- ment classified as low-risk and carried out according to the requirements of the Guidelines, events (a) through (h) might be required to yield a hazardous situa- tion. Available data might permit ass&n- ment of probabilities of : 1 for (a) ; of lo4 (1 in 100) for fb) ; of l(r (1 in lo.- 000) for (19; and of lOa (1 ln a mfl- lion) for (d). La& of any pertinent knowledge concerning events (e) through (h) would make assignment of probablll- ties impossible. Even assuming a proba- bility of one for each event (e) through (h) , the overall probability of a deieterl- ous effect on a member of a species at risk in this hypothetical situation would then be the product of ail probabihtiee (a) through (h), namely lOmu (one in a trillion). This probability then needs to be compared with the number of orga- nisms grown for the experiment. ml- tally, bacteria are grown ln liquid mix- tures to a concentration of between 1V and 10" organisms per ml. The probabil- ity will also need to be corrected for the length of time over which the experiment ls to be conducted In reality, it may fre- quently be difficult to assess the relevant probabilities. It is currently impossible to assign specific probabllltles for many exneri- ments, although crude estimates ~821 often be made from current knowledge of laboratory-acquired infections, from prototype experiments set up to measure bacterial or viral escape (4). and from knowledge concerning the stability of or- ganisms and DNA. NlH is currently SUP- porting research designed to improve the ability to evaluate certain of these prob- abilities. b. Other considerations. The foregoing descriptions of the klnds of possibly hazardous situations that might arise from organisms obtained through recom- binant DNA experiments must be con- sidered ln the light of certain more gen- eral issues. (1) Monitoring for r&XZ.W of Org& nisms containing recombined DNA. Con- trol of the spread of any agent outside of an experimental situation to laboratory workers or the outside environment ti greatly assisted by adequate means for monitoring the agent in question. A per- tinent example is the monitoring for spli- lage and spread of radioisotopes. The presence of radioisotopes is readily meas- ured, and the exposure of laboratory per- sonnel or the environment to radiation can be quantified. The situation is funda- mentilly different in the case of orga- nisms or viruses containing recombined DNA. No simple general procedure exlsts for identifying an organism released from the Shotgun experiments. (1) Eukaryotlc DNA recomblnants. (ii) Prokaryotlc DNA rewmblnants. (iii) Characterized clones of DNA recom- blnants derived from shotgun experiments. Purified cellular DNAs other than -plasmids, bacteriophages, and other vlrusea Plasmlds, bacteriophages, and other viruses. (1) Animal viruses. (ii) Plant viruses. (iii) Bukaryotlc organelle DNAs. (iv) Prokaryotic plasmld and phage DNA% 3. Experiments wit.h other prokaryotic host- vectors. 4. Experiments with eaukaryotlc host- vectors. Animal host-vector systems. Plant host-vector systems. Fungal or similar lower eukeryotic systems. Roles and Responsibilities. A. Principal investigator. B. Institution. C. NIR Initial Review Group (Study Sec- tlons) . D. NM Recombinant DNA Molecule Pre gram Advisory Committee. E. NIH Staff. V. Footnotes. VI. References. VII. Members of the Recombinant DNA Molcc-1:e Program Advisory Committee. APPENDXCES A. Statement on the use of Bacillus subtdti in recombinant molecule technology. B. Polyoma and SV40 Virus. C. Summary of Workshop on the Design & Testing of Safer Prokaryotic Vehicles & Bacterial Hosts for Research on Re- combinant DNA Molecules. D. Supplementary Information on Phrhiinl Containment (Including De? ,! ilcd Colltents). I. INTRoDnCTIoN lhc p:irpo.se of these guidelines ih tn rei- omniend safeguards for research on recom- binant DNA molecules to the National In- stitutes of Health and to other institutions that support such research. In this context we define recombinant DNAs as molecules t.hat consist of different segments of DNA which have been joined together in cell-free systems, and which have the capacity to in- Sect and replicate in some host cell, either autonomously or as an integrated part of the host's genome. This is the first attenint to Drovide a de- tailed set of guidelines for use by study sec- tions as well as practicing scientists for ev:ii- uating research on recombinant DNA moie- culen. We cannot hope to anticipate a11 PO+ sible lines of imaginative research that are possible with this powerful new method-. ology. Nevertheless, a considerable volume of written aud verbal contributions from sci- entifits in a variety of disciplines has been received. In many instances the views pre- sented to us were contradictory. At pre>er?t, the hazards may be guessed at, speculated abo;:t.. or vo:ed upon, but they cannot be FEDERAL REGISTER, VOL. 41. NO. 176--THURSDAY, SEiTEMBER 9, 1976 :, i&M52 `NOTEES known absolutely in the absence of firm ex- perimental dst-nd. unfortunately, the needed data w&e, more often than not, un- available. Our DrOblem then has been to con- struct guidelldes that allow the promise o! the methodology to be realized while advocat- ing the considerable caution that is de- mand@ by what we and others view as po- tential hazards. In designing these guidelines we. have adopted the following principles. which are consistent with the general conclusions that were formulated at the International Con- ference Center, Pacific Grove, California, in February 1075 (3) : (1) There are certain ex- periments for which the assessed potential hazard ls so serious that they are not to be attemnted to the oresent time. (iii The re- mainder can be undertaken at thk present time provided that the experiment is justlfl- able on the basis that new knowledge or beneflls to humankind will accrue that can- not readily be obtained by use of conven- tional methodology and that appropriate safeguards are incorporated into the design and execution of the experiment. In addl- tlon to an insistence on the practice of good mlcroblologlcal techniques. these safeguards consist of providing both physical and blolo- glcal barriers to the dissemination of the po- tsntlallv hazardous aeents. (ill) The level of conU&ment provided by these'barriers is to match the estimated potential hazard for each of the different classes of recombinants. For projects in a given class, this level is to be highest at initiation and modlfled subse- quently only if there is a substantiated change in the assessed risk or in the applied methodology. (iv) The guidelines will be sub- jected to periodic review (at least annually) and modifled to reflect improvement% 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 bio- logical containment and for the classes of experiments to which they apply. We reaog- nlze that these definitions do not take into account existing and anticipated special pro- cedures and information that will allow psr- tlcular experiments to be carried out under different conditions than indicated here without sacrifice of safety. Indeed, we urge that individual investigators devise simple and more effective containment procedures and that study sections give consideration to such procedures which may allow change In the dontainment levels recommended here. It is recommended that all publications dealing with recombinant DNA work include a description of the physical and biological containment procedures practiced, to aid and forewarn others who might consider repeat- ing the work. II. CONTAINiVENT Effective biological safety programs have been operative in a variety of laboratories for many years. Considerable information there- fore already exists for the design of physical containment facilltles and the selection of laboratory procedures applicable to orgs- nisms carrying recombinant DNAs (4-17). The existing programs rely upon mechanisms that, for convenience. can be divided into two categories: (i) A set of standard prac- tices that are generally used in mlcrohiolog- ical laboratories. and (ii) sUecia1 orocedures. equipment, and `labor&&y install~tlons that provide physical barriers which are applied in varying degrees according to the estimated biohazard. Experiments on recombinant DNAs by their verv natUre lend themselves to a third contalnnient mechanism-namely, the ap- pllcatlon of highly speclftc biological barriera In fact, natural barriers do exist which either limit the infectivtty of a vector or vehicle (plasmid. bacteriophage or virus) to specific hosts, or its dissemination and survival in the environment. The vectors that provide the means for replication of the recombinant DNAs and/or the host cells ln which they replicate can be genetically designed to de- crease by many orders of magnitude the probability of dissemination of recombinant DNAs outside the laboratorv. As these three means ofVcontainment are complementary, different levels of contain- ment appropriate for experiments with dif- ferent recombinants can be established by applying different combinations of the physi- cal and biological barriers to a constant Use of the standard uractices. We consider these categories of containment separately here in order that such combinations can be con- veniently expressed in the guidelines for re- search on the different kinds of recombinant DNAs (Section III). A. Standard practices and training. The first principle of containment is a strict ad- herence to good mlcroblological practices (4- 13). Consequently, all personnel directly or indirectly involved in experiments on re- combinant DNAs must receive adequate in- struction. This should include at least traln- ing in aseptic tecbnlques and instruction in the biology of the organisms used in the ex- periments so that the potential biohazards can be understood and appreciated. Any research group working with agents with a known or potential biohazard should have an emergency plan which describes the DmCedUres to be followed lf an accident con- taminates personnel or environment. The principal investigator must ensure that everyone in the laboratory is famlllar with both the potential hazards of the work and the emergency plan. If a research group is working with a known Dathoeen for which an effective vaccine ls a&iabie. all workers should be immunized. Serological monitor- ing. where appropriate, should be provided. B. Physical contatnment levels. A variety of combinations (levels) of special practices, equipment, and laboratory installations that provide additional physical barriers can be formed. For example, 31 combinations are listed in "Laboratory Safety at the Center for Disease Control" (4) ; four levels are assocl- ated with the "Classification of Etlologlc Agents on the Basis of Hazard" (5). four levels were recommended in the "Summary Statement of the Asllomar Conference on Recombinant DNA Molecules" (3); and the National Cancer Institute Uses three levels for research on oncogenlc viruses (6). We emphasize that these are an ald to. and not a substitute for, good technique. Personnel must be competent in the effective Use of all equipment needed for the required contaln- ment level as described below. We deflne only four levels of physical containment here, both because the accuracy with which one can presently assess the biohazards that may result from recombinant DNAs does not war- rant a more detailed classlflcatfon, and be- cause additionai flexibility can be obtained by combination of the physlcai wlth the bio- logical barrlers. Though different in detail, these four levels (Pl should also be done using a secondary recipi- ent that is restrictive for the plasmld vector as well as with primary donors possessing re- pressed conjugative plasmlds with incom- patibility group properties like those com- monly found in enterlc mlcrocrganlsms. Since a common route of escape of plasmid- hoat systems in the laboratory might be by accidental ingestion, it is suggested that the same types of experiments be conducted in suitable animal-model systems. In addition to these tests on survival of the vector audj or a cloned DNA framnent. it would be useful to determine the s&lval' of the host strain under nongrowth conditions such as in water and as a function of drying time after a culture has been spilled oti a-lab bench. For EK2 host-vector svstems in which ihe vector is a phage, no more than one in 10" phage particles should be able to perpetuate itself and/or a cloned DNA fragment under non-permissive conditions designed to repre- sent t,he natural environment either (aj as a DroDhage or Dlasmid in the laboratorv ho&t u&d ior phage-propagation or (b) by &vlv- ing in natural environments and transferring Itself and/or a cloned DNA fragment to a host (or its resident lambdoid prophage) with properties common to those in the ~a- tural environment. In terms of potential EK2 X-host systems. the following types of genetic modification should reduce survival of cloned DNA. The examples given are for illustrative purposes and should not be construed to encompass all possibilities. The probability of establishing X lysogeny in the normal laboratory host should be reduced by removal of the phage att site, the Int function, the reprea5or gene(s) and adding virulence-enhancing mutations. The trequency of plssmld forma- tlon, although normally already less than 10 *, could be further reduced by defecte ir; FEDERAL REGISTER. VOL. 41, NO. 176--THURSDAY, SEPTEMBER 9, 1976 38156 NOTICES the PR-Q region, including mutations such as vir-s. cro(TS), ~17, fi=, O(TS), P(TS) , and nin. Moreover, chloroform treatment used routinely following cell lysis would reduce the number of surviving cells, including pos- sible lysogens or plasmid carriers, by more than 10". The host may also be modified by deletion of the host Xaft site and inclusion of one or more of the mutations described above for nlasmid-host svstems to further reduce the ^chanoe of form&ion and survival of any lysogen or plasmid carrier cell. The survival of escaping phage and the chance of encountering a sensitive host in nature are very low, as discussed for EKl sys- tems. The infectivity of the phage particles could be further reduced by introducing mu- tations (e.g., suppressed ambers) which would make the phage particles extremely unstable except under special laboratory con- ditions (e.g., high concentrations of salts or putrescine j . Another meana would be to make the nhaee itself a two-COmDOnent SYS- tern, by eiin&ating the tall genes and ie- producing the phage as heads packed with DNA: when necessary and under specially controlled conditions, these heads could be made infective by adding tail preparations. An additional safetv factor in this reaimen is the extreme Instability of the heads, &less they are stored in 1OmM putrescine, a con- dition easy to obtain in the laboratory but not in nature. The propagation of the escap- ing phage in nature could further be blocked bv addine various conditional mutations which would permit growth only under spe- cial laboratory conditions or in a special permissive laboratory host with suppressor or gro-type (mop, dnaB, rpoB) mutations. An additional safety feature would be the use of an r- m- (hsdS) laboratory host, which produces phage with unmodified DNA which should be restricted in r+ m+ bacteria that are probably prevalent in nature. The likeli- hood of recombination between the ,J vector and lambdold prophages which are present in some E. culi strains might be reduced by elimination of the Hed function and the pres- ence of the recombination-reducing Gam function together with mutations contribut- ing to the high lethality of the X phage. However, these second-order precautions might not be relevant if the stability and infectivity of the escaping h particles are reduced by special mutations or by propa- gating the highly unstable heads. Despite multiple mutations in the phage vectors and laboratory hosts, the yield of phage particles under suitable laboratory conditions should be high (10'0 -10" partl- cles/ml). This permits phage propagation in relatively small volumes and constitutes an additional safety feature. The phenotypes and genetic stabilities of the mutations and chromosome alterations included in these A-host systems indicate that cohtainment well in excess of the required lo* or lower survival freauencv for the h &or with or without a Eloneci DNA frag- ment should be attained. Obviously the pres- ence of all mutations contributing to this high degree of biological containment mUSt be veri5ed periodically by appropriate tests. Laboratory tests should be performed with the bacterial host to measure all possible routes of escape such as the frequency of lysogen formation, the frequency of plasmid formation and the survival of the lysogen or carrier bacterium. SfmIlarly. the potential for perpetuation of a cloned DNA fragment car- ried by lnfectlous phage particles can be tested by challenging typical wild-type E. coli strains or a X-sensitive nonpermissive labora- tory K-12 strain, especially one lysogenic `for a lambdold phage. In view of the fact that accurate assess- ment d the probabilltiea for escape of in- fectious A grown on r- m- Su. hosGs is de- pendent upon the frequencies of r-, Su-, and X-sensitive strains in nature, investigators need to screen E. coli strains for these prop- erties. These data wlll also be useful in pre- dicting frequencies of successful escape of plasmid cloning vectors harbored in r. m- Su- strains. When any investigator has obtained data on the level of containment provided by a proposed EK2 system, these should be re- ported as rapidly as possible to permit gen- eral awareness and evaluation of the safetv features of the new system. Investigators are also encouraged to make such new safer clon- ing systems generally available to other scien- tists. NIH will take appropriate steps to aid in the distribution of these safer vectors and 1lOSt.s. EK3 host-vectors. These are EK2 systems for which the specified containment shown by laboratory tests has been independently conArmed by appropriate tests in animals, in- cluding humans or primates. and in other relevant environments in order to provide additional data to validate the levels of con- tainment afforded by the EKP host-vector systems. Evaluation of the effects of indi- vidual or combinations of mutations con- tributing to the biological containment should be performed as a means to con5rm the degree of safety provided and to further advance the technology of developing even safer vectors and hosts. For the time being, no host-vector svstem will be considered to be a bona tide Ei(3 host-vector system, until it is so certified by the NIH Recombinant DNA Molecule Program Advisory Committee. 2. Classfflcatfon of experiments using the E. coEi K-2 containment systems. In the fol- lowing classl5cation of containment criteria for different kinds of recombinant DNAs, the stated levels of physical and biological con- tainment are minimums. Higher levels of biological containment (EKS>EK2>EKl) are to be used if they are available and are equally appropriate for the purposes of the experiment. Shotgun experiments. These experi- ment.? involve the production of recombinant DNAs between the vector and the total DNA or (preferably) any partially purified fraction thereof from the specified cellular source. (i) Eukaryotic DNA recombinants-Prf- mates. P3 physical contalnment+an EK3 host-vector. or P4 physical containmentfan EK2 host-vector, except for DNA from un- contaminated embryonic tissue or primary tissue cultures therefrom, and germ-line cells for which P3 physical contalnment+an EK2 host-vector can be used. The basis for the lower estimated hazard in the case of DNA from the latter t&sues (if freed of adult tissue) is their relative freedom from hori- zontally acquired adventitious viruses. Other mammals. P3 physical containment +an EK2 host-vector. Birds. P3 physical containment J an EKP host-vector. Cold-blooded vertebrates. P2 physical con- tainmentfan EX2 host-vector except for embryonic or germ-line DNA which require P2 physical containment+an FXl host- vector. If the eukaryote is known to produce a potent toxin, the containment shall be increased to P3+EK2. Other cold-blooded animals and lower eukaryotes. This large class of eukaryotes is divided into the following two groups: (I) Species that are known to produce a potent toxin or are known pathogens (i.e., an agent listed in Class 2 of ref. 6 or a plant pathogen) or are known to carry such patho- genic agents must us-e P3 physical contain- ment+an EK2 bc&-vector. Any species that has a demonstrated capacity for carrying par- ticular pathogenic agents is included in this group unless it has been shown that those organisms used as the source of DNA do not contain these agents; in this case they may be placed in the second group. (2) The remainder of the species in this class can use FZtEKl. However, any insect In this group should have been grown under labomtory conditions for at least 10 genera- tions prior to its use as a source of DNA. Plants. P2 physical containment-&an EKl host-vector. If the plant carries a known pathogenic Rgent or makes a product known to be dangerous to any species, the contain- ment must be raised to P3 physical contain- ment L- an EK2 host-vector. (ii) Prokaryotes DNA recombinants- Prokaryotes tlLat exclrange genetic infor- matio with E. coli I. The level of physical containment IS di- rectly determined by the rule of the most dangerous component (see introduction to Section III). Thus Pl conditiona can be used for DNAs from those baoteria in Class 1 of ref. 6. ("Agents of no or minimal haz- ard o o *") which naturally exchange genes with E. co% and P2 conditions should be Used for such bacteria if they fall in Class 2 Of ref. 6 ("Agents of ordinary potential hazard o o *"). or plant pathogens or sym- blonts. EKl host-vectors can be used for all experiments requiring only Pl physical containment; in fact, experiments in this category can be performed with E. calf K-12 vectors exhibiting a lesser contaiment (e.g.. conjugative plasmids) than EKl vectors. Ex- periments with DNA from species requiring pd physical containment which are of low pathogenicity (for example, enteropathogenic Escherfchfa colt, Salmonella typhfmurfum, and Klebsfella pneumonfae) can use EKl host-vectors, but those of moderate path- ogeniclty (for example, Salmonella typhf, Shigella dysenteriae type I, and Vfbrfo cholerae) must use EK2 host-vectors.* A specific example of an experiment with a plant pathogen requiring P2 physical con- tainment-&an EK2 host-vector would be cloning the tumor gelle of Agrobacteriztnr tumefacfens. Prokaryotes that do not exchange genetic information with E. coli.The minimum con- tainment conditions for this class consist of P2 physical containmenttan EK2 host- vector or P3 physical containment+an EKl host-vector. and apply when the risk that the recombinant DNAs will increase the path- ogenicity or ecological potential of the host is judged to be minimal. Experiments with DNAS from pathogenic species (Class 2 ref. 6 plus plant pathogens) must use P3 f EK2. (iii) Characterized clones of DNA tecombi- nants derfved Jrom shotgun experfments. When a cloned DNA recombinant has been rigorously characterized' and there is suffi- cient evidence that it is free of harmful genes,' then experiments involving this re- combinant DNA can be carried out under Pl+EKl conditions if the inserted DNA is from a species that exchanges genes with 8. co& and under P2+EXl conditions if not. Purified cellular DNAs other than plasmids, bacteriophages; and other r:iruses. The formation of DNA recombinants from cellular DNAs that have been enrichedc by physical and chemical techniques (l.e., not by cloning) and which are free of harmful genes can be carried out under lower con- tainment conditions than used for the COP- responding shotgun experiment. In general, the containment can be decreased one step in physical containment (P4+P3+P2+Pl) while maintaining the biological contain- ment specified for the shotgun experiment. or one step in biological containment (EK3-r EK24EKl) whlle maintaining the speci5ed physical containment-provided that t.he See foot,notes on p. 38459. FEDERAL REGISTER, VOL. 41, NO. 17dTHURSDAY. SEFTEMBER 9, 1976 NOTICES 38437 new condition is not less than that specified above for characterized clones from shotgun experiment8 (Section -ill). cc> PZasmfds, bacteriophages, and other utfuses. Ftecom~binanta formed between EK- type vectors and other plasmid or virus DNAs have In common the potential for acting aS double vectors because of the replication functions in these DNAs. The containment condltlons given below a.pply only to propa- gation of the DNA recomblnants in E. coZi K- 12 hosts. They do not apply to other hosts where they may be able to replicate 83 a re- sult of functions provided by the DNA ln- serted into the EK vectors. These are con- sidered under other host-vector Systems. (1) dnfmal uftuses. P4+EK2 or P3+EK3 shall be used to isolate DNA recombinanti that include alI or part of the genome of an animal virus. This recommendation applies not only to experiments of the "shotgun" type but also to those involving psrtlally characterized subgenomic segments of viral DNAs (for examole. the eenome of defective vlruaes, DNA %g&eniX i$olated after treat- ment of viral genomes with restriction enzymes, et&). When cloned recomblnants have been shown by Suitable biochemical and biological t&a tc be free of harmful regions. thev can be handled in .P3+EX2 conditions. In iae case of DNA vlru~e& `harmlesS regions include the late region of the genome: ln the csse of DNA cupies of RNA viruses, they might include the genes coding for capsld proteins or envelope proteins. (U) PZant vfruses. F?+EKl or PZ+EK2 cond1t.ion.s shall be used to form DNA re- combinanta t)rcrt include all or part of the genome of a plant virus. (ffl) Eukaryotic ofganelle DNA.% The con- tainment conditions given below apply only when the oreanelle DNA has been ~urlfled* from &olate$ organelles. Mitochon&al DNA from primates: P3qEKl or P2+EK2. Mlto- chondrial or chloroplast DNA from other eukaryotes: P2+EKl. Otherwise, the condi- tions given under shotgun experiments apply. (iv) Prokaryotfc p&mid and phage DNAa. PZasmkls and phage from hosts that ex- change genetfc-fnformatfon wfth E. calf. Experiments with DNA recombinanti formed from plasmlds or phage genomes that have not been characterized with regard to presence of harmful genes or are known tc contribute slgniflcantly to the pathogerdclty of their normal boats must use the contaln- ment conditions specitled for shotgun experi- ment6 with DNAs from the respective host. If the DNA recombinanti are formed from pla~mids or phage that are known not to con- tain harmful genes, or from purified a and characterized plafcnld or phage DNA segments known not to contain harmful genes, tqe experiments can be performed with PI physl- cal containment fan EKl host-vector. PZasmfdp and phage from hosts that do not ezchange genetfc fnformation with E. calf. The rules for shotgun experiments with DNA from the host. apply to their plas- mlds or phages. The minimum contaln&en~ conditions for this cateeorv IP2 CEKP. or P3+EKl) can be used-f"; &s&i a&i phage, or for purified* and characterized segments of plasmld and phage DNAS, when tie risk that the recombinant DNAs will Increase the pathogenlclty or ecological po- tential of the host is iudeed to be minilnal. NOTE : Where appilcaile, cDNAY (i.e., complementary DNAs) synthesized in vitro from cellular or viral RNAs are included within each of the above classlflcat~ions. For example, cDNAs formed from cellular RNAS that are not purified and character- ized are included under . shotgun ex- periments; cDNAa formed Urom purified and See footnotes on p. 33459. characterized RNAs are included under ; cDNAs formed from viral RPv'Pls are included under ; etc. 3. Experiments with other prckaryotic host-vectors! Other prokaryotic ho&-vector systems are at the speculative, planning. or developmental stage, and consequently do not warrant detailed treatment here at this time. However, the containment crl- teria for different types of DNA recombi- nants formed with E. CoZf K-12 host-vectors can, with the aid of some general principles given here, serve as a guide for containment conditions with other host-vectors when appropriate adjustment is made for their different habitats and characteristics. The newly developed host-vector systems should offer Some distinct advantage over the E. coli K-12 host.-vectors-for instance. ther- mophilic organism or other host-iectors WhQSe major habitats do not include humans and/or economically important animals and plants. In general, the straln of any pro- karyotic species used 86 the host fs to con- form to the definition of Class 1 etiologlc agents given in ref. 5 (Le., "Agents for no or minimal hazard o * o "), and the plasmid or phage vector should not make the host more hazardous. Appendix A gives a de- tailed discussion of the B. aubtflfs system, the most, promising alternative to d&e. At the initial stage, the host-Ve&Qr mu& exhibit at least a moderate level of biological containment comparable to EKl Systems, and should be capable of modification to ob- tain high levels of containment comparable t0 EK2 and EK3. The tvne of confirmation test(s) required to movka host-vector from an EK2-type classification to an EKS-type Will clearly depend upon t e habitat of the host-vector. +Fu preponderant r example, ii the unmodified host-vector propagates mostly in, on, or arQURd higher plants, but, not appreciably in warm-blooded animals, modidcatlon should be designed to reduce the probability that the host-vector can e.s- cape to and propagate in, on, or around Such plants, or transmit recombinant DNA to other bacterial hosts that are able to occupy these ecological niches, and it is these lower probabilities which must be confirmed. The following principles are to be followed ln using the containment criteria given for ex- periments with E. calf K-12 host-vectors ss a guide for other prokaryotlc systems. Experl- ments wit+ DNA from prokaryotes (and their plasmids or viruses) are classified accord- ing to whether the prokaryote in question exchanges genetic information with the host- vector or not, and the containment condi- tions given for these two cla.%ses with E. calf K-12 host-vectors applied. Experiments with recombinanti between plasmld or phage vec- tors and DNA that extends the range of re- sistance of the recipient species to thera- peutically useful drugs muse use P3 physical containment + a hose-vector comparable to EKl or P2 Dhv&Cal containment + a host- vector compa&ble to EKZ. Transier of re- combinant DNA to plant pathogens can be made safer by using nonreverting, doubly auxotrophic, non-pathogenic variants. Ex; Derimentz3 usine: a DlaDt, Dat.hok%n that af- ?ects an element oi the focal gora will re- qullp more stringent containment than if carried out In areas where the host plant is not common. Experiments with DNAs from cukargotes (and- their plssmlds or viruses) can also fol- low the criteria for the corresponding experi- ments with E. colt K-12 vectors if the major habitats of the given host-vector overlap those of E. colt. If the host-vector has a major habitat that does not overlap those of E. coli (e.g., root nodules in plants), then the containment conditions iOr some eUkaryQtiC recombinant DNAs ned to be increased (for instance. higher p1ant.S and their viruses in the preceding example) while others can be reduced. 4. Experiments with eukaryotfc host-vec- tors AnfmaZ host-vector systems. Be- cause host cell lines generally have little if any capacity for propagation outside the laboratory, the primary focus for contain- ment is the vector, although cells should also be derived from cultures expected to be of minimal hazard. Olven good microblologlcal practices, the most, likely mode of escape of recombinant DNAs from a physically con- talnecl laboratory is carriage by humans; thus rectors should be chosen that have llt- tie or no ability to replicate in human cells. To be used as a vector in a eukarvotic host. a DNA molecule needs to display ail of the f&- lowing properties: (1) It shall not consist of the whole ge- nome of any agent that is infectious for hu- mans or that replicates ta a significant ex- tent, in human ce124 in ttgsue culture. (2) Its functional anatomy should be known-that is, there should be a clear idea of the location within the molecule of: (a) The sites at which DNA synthesis originates and terminates. (b) The sites that are cleaved by restric- tion endonucleases. * (c) The template regions `for the major gene products. (3) It should be well studled genetically. It is desirable that mutants be available in adequate number and variety. and that Quantitative studies of recombination have &en performed. (4) The recombinant must be defective. that is, its propagation as a virus is depend- ent upon the presence of a complementing helper genome. This helper should either (a) be integrated into the genome of a stable line of host cells (a situation that would effectively limit the growth of the vector to that particular cell line) or (b) consist of a defective genome or an appropriate condi- tional -lethal lnutant virus (in which case the experiments would be done under non- permissive conditions), making vector and helper dependent upon each other for prop- agation. However, if none of these is- avail- able, the use of a non-defective genome as helper would be acceptable, Currentlv onlv two viral DNAs can be con- sidered as &e&g these requirements: These are the genomes of polyoma virus and SV40. Of these, polyoma virus is highly to be preferred. SV40 is known to propagate in human cells, both in viva and in vitro, and tQ infect laboratory personnel, as evidenced by the frequency of their conversion to pro- ducing SV40 antlbodles. Also, SV40 and re- lated viruses have been found in association with certain human neurological and ma- lignant diseases. SV40 shares many prop- erties, and gives complementatlon. with Yhe common human papova viruses. By contrast, there is no evidence that polyoma infects humans, nor does it replicate to any signifi- cant extent In human cells in uftro. How- ever, this system still needs to be studled more extensively. Appendix B gives further details and documentation. Taking account of all these factors: 1. Polyoma tiirus. a Recombinant DNA molecules con&ting of defective polyoma virus genomes plus DNA sequences of any nonpathogenic organism, including Class 1 viruses (5), can be propagated in or used to transform cultured cells. P3 conditions are required. Appropriate helper virus can be used if needed. Whenever there is a choice, it is urged that mQuSe cells, derived preferably from embryos, be used 83 the source of eu- karyotic DNA. Polyma virus is a mouse virus and recombinant DNA molecules containing both viral and cellular sequences are already known to be present in virus stocks grown at a high multiplicity. Thus, recomblnants form6d i?~ ?ilro between polpoma virus DN.4 FEDERAL REGISTER, VOl, 41. NO. 176--THURSDAY, SEPTEMBER 9, 1976 33458 NOTICES and mouse DNA are presumably co: novel irom an evolutionary point of view. b. S!$h experiments are to be done under P4 conditions if the recombinant DNA con- tains segments of the genomes of Class 2 animal viruses (5). Once it has been shown by suitable biochemical and biological tests that the cloned recombinant contains only harmless regions of the viral eenome isee Section III%2-c-l) and that thi host ra&ge of the polyoma virus vector has not been a.l- iered, experiments can be conti!?ued under P3 condit,ions, 2. SV40 Virus. a. Defective SV40 genomes. with appropriate helper, can be used as a vector for recombinant DNA molecules con- z,aining sequences of any non-pathogenic organism or Class I vicsus (5). (I.e., a shot- gun type experiment). P4 conditions are re- quired. Establjshed lines of culturrd cells should be used. b. Such enperi&nl.s are to be carried out in P3 (Or P4) conditions if Tile non- SV40 DNA segment is (a) a purified 0 seg- ment of prokaryotic DNA lacking toxigenic genes, or (b) a segment of eukaryotic DNA whose function has been established. which does not code for a toxic product, and which has been previously cloned in a prokaryotic host-vector system. It shall be confirmed that the defective virus-helper virus sys- tem does not replicate significantly more eP- ficiently in human cells in tissue culture Wan does SV40, following infection at a mul- t.iplicity of infection of one or more helper SV40 viruses per cell. c. A recombinant DNA molecule conslst- ing of defective SV40 DNA lacking sub- stantial segments of t,he late region, plus DNA from non-pathogenic organisms or Class I virllses (6), can be propagated as an alaton- omous cellular element in established lines of cells under P3 conditions provided that there 1s no exogenous or endogenous helper, and that it Is demonstrated that RO infec- tious virus particles are being produced. Un- til this has been demonstrated, t.he appro- priate containment conditions specified in 2. a. and 2. b. shall be used. d. Recombinant DNA molecules consisting of defective SV40 DNA and sequences from non-pathogenic prokaryotic or eukaryotic organisms or Class I viruses (6) can be used to transform established lines of non-permls- sive cells under P3 conditions. It must be demonstrated that no infectious virus par- ticles are being produced; ,rescue of SV40 from such transformed cells by co-cultiva- tion or Wansfection techniques must be car- ried out in P4 conditions. 3. Efforts are to be made to ensure that all cell lines are free of virus pa.rUcles and myco- plasma. Since SV40 and polyoma are limited in their scope to act as vectors, chiefly because the amount of foreign DNA that the normal virlons can carry probably cannot exceed 2 X 10' daltons, the development of systems in which recombinants can be cloned and propagated purely in the form of DNA, rather than in the coati of infectious agents fs necessary. Plasmid forms of viral genomes or organelle DNA need to be explored as possible cloning vehicles in eukaryotic cells. Plant host-vectof s@ems. For cells in tissue cultures, seedlings, or plant parts (e.g., tubers, stems, fruits, and detached leaves) or whole mature plant.9 of small species (e.g., Arabidopais) the Pl-P4 contain- ment conditions that we have specified prevl- ously are relevant concepts. However, work with most plante poses additional problems. See footnotes on p. 38469. The greenhouse facilities accompanyfug p2 laboratory physical containment condition3 can be provided by: (1) Insect-proof green- houses, (ii) appropriate sterilization of con- t.aminated plants, pots, soil, and runoff water, and (iii) adoption of the other standard practices for microbiological work. P3 phyai- cal containment can be suRicient.ly approxi- mated by coniining the operations with whole plant5 to growth chambers like those nsed for work with radioactive tsotopes: Pro- aided, That (i) such chambers are modified to produce a negative pressure environment Y&h the exhaust air appropriately filtered, (ii) that other onerations with Infections materials are carried out undqr the specifiih P3 conditions, and (iii) to guard against in- advertent insect transmission of recombinant DNA, growth chambers are to be routinely fumigated and onIy used in insect proof roomy. The P2 and P3 conditions specified earlier are t,herefore extended to include these cases for work on higher plants. The host cells for experiments on re- combinant DN.4.s may be cells in culture, in seedling or plant parts. Whole plant.5 or plant. parts that cannot be adequately contained shall not be used as hosts for shotgun ex- periment,s at this time, and attempts to in- fect whole plants with recombicant DNA shall not be initiated until the effects on host cells in culture, seedlings or plant parts have been thoroughly studied. Organelle or plasmid DNAs and DNAs of viruses of restricted host range may be used `a9 vectors. In general, similar criteria for selecting host-vectors to those given in the preceding section on animal systems are to apply to plant systems. DNA recombinanti formed between the initial moderately contained vectors and DNA from cells of species in which the vector DNA can replicate, require P2 physical contain- ment. However, if the source of the NA is itself pathogenic or known to carry patho- genic agents, or to produce products dan- gerous .to plants, or if the vector is an un- modified virus of unrestricted hoat range, the experiments shall be carried out under P3 conditions. Experiments on recombinant DNAs formed between the above vectors and DNAs from other species can also be carried out under P2 if that DNA has been puriflede and de- termined not to contain harmful genes. Otherwise, the experiments shall be carried out under P3 conditions if the source of the inserted DNA Is not itself a pathogen, or known to carry such pathogenic agents, or to produce harmful products--and under P4 conditions if these conditions are not met. The development and use of host-vector systems that exhibit a high level of blologi- Cal containment permit 8 decrease of one step in the physician containment speclfled above (P4+P&P2+Pl). FvngaZ or similar lower euharyotk host-uector systems. The containment cri- teria for experiments on recombinant DNA8 using these host-vectors most, closely re- semble those for prokaryotes, rather than those for the preceding eukaryotes, in that the host cells usually exhibit a capacity for dissemination outside the laboratory that is similar to that for bacteria We therefore consider that the containment guidelines given for experiment& with B. c02i K-12 and other prokaryotic host-vectors (sections IIIB-1 and -2, respectively) provide adequate direction for experiments with tiese lower eukaryotic host-vectors. This is particularly true at this time since the development oi these host-vectors is presently in the specu- :ative stage. N. ROLLS AND R~s~o~sx~~~rrres Safety in research involving recombinant DNA moleculea depends upon how the re- search team applies these guidelines. Motiva- tion and critical judgment are necessary, in addition to specific safety knowledge, to en- hure protection of personnel, the public, and rile environment. The guidelines given here are to help &he principal investigator determine the nature of the safeguards t.hat should be fmple- mented. These guidelines will be incomplete 1x1 acne respects because all conceivable ex- oerimenls ivit,h recombinant DNAs cannct now be anticipated. Therefore, they cannot sl;bst:tut~e fc.r the investigator's own knou-l- edgenble and di%riminating evaluation WIlenever !his evaluation calls for an In- crease in cont,ainment over that indicated in t.he culdelines. tlx InvestiEator has a rcspon~~G`~lity to institute such an increw. 111 ,~o:`.trast. :he containment conditions called for in the guidelines should not be decreased without review and approval at the instiiutional and NIH levels. The following roles and resp%ibilities de- :ilit? an administrative framework in which hafety is an essent,isl and integrated func- tion of research involving recombinant DNA molecules. A. Prinecipal im:esliyulor. The principal i:l- vestigator has the primary responsibility for : (i) Determining the real and potential bic- hazards of the proposed research, (ii) dc- terniiniug the appropriate level of biological and physical containment, (iii) selecting the microbiological practices and laboratory techniques for handling recombinant DNA materials, (iv) preparing procedures for deal- ing with accidenta spills and overt personnel contamination, (v) determining the appll- cability of various precautionary medical practises, serological monitoring, and lm- munization, when available, (vi) securing approval of the proposed research prior tc initiation of work, (vii) submitting informa- tion on purported EK2 and EK3 systems to the NIH Recombinant DNA Molecule Pro- gram Advisory Committee and making the st.rains available to others, (viii) reporting to the institutional biohazards committee, and the NIH Offlce of Recombinant. DNA Ac- tivit,ies new information bearing on the guidelines, such as technical information re- lating to hazards and new safety procedures or innovations, (ix) applying for approval from the NIH Recombinant DNA Molecule Program Advisory Committee for large scale experiments with recombinant DNAs known ta make harmful products (Le., more than 10 liters of culture), and (x) applying to NIH for approval to lower containment levels when a cloned DNA recombinant derived from a shotgun experiment has been rfgor- ouslv characterized and tbere is sufecieni evi8nce that tt is free of harmful genes. Before work is begun, the principal in- vestigator is responsible for: (i) Making available to program and support staff copies of those portions of the approved grant ap- plicatlon that describe the biohazards and the precautions to be taken, (11) advIsIng tba program and support staff of the nature and assessment of the real and potential bio- hazards, (ill) instructing and training this staf7 in the practfces and techniques required to ensure safety, and In the pfoceduti for dealing with accidentallv created bloh-ds. and (ii) informing the-staff of the-reasons and provisions for any advised or requesfed precautionary medical practlses, vaccina- tiona, or serum collection. During the conduct of the research, tbs principal investigator is responsible for: (rj FEDERAL REGISTER, VOL 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976 NOTICES Supervising the safety performance of the staff to ensure that the required safety prac- t&es and techniques are employed, (ii) ln- vestlgatlng and reporting in writing to the NIH Offfce of Recombinant DNA Activities and the institutional biohazards committee anv serious or extended illness of a worker or any accident that results in (a) inocula- tion of recombinant DNA materials through butaneous penetration. (b) ingestion of recombinant DNA materials, (c) probable inhalation of recombinant DNA materials following gross aerosolization, or (d) any incident causing serious exposure to per- sonnel or danger of environmental contaml- nation, (111) lnveslgatlng and reporting in writing to the NIH Office of Recombinant DNA Activities and the institutional bio- hazards committee any problems pertaining to operation and implementation of blo- logical and physfcal containment safety prac- tices and procedures, or equipment or facil- ity failure, (iv) correcting work errors and conditions that may result in the release of recombinant DNA materials, and (v) ensur- ing the integrity of the physloal contaln- ment (e.g-. biological safety cabinets) and the biological containment (e.g., genotyplc and phenotyplc characteristics, purity, etc.). B. Institution. Since in almost all cases, NIH grants are made to institutions rather than to individuals. all the reeponslblllties of bhe principal lnvestlgator listed above are the responslbllltles of the institution under the grant, fulfilled on its behalf by the prin- cipa) investigator. In addition, the institu- t:on ls responslble for establishing an lnsti- tutional biohazards committee' to: (1) Ad- vise the institution on policies. (ii) create and maintain a central reference file and library of catalogs, books, articles, newslet- ters. and other communications as a source of advice and reference regarding, for exam- ple, the avallabillty and quality of the safety equipment, the avalIabfl1t.y and level of blo- logical containment for various host-vector systems, sult.able training of personnel and data on the potential biohazards associated with certain recombinant DNA& (111) de- velop a safety and operations manual for any P4 faclllty maintained by the institution and used in support of recombinant DNA research, (iv) certify to the NIH on appllca- tlons for research support and annually thereafter, that facilities, procedures, and practices and the training and expertise of the personnel involved have been reviewed and approved by the inst~itutional biohazards committee. The biohazards commtttee must be sufff- clently qualified through the experience and exoertise of its membershin and the diversity of its membership to ensure respect for its advice and counsel. Its membership should include individuals from the institution or consultants, selected so as to provide a diver- site of dlsclollnes relevant to recombinant DNA technology. biological safety, and engi- neering. In addition to possessing the profes- sional competence necessary to assess and review speclflc activities and facilities, the commlt.tee should possess or have available to it, the competence to determine the ac- ceptabllity of lte flndlngs in terms of ap- pllcnble laws, regulations, standards of prac- tires, community attitudes, and health and enrironmental conslderatlons. Minutes of the meetings should be kept and made available for public inspection The institution is re- sponsible for reporting names of and relevant background information on the members of ita biohazards committee to the NIH. C. NIH Initial Review Groups (Study Sec- tions) . The NIH Study Sect,ions. in addition to reviewing the scientific merit of each grant application involving recombinant DNA molecules, are responsible for: (1) Making an independent evaluation of the real and potential biohazards of the pro- posed research on the basis of these guide- lines, (11) determining whether the proposed physical cont,ainment safeguards certified by the institutional biohazards commit,tee are appropriate for cont,rol of these biohazards. (iii) determining whether the proposed blo- logical containment safeguards are appro- priate, (iv) referring to the NIH Recombl- nant DNA Molecule Program Advisory Com- mittee or the NIH Oi_ce of Recombinant DNA Act,lvltles those problems pertaining to assessment of biohazards or safeguard deter- mination that cannot be resolved by the Study Sections. The membership of the Study Sections will be selected in the usual manner. Biological safety expertise, however, will be available to the Study Sections for consultation and guidance, D. NIH Recombinmzt DNA Molecule' PTO- gram Advisory Committee. The Recombinant DNA Molecule Program Advisory Committee advises the Secretary, Department of Health, Education, and Welfare, the Assistant Secre- tary for Health, Department of Health, Edu- cation, and Welfare, and the Director, Na- tional Institutes of Health, on a program for the evaluation of potent&l biological and ecological hazards of recombinant DNAs (molecules resulting from different segments of DNA that have been Joined together in cell-free systems, and which have t.he capac- ity to infect and replicate ln some host cell. either autonomously or as an integrated part of their host's genome) , on the development of procedures which are designed to prevent the spread of such molecules within human and other populations, and on guidelines to be followed by investigators working with potentially hazardous recombinants. The NIH Recombinant DNA Molecule Pro- gram Advisory Committee has responsiblllty for: I II Revlslne and undatlne euldelines to be `followed by~lnvestlg&ors Go&ring with DNA recomblnante, (ii) for the time being, receiving information on purported EKZ and EK3 systems and evaluating and certifying that host-vector systems meet EK2 or EK3 criteria. (ill) resolving questions ooncemlng potential biohazard and adequacy of con- talnment capability if NIH staff or NM In- lt.ial Review Group so request. and (Iv) re- viewing and approving large scale experiments with recombinant DNAs known to make harmful products (e.g., more thsn 10 liters of culture). E. NIH Staff. NH-I Staff has respolitlbillty for: (i) assuring that no NIH grants or con- tracts are awarded for DNA recombinant re- search unless they (a) conform to these guidelines, (b) have been properly reviewed and recommended for approval, and (c) in- clude a properly executed Memorandum of Understanding and Agreement, (ii) revlew- lng and responding to questions or problems or reports submitted by institutional blo- hazards committees or principal lnvestiga- tars. and disseminating findings, as appro- priate, (iii) receiving and reviewing appllca- tlons for approval to lower containment levels when a cloned DNA recombinant de- rived from a shotgun experiment has been rigorously charact.e&ed and there is sum- clent evidence that it is free of harmful genes, (iv) referring items covered under (il) and (111) above to the NIH Recombinant DNA Molecule Program Advisory CommIttee, as deemed necessary, and (v) performing site inspections of all P4 physical containment facllltles, engaged In DNA recombinant re- search, and of ot.her facilities a.? deemed necessary APPENDIX D V. Foororxs 1 Blological Safety Cabinets referred to in this section are class~ed as Class I. Chsa II or Class III cabinets. A Ckrss I cablnet ls a ventilated cabinet for personnel protection having an inward flow of air away from the operator. The exhaust air from thls cablnet is filtered through a high efflclency or high ef- ficiency particulate air (HEPA) filter before discharged to the outside atmosphere. This cabinet is used in three operational modes: (1) with an 8 inch high full width open front, (2) w1t.h an installed front closure panel (having four eight inch diameter open- ings) without gloves, and (3) with an ln- stalled front closure panel equipped with arm length rubber gloves. The face velocity of the inward flow of air through the full width open front is 75 feet per minute or greater. A C&s II cabinet is a ventilated cabinet for personnel and product protection having an open front with inward air flow for personnel protection. and HEPA filtered mass recirculated alr flow for product protec- tion. The cabinet exhaust air is filtered through a HEPA filter. The face velocity of the inward flow of air through the full width open front 1s 75 feet per minute or greater. Design and performance speciflcatlons for Class II cabinets have been adopted by the National Sanitation Foundation, Ann Arbor. Michigan. A Class III cabinet is a closed front ventilated cabinet of sas tieht construc- tion which provides the highest-level of per- sonnel protect.ion of 811 Biohazard Safet.y Cabinets. The interior of the cabinet is pro- tected from contaminants exterior to the cabinet. The cablnet is Atted with arm length rubber gloves and is operated under a nega- tive pressure of at least 0.5 inches water gauge. All supply air is filtered through HEPA filters. Exhaust air is Altered through HEPA fllters or incinerated before belng dip- charged to the outside environment. "Defined as observable under optimal lab- oratory conditions by transformation, trans- duction, phage infection and/or conjugation with transfer of phage, plssmid and `or chro- mosomal genetic information. `The bacteria which constitute Class 2 of ref. 5 ("Aeents of ordlnarv notentlal hazard . _ .") rep&sent a broad sp&`um of etlologir agents which possess different 1eveIs of vir- ulence and degrees of communicablllty. We think it appropriate for our specific purpose to" further subciivlde the agents of Class 2 into those which we believe to be of rela- tively low pathogeniclty and those which are moderat.ely pathogenic. The several specific examples given map surnc-e to illustrate t.he principle. 4 The term "characterized" and "free of harmful genes" are unavoidably vague. But in this instance. before containment condi- tions lower than the ones used to clone the DNA can be adopted, the lnvestlgator must obtain approval from the National Institutes of Health. Such approval would be contln- gent upon data concerning: (a) The absence of potentially harmful genes (e.g.. sequen- ces contained in indigenous tumor vlru.ses or which code for toxic substances), (b) the relat.ion between the recovered and de- sired segment (e.g., hybridization and re- striction endonuclease fragmentation anal- ysis where applicable), and (c) maintenance of the biological properties of the vector. 6 A DNA preparation 1s defined as enriched lf the desired DNA represents at least 99% (w/w) of the tot.al DNA ins the preparation. The reason for lowering the containment level when this degree of enrichment has been obtained is based on the fact that the total number of clones that must be ex- amined to obtain the desired clone ia FEDERAL REGISTER, VOL. 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976 38460 NOTICES markedly reduced. Thus, the probability of cloning a harmful gene could, for example, be reduced by more that IWfold when a non- repetitive gene from mammals was being sought. Furthermore, the level of purity spe- cified here makes it easier to establish that the desired DNA does not contain harmful genes. *The DNA preparat,lon Is deflnsd as purl- fled if the desired DNA reuresenta at least 99 percent (w/w) of the t&Z DNA in the preparation, provided that it was verified by more than one procedure. `In special circumstances, in consultation with the NIH OfBee of Recombinant DNA Ac- tivities, an area biohazards committee may be formed, composed of members irom the institution and/or other organizations be- yond its own staff, as an alternative when additional expertise outside the instit.ution is needed for Bhe indicated reviews. V. REFERENCES 1. Berg, P., D. Baltimore, H. W. Boyer, S. N. Cohen. R. W. Davis, D. S. Hogness, D. Na- than& R. 0. Roblln. J. D. Watson, S. Welss- man, and N. D. Zlnder (1974). Potential Bfo- hasards oj Recombinant DNA Molecules. Scl- ence 185, 303. 2. Advisory Board for the Research Coun- cils. 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An In- troduction to Experimental Aerobiology. John Wiley & Sons, New York, pp. 194-263. 18. Grunsteln, M. and D. S. Hognsss (1975). Colony Hybrtdieation: A Method for the Iso- lation of Cloned DNtis That Contain a Spe- cific Gene. Proc. Nat. Acad. Sci. V.8.A. 72, 39613965. 19. Morrow, J. F.. 6. N. Cohen, A. C. Y. Chang, H. W. Bayer, H. M. Goodman and R. B. Helling (1974). Replication and Trans- cription ot Eukaryotic DNA `In Escherichia COZi. Proc. Nat. Acsd. Sci. USA 71, 1743-1747. 20. Hershfield, V., H. W. Boyer, C. Yanofsky, M. A. Lovett and D. R. Helinskl (1974). Plas- mid CoIEl as a Molecular Vehicle for CZon- ing and Amplification of DNA. proc. Nat. Acad. Sci. USA 71,345&3459. 21. Wensink, P. C., D. J. Finnegan, J. E. Donelson, and D. S. Hogness (1974). A Sys- tern @r Mapping DNA Sequences 5n the Chromosomes of Drosoulzila melanooaster. Cell 3, 316525. ' L < 22. Timmis, K., F. Cabello and S. N. Cohen (1974). Utilization of Ttoo Distinct Modes ot Replication by a Hybrid Plasmid Constructed in Vitro lrom Separate ReQliconS. proc. N&t. Acad. Scl. USA 71,4&X%4560. 23. Glover, D. M., R. L. White, D. J. Fiune- gan and D. 8. Hogness (1975). Chnracteriza- lion Of Six Cloned DNAs from DrosoQhfZa melanogaster. Including one that Contains the Geaes lor rRNA. Cell 5, 149-155. 24. Kedes, L. H., A. C. Y. Chang, D. House- man aud S. N. Cohen (1975). Isolation ol Histone Genes from Unfraclionated Sea Urchin DNA by Subculture Cloning in E. colt Nature 255, 533. 25. Tanaka, T. and B. Weisblum (1975). Construction of a Colicin EZ-R Factor Com- posite Plasmid in Vitro: Means for Ampli- floation of Deowibonucleic Acid. J. Bacte- rlol. 121, 354-362. 26. Tanaka, T, B. Welsblum, M. Schnoss and R. Inman (1976). Construction and Characterization of a Ohfmerfc Plasmid Com- posed ot DNA from Eschertchfa col and DrosophiZa meZanogastt?r. Biochemistry 14. 2064-2072. 27. Thomas. M., J. R. Cameron and R. W. Davis (1974). ViabZe Molecular Hybrids o/ Bacteriophage Lambda and Eukaryotic DNA. Proc. Nat. Acad. Scl. USA 71, 46704683. 28. Murray, N. E. and K. Murray (1974), Manfpulation of Restttction Targets tn Phage A to jorm Re&?ptor Chromosomes for DNA Fraoments. Nature 251. 476-481. 25. Rambach, A. a& P. Tiollals (1074). Bacteriophage X Having EcoRZ EndonucZease Sites only in the Non-essential Region oj the Genome. Proc. Nat. Acad. Scl. USA 71, 3927- 3930. 30. Smith, H. W. (1975) StuufuaZ of Orally- Administered Escherichia eoZf Kit tn the Alimentary Tract 01 Man. Nature 255, 600- 502. 31. Anderson, E. 8. (1975). ViabiZttg of, and Transjer of a Plasmid from Escherichia coli Ki2 in the human intestine. Nature 255, 602-604. 32. Falkow, S. (1076). Unpublished experl- ments quoted in Appendix D of the Report oj the Organizing Committee of the AsUo- mar Conference on Recombfnunr DNA Mole- cules (P. Berg, D. Baltimore, S. Brenner, R 0. Roblin and M. Singer. eds.) submitted to the National Academy of Sciences. 33. R. Curtiss III. oersonal communtca- I * tlon. 34. Novick, R. P. and S. I. Morse (1967). In Viva Transmission of Drug Resistance Factors between Strains ol Staphylococcus aweus. J. Exp. Med. 125,46-59. 36. Anderson, J. D., W. A. Gillespie and Ed. H. Richmond. 1974. Chemotherapy and Antibiotic Resistance Transfer between En- terobacterta in the Human Gaatrointestinol Tract. J. Med. Mlcroblol. 6, 461-473. 36. Ronald Davis, personal communication 97. K. Murray, personal communication: W. Szybalskl, personal communication. 88. Manly. K. R., E. R Signer and C. M Raddlng (1969). Nonessential Functfolu of Bactetiophage A. Virology 37 177. 30. Gottesman, M. E. and R. A. WeLsberg (1971). Prophage Insertion and Ezcisfon. In The Bacteriophage Lambda (A. D. Hershey. ed.) . Cold Spring Harbor Laboratory pp. 113- 138. 40. Shimada, K., R. A. Weisberg and M. E Gottesman (1972). Prophage Lambda at Un- usual Chromosomal Locatfons: I. Location oj the Secondary Attachment Sites and the Properties of the Lysogens. J. Mol. Biol. 63. 483-503. 41. SlMnner, E. (1969). PZasmid Formcrfio~. A New *ode of L'ysogeny by Phage A. Nature 223. 15f&-180. 4& Adams, M. H. (1959). Bacteriophages. Intersciences Pub&hers, Inc.. New York. 43. Jacob, F. and E. L. Wollman (1956). Sut I-es Processus de Conjuqaison et de Rf- combinasfon chez Escherichia coli. I. L'tndilc- tfon par Conjugafson ou Induction Zygoti- que. Ann. Inst. Pasteur 91, 486-510. 44. J. 5. Parkinson as cited (p. 8) by Her- shey, k D. and W. Dove (1971). Introdug:- tfon to Lambda. In: The Bacteriophage 1. A. D. Hershey, ed. Cold Spring Harbor ~nl-,,- ratory. New York. CYAIRMAN STETTEN. Hewitt, Jr., M.D., Ph.D., Dcp~.l~y Director for Science, National Il;sti: .:tes of Health VICE CEAIRMAN J,4COBS, Leon, Ph.D., Associate Dlrectclr i;~r Collaborative Research, National Instltu:<+ of Health. ADELBERG, Edward A., Ph.D.. Professor. De- partment of Human Gene&s, Scho& of Medlclne, Yale University. CHU, Ernest. K Y, Ph.D., Professor, De- partment of Human Genetics, Medica School, University of Michigan CURTIS& Roy, III, Ph.D., Professor. Dr- partment or Microblolo,T, School or hwdi- clne, University of Alabama. DARRELL, James E., Jr.. M.D.. Pr0fe.ssc.r. DepartmerIt of Molecular Cell Biology. Rockefeller University. HEXJNSKI. Donald R.. Ph.D., Professor DF- partment of Blolocv. Universitv of Calm- fornia, San Diego. --. HOGNESS. David 5.. Ph.D., Professor. Gr- partment of Biochemistry, Stanford UT-,!- vend tv. KUTTER, Elizabeth M., Ph.D., Member of the Faculty in Biophysics, The Evergrttn State College. LI'ITLEFIELD, John W., M.D., Professor & Chairman, Department of Pediatrics, Chll- dren's Medical & Surgical Center, Johns Hopkins Hospital. - REDFORD, Emmett& S., Ph.D.. LL.D., Ash&I Smith Professor of Government and Pub- 110 Aftairs, Lyndon B. John&ma School OL Publ!c Affalq Eniversity of Texas at hxa- 1m FEDERAL REGISTER, VOL. 41, NO. I76-THURSDAY, SEPTEMBER 9: 1976 ROWE, Wallace P, MD, Chief, Laboratory of Viral Diseases, Natlonal Institute of Al- lergy & Infectloua Diseases, National In- stitutes of Health. SETLOW. Jane K, Ph.D., Biologist, Brook- haven National Laboratory. SPIZIZEN, John, Ph.D., Member and Chair- man, Department of Microbiology, Scripps Clinic & Research Foundation. i3ZYBALSK.I. Waclaw, D.Sc.. Professor o! Oncology. McArdle Laboratory, Unlver- slty Of wiiconsin. THOMAS, Charles A, Jr., Ph.D., ProfesSo?. Department of Biologlcd Chemistry, Har- vard Medical School. GARTLAND, Willlam J, Jr., Ph.D., Health Scientist Administrator, National Institute of General Medical Sciences, National In- stitutes of Health. LIAISON REPRESENTATNES HEDRICH'. Richard. Ph.D., Coordination Pro- gram of Science Technology S Human Value. National Endowment for the Hu- -itieS. LEWIS, Herman W.. Ph.D., Division of Bio- logical and Medical Sciences, National Sci- ence Foundation. NIGHTINGALE, Elena 0.. Ph.D., Assembly of Life Sciences, National Academy of Sciences. BHEPHERD. George R.. Ph.D., Division of Bio- medical and Environmental Research, En- ergy Research and Development Adminis- tratlon. APPRNDIX A TO APPENDIX D IN RECOMBINANT MOLECULE TECIINOI.O(iY Unquestionably. Escherichfu calf 1s the most well characterized unicellular organism Years of basic research have enabled lnvestl- gators to develop a well characterized genetic map. to obtain detailed knowledge of v&u- lent and temperate bacteriophages, and to explore the physiology, genetics, and regula- tion of plasmlds. More recently, the develop- ment of DNA-mediated transformation has permitted exogenous fragments or molecules of DNA to be incorporated into the genome or to reside as self-replicating unite. The dis- coverv of transformation of Bacillus subtiZfs by S~iazen (1) stimulated the development of an alternative model system. The purpose of this report is to summarize the current status of this genetic system and to describe the actual and potential vectors and vehicles available for recombinant molecule technol- WY. A. Current knozD&dge of the chromosomal archftecture and mechanisms ot genetic ax- ehanae in B. subtilis. Two mechanisms o! geneilc exchange have been utIlLzed to estab- lish the linkage map of B. subtilis, DNA- mediated transformation (capable of trams- ferrlng approximately 1 percent of the gen- ome) and transduction with bacteriophage PBS1 (capable of transferring b-8 percent of the chromosome). Recent detailed genetic atudles with PBS1 by Lepesant-Kejzlorova et aI. (2) have resulted in the development of a circular genetic map for this organism The current edition of the map (3) contains 196 loci. Blophysical analyses have estab- lished that the chromosome is circular (4) and replicates bidlrectionally (6). Transformation with purlfled fragments of DNA is a highly efllcient process in B. subtflis with frequencies of 1 to 4 percent usually attained for any auxotrophlc or antibiotic resistance markers. Frequencies of approxi- mately X0 percent transformation can be achieved with DNA prepared from gently lysed L-forms or protoplasm (8). These large fragments of DNA are readily incorporated by the recipient cell. Qeneralised transduc- tion occurs with bacteriophages SPlO (7). PBS1 (8), and SPPl (9). while a low fre- ouencv of soeciallzed transduction has been -. report& wib bacteriophage 0195 (10). Although transformation is most efficient in homologous crosses (B. subtilis into B. subtilis), it has also been possible to ex- change DNA among closely related species (11). The most extensively studied members oi the B. subtilis genospecies include 8. Zichenijormis, B. pumilus, B. amylolique- faciens, and B. globigif (refer to reference 12 for a review and references 13-15 for exam- ples of this heterologous exchange). This ex- change occurs even though there is a sur- prisingly wide discrepancy between DNA- DNA hybridization among these organisms (16). Even though the frequency of trsns- formation 1s low In the heterologous cross [e.g.. B. amyloliquefaciens (donor) jB sub- tilia (recipient) 1, the newly acquired DNA from B. wnyloliquefaciens in the B. subtilis background can be readily transferred at high efficiencies to other recipient strains of B. subtilis (14). Therefore, the extremely high frequency of transformation permits the recognition and selection of rare events. B. Current and potential vectors for re- combinant molecule experiments. Lovett and coworkers have recently described crypti plasmids in B. pumilus (17) and B. subtilis (18). Of these organisms, B. subtilti ATCC 7993 appears to be the most useful since it carries one to two Copies of a plasmid wit,h a molecular welght of 48 x 1CP. This strain is aho closely related to B. subtilis 168. An- other strain of B. subtflis (ATCC 15841) con- tains 16 copies of a plasmid with a molecular wetoht of 4.6 X 108. Currentlv it is not known .---w- ~~ whether genetic markers can be readily ln- traduced into these plasmids. To date it has not been possible to readily stabilize plas- mids derived from E. pumilus in B. subtilis even with heavy selective pressure I P. Lovett, personal communication), Two temper&e bacteriophages are under development. as veotors in B. subtilis. +3T and SPOZ. Lyeogeny of thymine auxotrophs (strdns caxrying thyd thyB) by beoterio- phage Q3T results In "conversion" to a Thy+ phenotype. The attachment sita for this baateriophage and the bacteriophage gene for thymldylate synthetase (thyP) map be- tween the bacterial thyA and thyB loci in the terminal region of the chromosome of B. subtilis (19) :The viral genome is readily oleaved by the aite-specific endonuclease, Barn 1 (20). to produce 6 fragments (one of which carriee the thyP gene). The thyP carryiq gene can be integrated into the bacterial genome in the absence of the intact viral genome. Because deletions are available that include the thyP region, it is theoreti- eallv nossible to introduce thvP at manv W&i on the chromosome. The thyP ge& can be readily purlfmd for insertion ix& plesmide or utilized BS a scafiold to integrate other heterologous DNA lnt.o the ohromo- some of B. subtflia. Alternatively, ie la pas- eible to purify fragments of the chromcczome bv ael electroohoresis (21. 22). for insertion h& be&eriophage 48T or S&2. At present, unfortunately, only the former carries a selective marker, i.e., the gene for thymidyl- de syntietase, thyP. C. Development ol vehicles. B. subtilis h a Gram-positive sporu.lat.ing rod that usually inhabits solI. Although tt oan exist on cutaneous surfaces of man (23) and experi- mental animals, it r&rely produces disease. To develop a suitable vebiole it is imperative to have a hoot that Ss aeporogenic. The most appropriate deletion mutation is deletion !IQ (c.it D). In addition to a deficiency in epmlatin thb mutant rapidly lyres when # has reached the end of its growth cycle. Presumably thle is due to tie failure fo inwtlvate 0~9 of the fbut~lytlc enzymes (24) Through the introduction of a D-alanine requirement (34 ug/ml) it is possible to block transport of compounds that are transported by active transport (25.28). The further introduction of thymlne anxotrophy (defects ti the thyA thyB loci) wiU enable the strain to survive only with a plasmid vector carrying the purified thyP gene from bactWiODhi%?e +3T or a defective bacterio- phage &3T -&rying the thyP gene but at.- tached to the chromosome at an alternative site (due to the presence of deletion 29 in the host). We have recently isolated tem- perature-sensitive thyP mutants. Ii we ran isolate a temperature-dependent lysogen that will grow only at 48oC lt should be possible to make an unusual vehicle. D. Site-specific endonucleases. Recently two restriction modification systems hare been observed between B. subtilis 168 and other bacilli. Trautner et al. have isolated an effective system that Mibits infection of the R strain of I?. subttlti by bacteriophage SPPI propagated on 8. subtilis 168 (27). The site-specific nnrlease recognizes the sequence GGCC. CCGG o Young, Radnay, and Wilson ObSeNSd II restriction modificaiton system between B. amyloliquefaciens and B. subtilis 168 (28). The endonuclease from B. amyLoZiquefaciens (20) recognizes the sequence GGTAC (29). CCTAGG bfore recently, two additional enzymes have been isolated from B. gZobigii (30). The recog- nition sequence is not known. E. Adrantcrgm and liabilities of the B. sub- tilis system-a. Advantages. 1. B. subtilis b nonpathogenic. Asporogenic deletion mu- tants are available to preclude the problem of persistence through sporulation. 2. The circular chromosomal map is well defined. At leir?t 196 10~1 have been posl- tioned. 3. The organism is commercially important In the fermentation industry. 4. Large numbers of organisms can be dis- posed of readily with minimal environmental impact. 5. Unlike E. co;i, it lacks endotexin in the cell wall. Therefore the cells can be used as a single cell protein source. 6. The frequency of transformation is very high, facilitating the detection of rare events. 7. A unique bacteriophage, d3T. exists that carries a gene that can be readily puritled for "scaRoldlng" experiments. b. Disadvantages. 1. The knowledge of genetics and physiology of plasmlds and viruses is prlmitlve compared with B. wolf. 2. High-frequency, spec&lized transduc- tion is not available as a means of gene enrichment. Based on its promise, it seems appropriate, and not chauvinistic, to urge development of this system, Prepared by : Dr. Frank Young, University of Rochester. REPERENCES 1. Spieizen. J. 1958. Transformation of bio- chemically deficient strains of Bacillus sub- tilis by deoxyribonucleate. Proc. Nat. Acad. Sci. U.S.A. 44: 1072-1978. 2. Lepesant-Kejzlarova, J., J. A. Lepesant, J. Walle. A. Billault, and R. Dedonder. 1978. Revision of the linkage map of Bacillus sub- tilis 168: indications for circularity of the chromosome. J. Bacterial. 121:828-884. 3. Young, F. E. and 0. A. Wilson. 1978. Chromosomal map of Bacillus subtilfs p. 696-814. In P. Gerhardt, R. N. Costllow, and H. L. Sadoff (ed.), Spores VI. American S+ eiety for Microbiology, Washington, D.C. 4. Wake, R. G. 197% Termination of Bb- tillus sxbtilis chromosome repliratlon (L~I FEDERAL REGISTER, VOL. 41, NO. I76-THURSDAY, SEPTEMBER 9, 1976 NOTICES vlsusllzed by autoradiography. J. hIo1. Blol. 86:223-231. 5. Harford, N. 1976. Bidirectional chromo- some replication in Bacillus subtills. J. Bao- i,eriol. 121:835-847. 6. Bettinger, G. E. and F. E. Young. 1976. `Transformation of Bacillus subtflis: Trans- forming ability of deoxgrlbormcleic acid in lysates of L-forms or protoplasts. J. Bacter- iol. 222:987-993. 7. Thorne, C. B. 1962, Transduction in Rmillus subtilis. J. Bacterlol. 83: 106-111. 8. Takahashi, I. 1961. Genetic transduc- tion in Bacillus s-ubtilis. Blochem. Biophys. Res. Commun. 5: 171-176. 9. Yasbin, R. E. and F. E. Young. 1974. Transduction in Bacillus subtilfs by bac- teriophage SPPI. J. Viral. 14:X%3-1348. 10. Shapiro, J. A., D. H. Dean and H. 0. Halvorson. 1974. Low-frequency specialized transduction with BmiZZus subtizis bacterio- phage @IO& Virology 62:393-403. 11. Marmur, J., E. Seaman, and J. Levine. 1963. Interspecific transformation in B&Z- Zus. J. Bacterial. 85:461-467. 12. Young, F. E. and G. A. Wilson. 1972. Genetics of Bacillus subtilis and other gram-positive sporulating bacilli, p. 77-106. In I-L 0. Halvorson, R. Hanson, and L. L Campbell (ed.), Spores V. American Society for Microbiology, Washington, D.C. 13. Chilton, M. D., and B. J. McCarthy. 1969. Genetic and base sequence homologies In bacilli. Genetics 62:697-710. 14. Wilson, G. A. and F. E. Yonng. 1972. Intergenotic transformation of the Bacillus :;ubtiLis genospecies. J. Bacterial. 112:705- 716. 15. Yamaguchi, K., Y. Nagata, and B. hIaruo. 1974. GeneOic control of the rate of an amylase synthesis in Bacillus subtt?is. J. Bactsriol. 119:410416. 16. Lovett, P. S., and F. E. Young. 1969. Identification of Bacillus subtilis NRRL B- 3276 as a strain of BacilEus plc?nilus. J. Bac- teriol. 100:65%661. 17. Lovett. P. S., and M. G. Bramuccl. 1975. Plasmid deoxyritonuclelc acid in BaciZlw subtilfs and Bacillus pur~flus. J. Bacterial. 124:484490. 18. Lovet.t, P. S. 1973. Plasmid in B. pumnt- Zus and the enhanced sporulatlon of plasmid neeative variants. J. Bscteriol. 115:291-298. 19. Young, F. E., M. T. Williams. and G. A. Wilson. Genebics ot Bacillus subtilfs. I?L D. Schlessinger (ed.) Mlcroblology 1976, in _ press. 20. Wilson, G. A. and F. E. Young. 1975. Isolation of a seauence-wecific endonu- clease (Barn 1) frdm BacfiZw amyiolique- faciens H. J. Mol. Biol. 97:123-125. 21. Brown. L. Recombination analysis with purified endonuclease fragments in the RNA polymer- region of Bacillus subtilfs. In D. Schlessinger ted.), Microbiology 1976, in press. 22. Harris-Warrlck. R. M., Y. Elkana, S. D. Ehrlich. and J. Lederberg. 1975. Klectro- phoretic separation of BaciZZw subtfli, genes. hoc. Nat. Acad. Sci. TJS.A. 72:2207- 2211. 23. Kloos, W. F., and M. S. Musselwhlte. 1975. Dlstributlon and pergistenca of Sta- phylococcus and Micfococcw species and other aerobic bacteria on human skin. Ap- plied Micro. 30:381-395. 24. Brown, W. C., and F. E. Young. 1970. Dynamic interactions between cell wall polymers, extracellular proteasea and auto- lytic enzymea B&hem. Biophpa Res, Commun. 38:664-i%% 25. Clarb. V. L. and I'. E Young. 1974. Active transport of D-alanine and related amino adds by whole cells of BacUZus sub- tilfa. J. Bacterlol. 120 : 108%109% as. c&r& v. L. and F. E. young. Actvs transport fn cells of B. subtflis 188: Lose of endogenously energized transport in auxo- trophs deprived of D-alanine or glycerol. Submitted to J. Bacterial. 27. Trautner, T. A., B. Pawlek. S. Bran, and C. Anagnostopoulos. 1974. Restriction and modification in B. subtilis: biologic as- pects. Mol. Gen. Genet. 131:181-191. 28. Young, F. E., E. Radnay. and G. A. Wilson. Manuscript in preparation. 29. Wilson, G. A. and F. E. Young. Unpub- lished data. 30. Wilson, G. A., R. Roberts, and P. E. Young. Unpublished data. 31. Wilson, G. A. and F. E. Young. Re- striction and modification in bacilli. la D. Schlessinger (ed.), Microbiology 1976, In press. APPSNOIX B TO APPENDIX D POLYOMA AND sv40 YIRU.5 Polyoma virus is a virus of mice, and in- fection of wild mouse populations is a com- mon events, for the virus has often been isolated from a high proportion of healthy adult animals, both wild and laboratory bred, of many colonies (Gross, L.. Proc. Sot. Exp. Biol. 88, 362568. 1965; Rowe, W. P, Bact. Rev. 25,18-31, 1961). As far as is known the virus almost never causes a disease in these animals. However, when large quanti- ties of the virus are inoculated into newborn or suckling mice or hamsters, a variety of solid tumors ls induced (Gross, L., Oncogenic Viruses, Second Edition, Pergamon Press, NY) . Polyoma virus grows lytfcally in mouse cells in tlssne culture. Thus mouse cells in rulture are probably transformed only by virus particles that contain certain kinds of defective geonmes. Cells of other rodent spe- cies, however, can be transformed by poly- oma virus particles that contain complete genomes (Folk, W., J. Viral., II, 42&-431. 1973). The virus does not replicate to a signlflcant extent in human cells in tissue culture (Eddy, B.E., Viral. Monogr., 7, l-114, 196% Pollack, R. E., Salas, J.. Wang R. Ku- San0 T.. and Green, H., J. cell Physiol. 77, 117-120, 1971). The resistance of the cells seems to be a consequence of the failure of the virus to absorb or uncoat. However even when naked viral DN.4 is introduced into the cells only an abortive cycle of replication en- sues; early viral proteins are made, there is induction of cellular DNA synthesis. but no expressjon of late viral proteins is de- tectable (Gruen, R., Grassmann, M. and Grassmann, A. Virology, 58, 290-293, 1974). There is no evidence that polyoma virus can infeot humans (Hartley, J., Huebner. R.. Parker, J. and Rowe, W. P., unpub&hed data). `Thus no antibodies to the virus have been detected in people living ln bulldings that are infested with virus-infected mlop. nor ln laboratory workers who have been ex- posed to the virus for a number of years. At most, a small segment of polyoma virus DNA shcvws weak homology with a portion of the late region of SV40 DNA (Ferguson, J. and Davis. R. W.. J. Mol. Biol., 94, 135-150, 1976). However, there appears to be no genetic interaction between the two viruses and there fs no immunological cross-reaction between the gene products of the two viruses. SV40 causes perisitent but apparently harmless infections of the kidneys of vir- tually all adult rhesus monkeyi (Hslung, G. D.. Bact. Revs. 32.185-205. 19881. it causes tumors when inje&ed lnto~ n&&n ham- sters (Glrardf A J, Sweet, B. H, Slotnick. V. B. and Hillemann, M. R.. Proc. Sot. Exp. Biol. Med., 205, 42042'7, 1964) and trans- forma cells of several mammalian speciea (fnoludlng human). SV40 is able to infeot humana eince antibodies to the virus are found in a small proportion of the hu- population (Shah, K. V, Goverdhsn, M. K. aad Ozer$ H. L., Am. J. Epld. 93, 291-298, 1X0) and serum conversions have been noted in many laboratory personnel who have been exposed to the vlrua (Horvath, L. B., Acta Mlcrobiol. Acta Scl. Hung. 22, 201-206, 1965). Isolations of SV40 have been reported from humans, twice from patients suffering from the rare demyelinating disease. progressive multifocal leukoencephalopathy (Weiner, L., Herndon, R., Narayon, 0.. Johnson, R. T, Shah, K., Rubinstein L. G. Preunlsl T. J. and Conley, F. K., New England J. Med 286, 385 390, 1972) and apparently from a tumor of a person with metastatic melanoma (Soriano, F., Shelburne, C. E. and Gokcen, M., Nature. 249, 421424, 1974). In other studies a non- structural antigen characteristic of papova- viruses. T antigen, has been detected in t,he nuclei of cells cultured from 2 meningioma.!. while another SV40-specific antigen, U antigen, has been found In the cells of a third tumor Of the same type (Weiss, A. F I Port.man. R., Fisher, H., Simon. J. and Zaz:~ K. D., hoc. Nat. Acad. Scl. USA 72, 609-613. 1975). Furthermore new papovaviruses have been isolated from the brains of patients with PML (JC virus-Padgett. B. L., Walker, D. L., zuRbein, G. M., Eckroade, R. I. and Des+% B. H., Lancet 1, 1257-1260, 1971), from the urine of a patient carrying a renal allograft (BK virus-Gardner, S. D., Meld, A. M., Cole- man, D. V. and Hulme. B. Lancet 1, 1253-1257. 1971) and from a reticulum cell sarcoma and the urine of patients with the sex-linked recessive disorder, Wiskott-Aldrich syndrome (Tskemoto, K. K., R&son, A. S., Mullarkey. M. F., Blaese, R. M. Garon, C. F. and Nelson. D. J., Nat. Cancer Inst., 53, 1205-1207, 1974) All of these viruses which are dlstrihuted widely throughout human populations share sntigenic and biological properties with 8V 40; the virus particles are identical in size and architecture (Madeley, C. R., In Virus Morphology, Churchill-Livingstone, London 134-135, 1972); the non-structural lntrace!- lular T antigen, which appears to be coded by the A gene of SV40 cross reacts extensively with antigens found ln cells infected (?r transformed by BK or JC viruses: both Ji' and BK viruses induce tumors in newborn hamsters (Walter, D. L., Padgett, B. L., zli- Rheln, B. M., Albert, A. E. and Marsh, R. F Science 181. 674-676, 1973: Shah, K. V., Daniel, R. W. and Strandberg, J.. J. Nat. Can- cer Inst. 54, 945-950, 1976): BK virus cause% transformation of hamster cells in culture (M&jor, E. D., and Dihlayorca. G., Proc. Nat. Acad. Scl. US 70, 3210-3212, 1973: Portolani. hf., Barbanti, A., Brodano, G. and Laplaca. M.J., Vlrol, 15, 420-422. 1975) and is able ti complement the growth of certain tempera- ture-sensitive mutants of SV40 (Mason, D. I% and Tak~.motn. K. K., submitted for publjca- tlon!, k`VRTHEIC WOBK At pr@e.nt, a potential eukaryotlc cecmr of choice is polyoma virus. And while avail- able information indicates that. It fulflile all the necessary criteria. we recommend that the following subfecta be further fn- vestigated : 1. The moleculsa mechanism of resis&r.re of human cells to the virus. 2. The extent of homology between polg- oma virus DNA and the DNA.9 of humKli papovaviruses. 3. The ability of human papovavlruses t.3 complement defective PolYo- virus genomee. Report of a Working `Group Conslstlng of: Dr. Bernard Fields, Harvard Unlverelty School of Medicine. Da. Thomae J. Kelly, Jr.. Johns Hopkins University School of Medicine. Dr. Andrew Lewis, National Institute c.f m- lergy and Infectious D&ease& Dr Malr-olm Martin, National Instituk @ FEDERAL REGBTER, VOL 41, NO. I76--THURSDAY, SEPTEMBER 9. 1976 Allergy and Infectious Diseases. Dr. Robert Martin. Natloual Institute of Arthritis, Metabolism, `and Digestive Dis- Drymer Pfefferkoan. Dartmouth Medical School. Dr. Wallace P. Rowe, National Institute of Allergy and Infectious Diseases. Dr. Aaron Shatkin, Roche Institute of Molecular Biology. Dr. Maxine Singer, National Cancer Instl- tute. Rapporteur: Dr. Joe Sambrook, Cold Spring Harbor Laboratory. APPENDS C TO A~p~r~nrx D mMMAIIY OIr THE WOBKSHOP ON THE DESIGN AND TESTING OF S`4FEP PaOKARYOTIC VEHICLES ANIl BACTERIAL HOSTS FOB RsssARCH ON RE- COMBINANT DN.4 MOLECULES Torrey Pines Inn, La Jolla, California The development of techniques for the cloning of DNA from both prokaryotic and eukaryotlc organisms in bacteria has had great impact on research in biology and medicine and promises extraordinary social benefits. The biohazards involved in the use of this technology in many instances are very difficult to assess. For this reason codes of practice are being formulated in the United States and other countries for the COnduCt of those experiments that present a potential biohazard. One of the requlre- merits for conducting certain cloning experi- ments is the use of safer vector (bacterio- phage or plasmid) -host systems, i.e.. vector- bacterium systems that have restricted cn- pacity to survive outside of controlled condi- tions in the laboratory. Approximately sixty scientists from the United States and several foreign countries participated in a workshop on the Design and Testing of Safer Prokarvo- tic Vehicles-and Bacterla~Hosts for Reseakh on Recombinant DNA Molecules at La Jolla, California. on December 1 to 3, 1975. The workshop was sponsored by the Research Resources Branch of the National Institute of Allergy and Infectious Diseases. The pur- poses of the meeting were the exchange of recent data on the development of safer prokaryotic host-vector systems, devising methods of testine the level of containment provided by these-systems and exploring the various directions that future research should take in the construction of safer bacterial Systems for the cloning of foreign DNA. The flrst session of the workshop, chaired by W. Szybalski (University of Wisconsin), was devoted to bacteriophage vectors. Szy- balskl outlined the main safety features of the two-component, phage-bacterial system, In which the host bacteria offer the safety feature of not carrying the cloned DNA, and the phage vectors cannot be propagated in the absence of an appropriate host. There are two primary escape routes for the clones of fore&n DNA carried bv the Dhane vector: (1) Est~bllshment of a stable prgphage or plasmid in the laboratory host used for phage propagation, and subsequent escape of this self replicating lysogen or carrier system, and (2). escape of the phage vector which carries the cloned DNA and its subsequent produc- tive encounter with a suitable host in the natural environment. The general consensus was that tc ensure safety, both routes should be blocked by appropriate genetic modifica- tions. For phage A, route (1) can be blocked by phage mutations that interfere with lyso- aenieation (ati-. fat-. cI-. cIII-. tir) and plasmid formation (i+, ?&R. US. ril, clT, Ots, trots) , and by mutations on the Mch.eri- chta coli host that affect these processes fottB-. dncAts1 and host survival. Route 12). (which is of low probability since A phages do not survive well in natural environments (no Xc1 phage was recovered after ingestion of lDs-10" particles), are kllled by deslcca- tion, and have a low chance to encounter a naturally sensitive hosi) can be blocked further by the following phage modifications: (a) Mutations which result in extreme insta- bility of the infectious phage particles under all conditions other than those specially de- sianed for Dhme nronaeation in the labora- t&y (e.g., highcdnc&t;ations of putrescine or some other compound), or (b) employing phage vectors in which the tail genes are deleted and which permit propagation of only the DNA-packed heads: only under lab- oratory conditions could such heads be made transiently infectious by rejoining them with separately prepared tails. The high in- stabilitv of the Dhaee would minimize the possibihty of tr&f& of the cloned genes into receptive bacteria found in nature. Moreover, the propagation of the phage can be blocked by many conditional mutations, which would be designed to block any sec- ondary route of escape, mainly depending on transfer of the cloned DNA into another phage or bacterial host. It was recommended further that the vector be designed in such a manner as to permit easy insertion and monitoring of the foreign DNA and rapid assay of the safety features and give a high yield of cloned DNA (not less than 10" mole- cules per ml). There also was general agree- ment that host-phage systems other than E. coli should be considered, especially those restricted to very rare and unusual environ- ments. Also, plasmids derived from phage vectors and which give very high DNA yields while exhibiting safety features, e.g., Xdvcrots, should be considered as vehicles for cloned DNA. Szybalski and S. Brenner (Cambridge Unl- versity) stressed that research on recombi- nant DNA molecules may lend itself to very simple and inexpensive mechanical con- tainment, e.g., a small sealed glove box, since all the vectors that carry such re- combinant molecules possibly can be both created and destroyed in such a box, while development of special methods might per- mit study of many properties of the recom- binant DNA, without ever removing it from the box. These safety features were reflected in the subsequent presentations. F. Blattner and W. Wflliams (Universiy of Wisconsin) described four specially constructed A-@36 phages which incorporate many of these safety features, and which they named Charon phages, for the mythical boatman of the river Styx. Some of these highly con- tained phages give yields of over lp parti- cles/ml.~R. Davis. J. Cameron and K. Strllhl (Stanford University) found that X phages that carry foreign DNA never grow as well as the parental vector, which would select against their survival in nature. They also reported that some eukaryotic genes could be exDreSSed in E. coli. nartiallv comnensat- ing for deficiencies in the hi&line pathway or in poZA or Zig functions. These investl- gators surveyed over 1000 strains of E. cdi isolated in the natural environment and did not find a sinale strain that could SUD- port propagation of the Xvii' vector. - V. Bode (Kansas State University) dls- cussed the possibility of growing tall-free X heads. Such heads, which are packed with DNA, are very fragile, unless stored in 0.1 M putrescine buffer. Head yields close to lw:ml could easily be attained and, when required, heads could be quantitatively rejoined with separately supplied tails under special lab- oratorv conditions. W. Arber. D. Scandella and J."Elliott (University of B&l) described bacterial host mutants that permit efficient infection only by phages with a full com- plement of DNA. This permits selecting for vectors that carry long fragments of forelgn DNA. K. Matsubara, T. Mukai and Y. Takagi (University of Osaka and Kyushu Unlver- sity ) 9 and 0. Hobom and P. Philllppsen (University of Frelburg and Stanford Univer- sity) described various defective A plasmids (Xdv) t.hat could be used as efeclent vectors Matsubara has shown that temperature- sensitive cro mutations permit obtaining be- tween 1000 and 3000 cloned molecules ner cell and at the same time result in killing of the carrier cells at body temperature. The mutations Ots and Pts were also evaluated as safety features. Phlllippsen described many new Adv plasmlds created by cutblng X DNA with HindIII and Barn1 restriction endonucleases followed by ligation. The final talk by F. Young, Cl. Wilson and M. Williams (University of Rochester) summarized the progress on the development of safer BaciElus subtilis host mutants and Dhaees. especially +3, as vectors. New res&lct& nucleeses, Bgl-1 and Bgl-2, were also de- scribed. The morning session on bacteriophage vectors wss followed by a session on plasmid vectors that was chaired bv D. Helinski (Uni- versity of California, S& Diego). He&ski presented the following properties as highly desirable characteristics of a safer plasmid vehicle: (a) Non-conjugative; (b) non- mobilizable or poorly moblllzable by a con- jugative plasmtd: (c) possesses little or no extraneous genetic information: (d) poorly recombines or does not recombine with the chromosome of the host cell: (e) provides no selective advantage to the host cell or the selective property is conditional; and (f) possesses mutations that restrict its mainte- nance to a specific host, prevent replication at mammalian body temperature and,`or provide with the capability of killing any cell to which it might be transmitted~other than the host cell. V. Hershfield fllniversitv of California, San Diego) described the prop- erties of a variety of derivatives of the ColEl derivatives, ColEl-trp, constructed in col- laboration with C. Yanofsky and N. Franklin (Stanford University) provides the means to use the tryptophan genes of E. culi as a se- lective marker in transformation with re- combinant DNA in situations where it is de- sirable to avoid antibiotic resistance genes. In addition, Hershfield described collabora- tive work with H. Boyer that resulted in the development of a mini-ColEl plasmld and derivatives of this plasmid (mini-ColEl-kan and mini-ColEl-trp) as cloning vehicles. Finally, she described the temperature-sensl- tivity properties of trp and km derivatives of a temperature-sensitive replication mu- tant of ColEl isolated by J. Collins (Moleeu- lar Biology Institute, Stockhelm) and hybrid ColEl plasmids carrying the EcoRI generated Cts fragment of bacteriophage X-trp61. J. Carbon (University of California. Santa Barbara) described a replica plating method that greatly facilitates the detection of E. coli clones bearing ColEl plasmids. The proce- dure, which utilizes the F, plasmid to pro- mote the transfer of a hybrid ColEl plasmid to a suitable auxotrophic recipient, was suc- cessful in identifying clones bearing hybrid plasmids carrying a number of different re- gions of the E. coli chromosome. The con- tributions of A. J. Clark and collaborators (University of California, Berkeley) were rel- evant to the problem of the mobilization and subsequent transfer of non-coniuestive pls.sn&ls carrying foreign DNA of a poten- tially hazardous nature. Clark described the variations in transmission frequencies be- tween the nonconjugative plasmids pSC101, pML31, pSC138 and a number of pSClO1 hy- brids containing various EcoRI fragments of F when the conjugal transfer of these plasmids was promoted by several different conjugative plasmids. I. C. Gunsalus and collaborators (Univer- sity of Illinois) and A. Chakrabarty (General Electric Research and Development Center) FEDERAL REGISTER, VOL. 41, NO. 176~THURSDAY, SEPTEMBER 9, 1976 NOTICES described the properties of a variety of plas- mids isolated from Pseudomonas putida. These contributions were followed by a dis- cussion on the merits of developing plasmid- host systems involving Pseudomonas strains that naturally exhibit unusual growth re- quirem.?nt.s. Similar studies with plasmids Isolated from Bacillz~ megaterium by 33. Carlton (University of Georgia) from B. sub- tilis by P. Iovett (University of Maryland) am other naturally occurring BCU?iZZus species by W. Goebel and K. Bernhard (Microbiology Institute, Wurzburg) were dfs- cussed and their further development as plasmid-host cloning systems was explored. It was clear from these presentations that considerable progress has meen made re- cently in the identification and characteriza- tion of a variety of plasmid elements that oc- cur naturally in Pseudomonas and Bacillus species. Several of the plasmlds described show considerable promise as plasmid cloning systems involving a host other Bhan E. coli. A third session on the ecology and epi- demiology of vector-host systems was chaired by S. Falkow (University of Washington). This workshop emerged, in part, from ex- pressed fears that microorganisms Coniam- ing cloned fragments of foreign DNA may potentially pose a threat to health or dis- rupt the normal ecological chain in some manner. Consequently, this session was de- voted to a review of currentlv available in- __--- .- ~~ formation on the ecology and epidemiology of E. coli and related bacterial species Since it was recognised that E. coli K-12 would be the prokaryotic host most commonly em- ployed in the cloning of DNA molecules in the immediate future. F. (Drskov (Escherichia Reference Center, Copenhagen) reviewed the sPate of E. cold serotyping and what has been learned about the distribution of E. coli types in health and disease. Only certain E. COZi types a,re generally recognized as good colo- nizers of the human aut and such strains come from a handful 2 the 160 well defined 0 (lipopolysaccharide) antigen types and in- variably possess K (acidic polysaccharide capsule) antigens. Some serotypee apparently have become disseminated worldwide and possibly represent the proliferation of a bac- terial clone because of, as yet unknown, selective pressures. In contrast, E. coli K-12 has no detectable 0 or K antigens and is considered to be rough. This may account, CLt least in part, for ite demonstrate poor ahilitv to colonize the human or animal aut. However, R. Freter (University of Michigan) pointed out that we still remain largely ignorant of the factors which control in- testinal E. coli populations. Freter also noted that while adherence to the mucosal surface of the small lnteetine is imoortant in the pathogenesls of E. coli diarrhkal disease, the `normal' long-lasting symbiotic relationship between a mammalian host and bacterium is established in the cecum and colon. It is in these locations that factors come into play to determine whether an E. ooZ4 strain passing through the intestine will become success- fully implanted or whether it will be quickly eliminated in the feoes. The factors control- ling implantation include competition for substrates, inhibitors and the physiological state of the organism when it reaches the large bowel. For example, ingested E. colt previously grown under usual laboratory conditions fare poorly while cells of the same strain `me-adauted' in Kh. nH. etc.. often colonize^ well. %reter has deieloped .a con- tinuous flow culture model which may be useful in studylng the mechanisms of im- plantation. Falkow reviewed the pathogenic- ltv of B. col. E. co24 Causes diarrheal disease either by direct invasion of the bowel epl- thelium or by elaboration of enterotoxln(s) . While invasive 8. coli appear to owe their pathogenicity to a constellation of at least five unlinked chromosomal gene clusters, toxlgenic E. coli species generally owe their pathogenicity to the possess lon of two aaecies. Ent and K. The introduction of Ent aid K `plasmids may be sufficient to convert a normal wild-type E. coli into a strain now capable of causing overt clinical disease. However, the introduction of these plaemids into E. coZi K-12 sublinas had no discernible effect on their ability to cause disease, al- though the K-12 strains could now better colonire calves. Despite the observat.ion that E. coli K--l2 did not appear to offer a signifl- rant hazard as a potential enteric pathogen even when it possessed well-defined deter- minant of pathogenicity it was emphasized by @rskov, Freter and Falkow that E. coli K-12 strains carrying recombinant DNA molecules could still act as effective genetic donors in vioo and still posed a significant problem requiring control. E. Geldreich (U.S. Enviromnent,al Protection Agency, Cincin- nat,i. Ohio) discussed the possible outcomes of the release of E. coli containing recom- binant DNA molecules into the aquatic en- vironment and concluded that total reliance cannot be placed on sewage treatment and the natural self-puriilcation capacity of re- ceiving waters to limit potential hazards. While these are realistic barriers to the dis- semination of E. co2i and associated fecal organisms via the water route, they are not infallible because of technological limita- tions, improper operational practices an's system overloading. Finally, M. Starr (Uni- versity of California, Davis) described the numerous genera of gram-negative bacteria found naturally occurring in the soil and on plants. He stated that most of these orga- nisms do not appear to be a reasonable alter- native to E. coli K-12 ss a host for recom- binant DNA molecules. Indeed, Starr pointed out that since such genera as Erwinia, Rhiz- obium and Agrobacterium are known to con- jugate with E. coli, the potential dissemina- tion of recombinant DNA molecule includes a greater spectrum of mimoorganisms than just enteric species. The fourth session of the workshop, chaired by R. Curtiss III (University of Ala- bama), was concerned with the construction of safer bacterial hosts for DNA cloning. The goals in constructing safer host strains enu- merated at the beginning of the session in- cluded introduction of mutations that would: (a) Preclude colonization in normal ecoloaical niches: (bl nreclude cell wall bio- synthesis except .in spkcially defined media; (c) cause degradation of genetic information in normal ecological niches: (d) cause vec- tors to be host-dependent; (e) minimize transmission of recombinant DNA to other strains in normal ecological niches: (f) in- crease usefulness for recombinant DNA molecule research; and (g) permit moni- toring. Most of the progress in developing safer hosts has been achieved with B. coli K-12, although F. Young described a B. s-ubtilis strain with a deletion for sporulation genes which readily undergoes autolysis. The strain also has defects in genes for purine and TTP biosynthesis and a mutation con- ferring a D-alanine requirement can be in- troduced to cause cell wall biosynthesis to be defective. This strain may be defective in transf matlon, however, and therefore might Tie useful only with a phage vector which has yet to be developed and/or dis- covered. A. I. Bukhari (Cold Spring Harbor Labora- torv) described the use of the daaD8 mum- tlon'in E. calf K-12 to block celi wall blo- synthesis and another non-reverting muta- tion which causes sensitivity to bile salts and detergents. The dapD8 allele is the most stable dap point mutation known, although it does revert at frequencies of 10-a to 10-O. Tbr mutation conferring bile salts 6ensivit.y was obtained after Mu-l infection of an Hfr strain and, although exhibiting the theoret- ioallv useful nronerties of ease of DNA hola- tion"and inadilit'g to survive in the intestinal tract, might be due to Mu insertion which would compromise its use for safe strain tonstruct10a Curtis6 reported on the work performed by him and his coworkers in constructing and `%?stlng numerous strains with different mu- tations, Survival of strains in vice was tested by feeding rats 10'0 cells in milk by stomach tube. Spur mutations did not reduce strain t,it.ers m feces whereas AthyA: AthyA drm, and Ltf1y.4 dra mutations gave lOa-fold, 109- fold and Iw-fold reductions, respectively, in strain t.iters in feces. Strains with AthyA mutations also exhibited thymineless death in in cifro tests. Since strains with dapD8 s!lele can revert to Dap+, strains were con- structed with both dupD8 and AbioH-usd mutations. These strains have not been ob- served to revert to Dap+ but can survive passage through the rat intestine and in growth media lacking diaminopimellc acid but containing NaCl and 0.6% usable car- bon ljources. This survival was due to the production of the mucopolysaccharlde, col- anic acid, which permits many of the cells to grow and survive as spheroplasts. A Agal- c?L~' mutation (also deletes ktt, bio and uurB aeileSl w&5 introduced which blocks colanic acid'biosynthesis and leads to no de- tectable survivors in media lacking diamino- pimelic acid or following passage through the rat intestine. The dapD8 AbioZf-asd AgaZ- chZ st,rains are more readily lysed, trans- form at higher freauencies and are coniu- gation-defective in- matings with donors possessing conjugative plasmids in the P, W and 0 incompatability groups but Con+ as recipients for F, I and T group nlasmids when COmDared to the da,w+ oaz* parent strain. - Strains with endA `m-ringe carefully to ulinimiLe air bubbles and frothing of the inoculum. 7. Expel excess air, liquid and bubbles from a syringe vert,ically into a cotton pledget moistened with the proper disinfectant, or into a small bottle of sterile cot.ton. 8. Do not use the syringe to expel force- fully a stream of infectious fluid into an open vial or tube for the purpose of mixing. MIX- ing with a syringe is condoned only if the tip of the needle fs held below the surface of the fluid in the tube. 9. If syringes are filled from test tubes, take care not to contaminate the hub of the needle, as this may result In transfer of in- fectious material to the fingers. 10. When removing a syringe and needle from a rubber-stoppered bottle, wrap the needle and stopper in a cotton pledget mois- tened with the proper disinfect.ant. If there is danger of the disinfectant contaminating sensitive experiments, a sterile dry pledget may be used and discarded immediately Into disinfectant solution. 11. Inoculate animals with the hand "behind" the needle to avoid punctures. 12. Be sure the animal is properly re- strained prior to the inoculation, and be on the alert for any u?lexpectz.i movements of the animal. 13. Before and after injection of an animal. swab the site of injection with a disinfectant. 14. Discard syringes into a pan of disin- fectant without removing the needle. The syringe first may be filled with disinfectant by Immersing the needle and slowly with- drawing the plunger, and finally removing the plunger and placing it separately into the disinfectant. The filling action clears the needle and dilutes the contents of the syringe. Autoclave syringes and needles in the pan of disinfectant. 15. Use separate pans of disinfectant for disposable and nondisposable syringes and needles to eliminate a sor:ing problem in the service area. 16. Do not, discard syringes and needles into pans containing pipettes or other glass- %-are that must be sorted out from the syringes and needles. C. Opening Culture Plates, Tubes, Bottles. and AmpouZes. 1. Plates. t.ubes and bottles of fungi may release spores in large nur hers when opened. Such cultures should be ma- nipulated in a B!o!ogical Safety Cabinet (6,15). 2. In the abse:m? of defini:e accidents or obvious spillage, it is not certain that open- ing of plates, tubes and bottles of other microorganisms has caused laboratory ln- fection. However, it !s probable that among the highly infective agentS, some infeCtiOns have occurred by this means and are repre- sented in the 80% for which no known act or accident is ascribable (3). 3. Water of syner&s In petrl dish cul- tures is usually infected and forms e film between the rim and lid of the inverted FRDERAL REGISTER, VOL. 41, NO. 176THURSDAY, SEPTEMBER 9, 1976 NOTICES 3SiG7 plate. Aerosols are dispersed w-hen this film is broken by opening the plate. Vented pIas- tic petri dishes where the lid touches the rim at only three points 8re less likely to offer this hazard (8,19). 4. The risk may also be minimized by us- ing properly dried plates, but even these (when incubated anaerobically) are likely to be wet 8fter removal from 8n anaerboci jar. Filter papers fitted into the lids reduce, but do not prevent, dispersal. If plates are obviously wet they should be opened in the 1 Biological Safety Cabinet (8). 5. Leas obvious is the release of aerosols when screw-capped bottles or plugged tubes are opened. This happens when 8 film of ln- fected liquid which may collect between the rim and the liner is broken during removal of the closure (8). 6. Dried. infected culture material mav also collect at or near the rim or neck of culture tubes and may be disposed into the air when disturbed (18). Containers of dry powdered hazardous materials. (e.g., Class 3 fungal agents in the spore phase of growth) should be opened only in a Biological Safety Cabinet (6. 14). 7. When the neck of an ampoule contain- ing liquid is broken after nicking with 8 file, the snapping action creates aerosols. The following methods have been recommended: (i) After nicking the ampoule with 8 fiIe, wr8p the ampoule in disinfectant-wetted cotton before breaking. Wear gloves (2). (ii) The bottom of the amnoule should be held'ln several layers of tlssde paper to pro- tect the hands, and a file mark made at the neck. A hot glass rod should be carefully applied to the mark. The glass will crack, al- lowing air to enter the ampoule and equalize the pressures. After a few seconds the am- poule should be wrapped in 8 few layers of tissue and broken along the crack. The tis- sues and ampoule neck can then be discarded into disinfectant, and the contents of the ampoule removed with a syringe. If the am- poule contains dried cultures, about 0.5 cm3 of broth should be added slowly to avoid blowing dried materi8l out. The contents may then be mixed without bubbling 8nd withdrawn into 8 culture tube (8). (iii) The researcher uses an intense, but tiny, gas-oxygen flame and heats the tip of the hard glass ampoule until the expanding internal air pressure blows 8 bubble. After allowing this to cool, he breaks the bubble while holding it in 8 large low temperature flame: this immediately incinerates any in- fectious dust which may come from the am- poule when the glass is broken (16). Prellm- inary practice with a slmulant ampoule of the same type actually in use is necessary to develop 8 technique that will not cause ex- plosion of the ampoule. (iv) A simple device has been recommend- ed consistinn of a sleeve of rubber tubing into which the ampoule is inserted before it is broken (17,18). D. Centrifuging. 1. A safety centrifuge cab- inet or saf&y centrifuge cup (3,7,8.14,22) may be used to house or safeguard all cen- trifuging of infectloua substances. When bench type centrifuges are used in 8 Bio- logical Safety Cabinet, the glove panel should be in place with the glove ports covered. The -centrifuge operation creates air currents that may cause escape of agent from an open cab- inet (z&3,4,13). 2. In some situations, in the absence of O- ring cap sealed trunnion cups, specimens can be enclosed in sealed plastic bags before cen- trifugation (12). 3. Before centrifuging, inspect tubes for cracks lnsoect the innside of the trunnlon cup fdr ro&h walls caused by erosion or ad- hering matter, and csrefully remove bits of glass from the rubber cushion (4,lO). 4. A germicidal solution should be added between the tube and trunnion cup to disin- fect the materials in case of accidental breakage. This practice also provides an ex- cellent cushion against shocks that might otherwise break the tube (4,lO). 5. Avoid decanting centrifuge tubes. If you must do so, afterwards wipe off the outer rim with 8 disinfectant; otherwise the ln- fectious fluid will spin off 8s an aerosol (4, 10). . 6. Avoid filling the tube to t,he point that the rim, cap or cotton plug ever becomes wet with culture (4, 10). 7. Screw caps, or caps which fit over the rim outside the centrifuge tube are safer than plug-in closures. Some fluid usually collects betwen 8 plug-in closure and the rim of the tube. Even screw-capped bottles are not without risk, however; if the Cim is soiled some fluid will escape down the out- side of the tube. Screw-capped bottles may jam in the bucket, and removing them is hazardous. Propping such bottles higher in the bucket with additional rubber buffers is mech8nlcally unsound (8). 8. Kitchen foil is often used to cap centri- fuge tubes. This creates more risk then the screw cap. Boll caps often become detached in handling and centrifuging (8). 9. The balancing of buckets is often mis- managed. C&xre must be taken to ensure that matched sets of trunnios. buckets and plastic inserts do not become mixed. If the components are not inscribed with their weights by the manufacturer, colored st.ains can be aDDlied to avoid COnfUSion. When the tubes-are balanced, the buckets, trunnions and inserts should be included in the procedure; and care must be taken to ensure that the centers of gravity of the tubes are eouidistant from the axis of rota- tion. To lllistrate the importance of this, two identical tubes containing 20g of mer- cury and 20g of water respectively will bal- ance perfectly on the scales; but their performance in motion is totally different, leading to violent vibration with all its at- tendant hazards (5). 10. Fill and open centrifuge tubes or t.runnion cups in 8 Biological Safety Cabinet (10). E. High-Speed Centrifuges (22). 1. In high- speed centrifuges the bowl is connected to a vacuum pump. If there is 8 bre8kage or accidental dispersion of infected particles the pump 8nd the oil in it will become con- tamin8ted. A high efficiency 5lter should be placed between the centrifuge and the p-p 03). 2. High speed rotor heads are prone to metal fatigue, and where there ia a chance that they asy be used on i&n-e than one machine each rotor should be accompanied by its own log book lndloatlng the number of hours run at top or de-rated speeds. Failure to observe this precaution can result in dangerous and expensive dlsintegr8tion. Fre- quent inspection, cleaning snd drying are important to ensure absence of corrosion or other traumata which may lead to creeping cracks. Rubber O-rings and tube closures must be examined for deterioration and be kept lubricated with the material recom- mended by the makers. Where tubes of dlf- ferent mate&& are provided (e.g., celluloid, polypropylene, stainless steel), care must be taken th8t the tibe closures designed specifio8lly for the type of tube in use are employed. These caps 8re often similar in appearance, but are prone to leakage if ap- plied to tubes of the wrong material. When properly designed tubes r+nd rotors 8re well maintained and handled. leakine should never occur (6) . 3. Cleaning and disinfection of tubes, rotors and other components requires con- siderable care. It is unfortunate that no single process is suitable for all items, and the various manufacturers' recommendations must be followed meticulouslv if fatigue. distortion and corrosion are td be avoided. This is not the place to catalogue recom- mended methods, but one less well appre- ciated fact is worthy of mention. Celluloid (cellulose nitrate) centrifuge tubes are not only highly inilammable and prone to shrinkage with age and distortion on boiling. but can behave 8s high explosive in an auto- clave (5) Large-scale sonal centrifugalion requires special attention (11). F. Blenders, ultrasonic disintegrators. colbid mills, ball milLs, jet mills, grinders, motar and pestk. All these devices release considerable aerosols during their operation. For m8ximum protection to the operator durine the blending of infectious materials. the f:llowing practices should be observed: 1. Operate blending and cell-disruption and grinding equipment in a Biological Safety Cabinet (9 ) 2. Use ssfetv blenders desianed to orevent leakage from the rotor bearing at the-bottom of the bowl (9). 3. In the absence of 8 leak-proof rotor, in- spect the rotor bearing at the bottom of the blender bowl for leakage orlor to oneration. Test it in 8 prelimin&y run with sterile aallne or methylene blue solution prior to use with infected material (9). 4. Sterilize the device and residual lnfec- tlous contents promptly after use. Use * towel moistened with disinfectant over the top of the blender (9). 5. Class blender bowls are undesirable for use with infectious material because of po- tential breakage. If used, they should be cov- ered with a polypropylene jar to prevent dispersal of glass (8). 6. A new machine, the Colworth Sto- macker (Enalsnd) . in which material Is homogeni`zed-in a piastlc bag in a closed con- tainer, would appear to be safer than some of the other blenders (8). 7. A heat-sealed flexible mastic film en- closure for 8 grinder or blender can be used, but It must be opened in 8 Biological Safety Cabinet (7). 8. Blender bowls sometimes require sup- plemental cooling to prevent destruction of the bearings and to minimize thermal effort.s on the product (`7). 9. Before opening the safety blender bowl, permit the blender to rest for at least one minute to allow settling of the aerosol cloud. 10. Clinical or other laboratories handling human blood should be aware of the aerosols produced by the microhaematocrit centri- fuge, the autaanalyzer stirrer, and the mico- tonometer, inasmuch as it Ferns that air- borne transmission of infectious heoatitis may occur in the laboratory (20). G. Miscellaneous precauticms and recom- mendations. 1. Water baths and Warburg baths used to inactivate, incubate, or test ln- fectious substances should contain a disin- fectant. For cold water baths, 70 percent propylene glycol is recomemnded (4, 10). 2. Deepfreeze, liquid nitrogen, and dry Ice chests and refrigerators should be checked and cleaned out periodically to remove any broken ampoules, tubes, etc., containing in- fectious material, and decontaminated. Use rubber gloves and respiratory protection dur- ing this cleaning. All infectious or toxic material stored in refrigerators or deep- freezes should be properly labelled. Security measures should be commensurate with the hszards (4,10,21). 3. Freeze-dried culture ampoules should al- ways be opened in 8 Biological Safety Gabl- net. The ampoule should be wrapped in 8 disinfectant-soaked swab before bresklne it open to minimize the risk of cutting ihe hands, and to a lesser extent of releasing FEDERAL REGISTER, VOL. 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976 8841% NOTICES aerosol of dried material. Whenever possible, ampoules should be filled with dry nitrogen after freeze-drying, thus avoiding implosion that may occur during the sealing as well as opening of evacuated ampoules. The whole process of freeze-drying itself should be per- formed in a Biological Safety Cabinet. Filtra- tion of the eilluent air from the vacuum pump is desirable either up (preferably), or dwcn stream of the pump (5). 4. Ensure that all virulent fluid culttires or viab!e powdered infectious materials in glass vessels are transported, incubated, and stored in easily handled, nonbreakable leak-proof containers that are large enough to contain all the fluid or powder in case of leakage or breakage of the glass vessel (4,lO). 5. All inoculated uetri elates or other ln- oculated solid media sho;ld be transported and incubated in leak-proof pans or leak- proof containers (4,lO). 6. Care must be exercised in the use of membrane filters to obtain sterile filtrates of infectious materials. Because of the fra- gility of the membrane and ot!ler factors, such filtrates cannot be handled as nonin- fectious until culture or other tests have proved their sterility (4.10). 7. Shaking machines should be examined carefullv for notential breakage of flasks or other &ntain&s being shaken:Screw Capped durable plastic or heavy walled glass flasks should be used. These should be securely fastened to the shaker platform. An addl- tional precaution would be to enclose the flask in a plastic bag with or without an absorbent material. 8. No person should work alone on an er- tremely hazardous operation (4,lO). Personal hygienic practices in the labora- tory are directed, in most part, toward the prevention of occupationally acquired phys- ical injury or disease. To a lees obvious ex- tent, they can raise the quality of the Iab- oratory work by reducing the possibilities for contamination of experimental materials. The reasons for many of the recommended precautions and practices are obvious, but, in some instances, ampiification will permit a better review of the applicability to any one specific laboratory. Consenuentlv. what might be forbidden in one laboratory might be only discouraged in another, and be permissible in a third. Nevertheless, adherence to safe practices that become habitual, even when seemingly not essential, provides a margin of safety in sit- uations where the hazard is unrecognized. The history of occupational injury is replete with examples of hazards unrecognized until too late. The following guidelines, recom- mendations, and comments are presented with this in mind: 1. Food, candy, gum, and beverages for human consumption will be stored and con- sumed only outside the laboratory (5, IO). 2. Foot-operated drinking fountains should bc the sole source of water for drinking by human occupants of the laboratory (27). 3. Smoking is not permitted in the lab- oratory or animal quarters. Cigarettes, pipes, and tobacco will be kept only in clean area.e (5, 10,26). 4. Shaving and brushing of iceth are net permitted in the laborat.ory. Pazors, tooth- brushes, toiletry supplies, and cosmetics are permissible only in clean change rooms or other clean areas, and should never be used until after showering or t!:orzKsh washing of the face and bands ( 27). 5. A beard may be undesirable in the lab- oratory in the presence of actual or potential airborne contamination, because it retains particulate contamination more persistently than clean-shaven skin. A clean-shaven face $s essential to the adequate facial fit of a face mask cr respirator when the work re- quires respiratory protection (10,27,31). 6. Develop the habit of keeping hands away from mout.h. no% eyes. face, and hair. This may prevent self-inoculation (10.27). 7. For product pratcction. prrxms with loxg hair should wear a suitable hair net or head cover that c.;n bc decontaminated. Tbis has long beeii a r-~:~iiremcnt in lies- pita1 operating room% and in t::e mnnufnc- ture of biologicsl l:hnrnace:!tical producm. A head cover also ni!! l%rotect the hair from fluid splashes, from YV. iugi:ig into Bunsen flames and petri dishes. and xv-i!1 reduce facial contamination caused by ha!,itual repetitive manual adjustment of the hair (5). 8. ILvl~`-flowin~ 7 _ hnir xiii ioose-iisi,piilg clothing are dnc~oroui in the pres?n!?o of open flnme or moving machinsry. Rings and wrist watches also are a mechnnical haenrd during operation of some types of machines (5, 10). 9. Contnzt :enses do not pr"sizle eye protec- tion, The capillary spsce between the con- &act lenses and the cornea may trap any ma- terial present on the surface of the eye. Caustic chemicals trapped in this space can- not be washed off the surface of the cornea. If the material in the eye is painful or the contact lens h displaced, muscle spasms ~-ill make it very difficult, if not impossible, to remove the lens. For this reason. contact lenses must not be worn by persons exposed to caustic chemicals unless safety glasses with side shields, goggles, or plastic face masks are also worn to provide full prOteC- tion. It is the resaotiSibilitv of suuervisow t0 identify employebs who wear contact lenses (25. 26). 10. Personal items, such as coats, hats, storm rubbers or overshoes, umbrellas, purses, etc., do not belong in the laboratory. These articles should be kept elsewhere (25). 11. Plants, cut flowers, an aquarium, and pets of any kind are undesirable sources of yeast, molds, and other potential microbial contaminants of biological experimental ma- terials (25). 12. Books and Journals returnable to the institutional library should be used only in the clean areas as much as possible (10,27). 13. When change rooms with showers are provided, the employer should furnish skin lotion (27). 14. When employees are subJect to poten- tlal occupational infection, the shower and/ or faceihand-washing facilities should be provided with germicidal soap (8,27). 15. Personal cloth handkerchiefs should not be used in the laboratory. Cleansing tls- sue should be available instead. 16. Hand n-ashing for persomtl protec- tion : (i) This should be done promptly after removing protective gloves. Tests show it i.e not unusual for microbial or chemical con- tamination to be present despite use of gloves, due to unrecognized small holes, abrasion.% tears, or entry at the a-rlst. (ii) Throughout the dsy, at intervals dictated br the nature of the worl:. the hands sho;d be washed. Pre:+nce cf a~wrist watch discourages adequsto vxhing of the wrist (1025). (iii) Hands should be washed after re- moving soiled protective clothing, before leaving the laboratory area, before eating, and before smoking. The provision of hand cream by the employer encourages these pr`ac- tices (5.8.19). (iv) A disinfectsnt xvaslsh cr dip msy be desirable in some cases, but Its uce must not be carried to the point of causing roughen- ing, desiccation or selwitizat:z,n of the skin 17. Anyone -wlt,h a fresh or healing cut. abrasion, or skin lesion should no7; work with infective material unless t.he injured area is completely protected ! 6.25). 13. Persons vac,nilis ted for smallpox may be shedders of vaccinia vu'us during the phase of cutaneous reaction. Therefore, vaccination requires permi,&on of the appropriate super- visor, because t-v:o u-eeks' absence may be necessary before returxing to work v;ith nor- mal cell cul?urcs or with susceptible animals, especially the normal mouse colony (25). 19. The surgeon's mask of gauze or filter paper is of little value for personal respira- tory protection 129). It is designed to prevent escape of droplets from the nose or mouth (23G). I: biohazards demand respiratory protection, then nothing but a full face res- pirnbr or ventilated hood will suffice. A half- mtik respirator does not protect the eyes, which are an unevaluated avenue of ixfec- tion through the conjuctiva and the naso- lacrimal duct (5.8) 20. Nompecific cont.aminstion by environ- mental organisms from humans, animals, equipment, containers for specimens or sup- plies, and outside air is a complication that may affect or invalidate the results of an experiment. The human sources of this con- tamination are evaluated as follows: (i) Sneezing, coughing and talking (23A. 24A). Sneezing, variously reported to gen- erate as many as 32,000 or 1,000,OOO droplets below 100 microns in diameter; coughing, which produces fewer and larger droplets; and talking, which has been reported to aver- age only 250 droplets when speaking 100 words. show great differences between per- sons in regard to the number of microorga- nisms aerosolized. As a general rule, it may be said that these actions by normal healthy persons may play a less important role in transmission of airborne infection to humans or experimental materials than does libera- tion of microorganisms from human skin. (ii) Dispersal of bacteria from human skin. There is a tremendous variation in the num- ber of bacteria shed from the skin by a clothed subject. For instance, In one study. the number varied from 6,000 to 60,000 per minute (23C). These bacteria were released on skin scales which were of a size that could penetrate the coarse fabric u.sed for the laboratory and surgical clothing in the test (23D). Dispersal of skin bacteria was several times greater from below the waist than from upper parts of the body (24D). Effective reduction is accomulished bv use of closely-woven or impervious clothing fitted tightly at the neck, wrists, and ankles to prevent the clothing from acting as a bellows that disperses air carrying skin scales laden with bacteria (23B). Such clothing sometimes is too warm to work in. It was found that a significant reduction in disper- sal of bacteria occilrred with the wearing of close-fitting and closely-woven underpants beneath the usual laboratory clothing (23D). The purpose of this summary is to alert laboratory personnel to the existence of this source of contaminatton (9) (ii!) Prolific dispersal of bacteria occurs from infected abrasions. small pustules, bolis, and skin disease (23F, 24B). Wa.sh!ng the lesions with aermicidal sono will sreatlv decrease the number of orcar;isnu on the skin and dispersal into the a&. Healthy nasal carriers who gem=rate aernsoli7ed staphp- lococci usually can be identified by the pres- ence of heavy contamination of their fin- gers, face. and hair (23E). This point may be useful in investigating the source-of staphylococcal contamination of ceil ii:ies. (iv) Footwear. In moderate and high risk situations, shoes reserved for only laboratory use have been recommended as a preeau- tion against transporting spilled infectious agents outside the laboratory. However, in experiments during which reduction of PO- tential contamination of experimental mate- rials is important, laboratory-only shoes can reduce the mi~rnbial load brought into the FEDERAL REGISTER, VOL. 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976 NOTICES 38469 laboratory each day by street shoes. Shoes are efllcient transporters. In one study, there were 4 to 850 times as many bacteria per squars centimeter on the laboratory foot- wear 88 on the floor itself (30). A. Care and handling. 1. Special attenbon must be given to the humane treatment of all laboraturv anfmat in accordance with the Animal Welfire Act ti 19'70. The implement- ing rules and regulations appear in the Code of Federal Regulations (UFR) Title 9, chap- ter I, Subchapter A, Parts 1, 2,o 3. Recom- mended provisions and practices that meet the requirements of the Act have been pub- lished by the U.S. Public Health Service (32). 2. There fu`e specific minimum requlre- ments (33,) concerning the caging, feeding, watering, and sanitation for dogs, cats, guinea pigs, hamsters, rabbits, and nonhu- man primates. To meet these requirements. the animal room supervisor must have a coap; yf 293CFR Chapter I, Subchapter A, . . . 3. Each laboratory should establish proce- dures to ensure the use of animals that are frse of diseases prefudicial to the proposed experiments and free from carriers of disease or vectors. such as sctoparasites, which en- danger other experimental animals or per- sonnel (10). B. Cages housing infected animaLs (lo). 1. Careful handling procedures should be employed to minimi= the dissemination of dust from cage refuse and animals. 2. Cages should be sterilized by autoclav- ing. Refuse. bowls and watering devices should remain in the cage during sterilira- tion. 3. All watering devices should be of the "non-drip" type. 4. Cage5 should be examined each morn- ing and at each feeding time so that dead animals can be removed. 5. Heavy gloves should be worn when fesd- 1% watering, handling, or removing in- fected snlmals. Bare hands should NEVER be placed in the cage ta move any object therein. 6. When animals are to be injected with biohazardous material, the animal caretaker should wear protective gloves and the labora- tory workers should wear surgeons gloves. Animals should bs properly restrained to avoid accidents that might result In dissem- inating biohasardous material, as well as to prevent injury to the animal and to per- sonnel. `7. Animals exposed to biohasardous aerosols should be housed in ventilated cages, in gas- tight cabinet systems, or in rooms designed for protection of personnel by use of ventl- let&d suits. 8. Animals inoculated by means other than by aerosols should bs housed in equipment suitable for the level of risk involved. 9. Infected animals to be transferred be- tween buildings should be placed in venti- lated cages or other aerosol-proof containers. 10. The oversize canine teeth of large monkeys present a particular biting hazard; these are important in the potential trans- mission of naturally-occurring, and very dangerous, monkey virus Infections. Such teeth should be blunted or surgically re- moved by a veterinarian. 11. Presently available epidemiological svi- dence indicates that infectious hepatitis may be transmitted from non-human primates (typically chimpanzees) to man. Newiy im- ported animals may be naturally infected with this disease, and persons in close con- tact with such animals may become infected. After six months residence in this country, chimpanzees apparently no longer transmit the disease. A record should be maintained for each newly imported animal. A sign should be posted at rooms housing these ani- mals to warn that the animals are potentially infectious. C. General Guidelines that Apply to Anlmal Room Maintenance (10). 1. Doors to animal rooms should be kept closed at all times BX- cept for necessary entrance and exit. 2. Unauthorized persons should not be per- mitted to enter animal rooms. 3. A container of disinfectant should be kept in each animal room for dlsinfe&ng gloves and hands, and for general decon- tamination, even though no infectious ani- mals are present. Hands, floors, walls, and cage racks should be washed with an ap- proved disinfectant at the recommended strength as @equently as the supervisor directs. 4. Floor drains in animal rooms, as wsli as floor drains throughout the building should be flooded with water or disinfectant periodically to prevent backup of sewer gases- 5. Shavines or other refuse on floors should not be washed down the floor drain because such refuse clogs the sewer lines. 6. An insect and rodent control program should be maintained in all animal rooms and in animal food storaee areas. 7. Special care should 6;? taken ta prevent live animals, especially mice, from finding their way into disposable trash. D. Necromu rules for fnfected animals (10). 1. Ne&opsy of i&ct.&i `animals should be carried out by trained personnel in Bio- logical Safety Cabinets with the hinged glass panel down. The glove port panel with or without attached gloves, and a respirator should be used at the discretion of the su- pervisor. 2. Surgeons gowns should be worn over laboratory clothing during necropaies. 3. Rubber cloves should be worn when ner- forming ne&psiea . 4. The fur of the animal should be wetted with a suitabls disinfectant. 5. Small animals should be pinned down or fastened on wood or metal in a metal tray. 6. Upon completion of necropsy, all poten- tially biohasardous material should be placed in suitable containers and sterilized imme- diately. 7. Contaminated instruments should be placed in a horizontal bath containing a suitable disinfectant. 8. The inside of the Biological Safety Cabinets and other potentially contaminated surfaces should be disinfected with a suie able germicide. 9. Grossly contaminated rubber gloves should be cleaned in disinfectant before re- moval from the hands, preparatory to sterili- zation. 10. Dead animals. should be placed in proper leak-proof containers, autoclaved and properly tagged before being placed outside for removal and incineration. VI. DECONTAMlNATION *ND DISPOS.4L (7. 10. 38-42) A. Introduction. Available data on the efllcacy of various decontaminsnts for etio- logic agents indicate that no major surprises will be forthcoming regarding the suscepti- bility of organisms containing recombinant DNA molecules. In the absence of adequate information, tests to determine the efficacy of oandidate decontaminants should be con- ducted with the specific agent of interest. The goal of decontamination is not only the protection of personnel and the environment from exposure to infectious agents, but also the prevention of contamination of experl- mental materials by a variable, persistent, and unwanted background of microorga- nisms. This additional factor should be con- sidered In selecting decontamination mate- rials and met.hods. B. Decontaminatfon Methods. Physical and chemical means of dscontaminatlon fall into four main categories: Heat: Liquid De&n- taminants; Vapors and Gases; and W Radia- UOll. 1. Heat. The application of heat, either moist or dry, is recommended as the most effective method of sterilization. Steam at 121 C under pressure in the autoclave is the most convenient method of rapidly achiev- ing sterility. Dry heat at 160 to 170 C for periods of 2 to 4 houm is suitable for deatruc- tion of viable age ts on impermeable non- P organic material uch as glass, but is not reliable in even shallow layers of organic or inorganic material that can act as insulation Incineration is another use of heat in the decontamination of microorganisms and also serves as an efllcient means for disposal. 2. Liquid Decontaminunts. In general, the liquid decontaminants find their most prac- tical use in surface decontamination and, at sufacient concentration, as decontaminanta of liquid wastes for final disposal in sanitary sewer systems. There are many misconcep- tions concerning the use of liquid decontami- nants. This is due largely to a characteristic capacity of such liquids to perform dra- matically in the test tube and to fail miser- ably in a practical situation. Such failures often occur because proper consideration was not given to such factors as temperature, time of contact, pH, concentration, and the presence and stats of dispersion, penetrability and reactivity of organic material at the site of application. Small variations in the above factors may make large ditrerences in effec- tiveness of decontmination. For this reason. even when used under highly favorable con: ditlons, complete reliance should not b-e placed on liquid decontaminants when the end result must be sterility. There are many liquid decontaminants available under a wide variety of trade names. In general, these can be categorized as halogens, acids or alkalies, heavy metal salts. auaternar7 ammonium comwunds. phenoli& compoiids, aldehydes. ketones. alcohols and &mines. Unfortunately, the more aotive the decontaminant the more likely lt is that the decontaminant will possess un- desirable characteristics, such as the posses- sion of corrosive properties. None is equally useful or effective under all conditions, 3. Vapors and Gases. A variety of vapors and gssee possess decontamination proper- ties. The most useful of these are formalde- hyde and ethylene oxide. When these can be employed in closed systems and under con- trolled conditions of temperature and hu- miditv. excellent decontamination can result. Vap& and gas decontaminants are primarily useful in decontaminating: (1) Biological Safety Cabinets and associated effluent alr- handling systems and air filters: (ii) bulky or stationary equipment that resists pentra- tion by liquid surface dscontaminants; (iii) instruments and optics that might be dam- aged by other decontamination methods; and (iv) rooms and buildinss and associated air- handling systems. - 4. Radiation. The usefulness of ultraviolet (W) irradiation as a decontaminant is limited by its low penetrating power. No ln- formation is available regarding the effect.lve- ness of W irradiation for decontaminating microorganisms containing recombinant DNA molecules. Dependence on W must be based on the results of experiments imitating particular anticipated environmental condi- tions and appllcatlons. Ultraviolet light is generally of limited application and is`pri- marlly useful In air locks and animal hold- ing areas for controlling low levels of air- borne contaminants. No one procedure or material will solve a11 decontamination problems. The only method of assuring the efficacy of selected method- ologies is to critically examine the results FEDERAL REGISTER, VOL. 41, N6. 176--THURSDAY, SEPTEMBER 9, 1976 34`m.l obtained in practical testi with the mlcro- organism(s) of interest. C. Laboratiy spills. A troublesome prob- lem that may occur In the laboratory is the decontamination of an overt biological spilt The occurrence of a spill pose5 less of a prob- lem if it occurs in a Biological Safety Cab- inet provided splattering to the outside of the cabinet does not occur. Direct applica- tion of concentrated liquid decontaminant and a thorough wipe down of the internal surfaces of such cabinetry will usually be ef- fective for decontaminating the work zone but gaseous decontaminants would be re- quired to rid the interior sections of the cabinet of contaminants. Each researcher must realize that in the event of an Overt a& cident, research materials such a5 tissue cul- tures, media, and annnals within such cabi- nets may well be lost to the experiment. The greater problem arises if the incident occurs in the open laboratory. All laboratory protocols should be designed to prevent such occurrences. The first action in the event of an overt laboratory spill is evacuation of the affected area to minimize the exposure of personnel involved. Next, the spill area must be isolated to prevent exposure of personnel and experimental materials beyond those in- volved in the immediate area of the spill. The procedures adopted must be rapidly ecec- tive and must not create additional aerosol or foster mechanical transfer of materials to unaffected areas. Personnel carrying out the procedures must be provided with pro- tective clothing and equipment, including respiratory protection. Consideration must be given to the safe disaosal of all materials and liquids resulting from cleanup proce- dures. Reentry of personnel to the area should be avoided until it can be reasonably established that the area has been effectively decontaminated. Further specific details are provided in Section VIII. D. Disposal. Decontamination and disposal In infectious disease laboratories are closely interrelated acts in which decontamination constitutes the introductory phrase of dis- posal. All materials and equipment used in research on recombinant DNA molecules will ultimately be disposed of; however, in the sense of daily use, only a portion of these will require actual removal from the labora- tory complex or on-site destruction. The re- mainder will be recycled for use either with- ln the same laboratory or in other labora- tories that may or may not engage in DNA recombinant research. Examples of the latter that immediately come to mind are: Re- usable laboratory glassware, instruments used in necropsy of infected animals, and laboratory clothing. Disposal should there- fore be interpreted in the broadest sense of the word, rather than in the restrictive sense of dealing solely with a destructive process. The principal questions to be answered prior to disposal of any objects or materials from laboratories dealing with potentially infectious microorganisms or animal tissues are: 1. Hare the objects or materials been effec- tively decontaminated by an approved proce- dure? 2. If not, have the objects or materials been packaged in an approved manner for immediate on-site incineration or transfer to another laboratory? 3. Does disposal of the decontaminated objects or materials involve any additional potential hazards, biological or otherwise. t0 personnel either: (1) Those carrying out the immediate disposal procedures or (ii) ThOSe who might come into contact with the objects or materisIs outside the laboratory Gnplex? Laboratory materials requlrlng disposal will normally occur as liquid, solid, and animal room wastes. The volume of these NOTJCES can become a major problem when there ls the requirement that all wastes be de- contaminated prior to disposal. It is most evident that a significant portion of this problem can be eliminated if the kind5 of materials initially entering the laboratory are reduced. In any case, and wherever pos- sible, materials not essential to the research should be retained in the nonresearch areas for disposal by conventional methods. Ex- amples are the packaging materials in which good5 are delivered, disposable carton-cages for transport of animals, and large carboys or tanks of fluids which can be left outside and drawn from a5 required. Reduction of this bulk will free autoclaves and other de- contamination and disposal processes within the laboratory for the more rapid and effi- cient handling of materials known to be contaminated. Inevitably, disposal of materials raises the quest.ion. "How can we be sure that the ma- terials have been treated adequately to as- sure that their disposal does not constitute a hazerd?" In the small laboratory, the prob- lcm is often`solved by requiring that each investigator decontaminate all contaminated materinls not of immediate use at the end of each day and place them in suitable con- tainers for routine disposal. In larger labora- tories where the mass of materials for dis- uosal becomes much greater and sterilization and decontamination bottlenecks occur, ma- terials handling and disposal will likely be the chore of personnel not engaged in the actual research. In either situation, a case can be made for establishing a positive method of dwsiznatinz the state of materials to be disposed if. ThiYs may consist of a tag- ging system stating that the materials are either sterile or contaminated. Disposal of materials from the laboratory and animal holding area5 will be required for research projects ranging in size from an ln- dividual researcher to those involving large numbers of researchers of many disciplines. Procedures and facilltie5 ti accomplish this will range from the simplest to the most elaborate. The primary consideration in any of these ki to dispel the notlon that labora- tory wastes can be disposed of in the same manner and with as little thought &9 house- hold wastes. Selection and enforcement of safe procedures for disposal of laboratory materials are of no less importance than the consideration given to any other methodol- ogy for the accomplishment of research objectives. Materials of dissimilar nature will be com- mon in laboratories studying recombinant DNA molecules. Examples are combinations of common flammable solvents, chemical car- cinogens, radioactive Isotopes, and concen- trated virus@ or nucleic acids. These may re- quire input from a number of disciplines in arriving at the most practical approach for their decontamination. E. Characteristics of chemical decontamf- nants in common use in laboratory opera- tions. Every person actively working with viable mlroorganisms, no matter how remote the field of specialization, will, from time to time, find it necessary to decontaminate by chemical methods work areas and materials. equipment. and specialized instruments. Chemical decontamination is necessary be- cause the use of pressurized steam, the most rapid and reliable method of sterilization. is not normally feasible for decontaminating large spaces, surfaces, and stationary equlp- ment. Moreover, high temperatures and moisture often damage delicate instruments, particularly those havihg complex optical and electronic components. Chemicals with decontaminant properties are, for the most part, available as powders, crystals, and llquld concentrates. These may be added to tap water ior application cs SW- face decontamlnants, and M)me, when added in sufficient quantity, flnd use as decontam- inants of bulk liquid wastes: Chemical de- contaminants that are gaseous at room tem- peratures are useful Ss space-penetrating decontaminants. Others become gases at rea- sonably elevated temperatures and can act as either aaueous surface or easeou5 soace- penetrating- decontaminants. - Inactivation of microorganism5 by chem- ical decontaminants may occur in one or more of the following ways: (1) Coagula- tion and denaturation of protein, (2) Lysis, (3) Binding to enzymes, or inactivation of an essential enzyme by either oxidation, binding, or destruction of enzyme substrate. The relative resistance to the action of chemical decontaminants can be substan- tially altered by such factors as: Concentra- tion of active ingredient, duration of con- tact, pH, temperature, humidity and pres- ence of extrinsic organic matter. Deuendina upon how these factors are manipulated, the degree of 5ucce5s achieved with chemi- cal decontaminants may range from mini- mal inactivation of target microorganism5 to an indicated sterility within the limits of sensitivity of the assay systems employed. There are dozens of contaminants arail- able under a wide variety of trade names. In general, these decontsminants can be classified as halogens, acids or alkalies, heavy metal salts, quaternary ammonium com- pounds, phenolic compounds, aldehydes. ke- tones, a!cohols, and amines. Unfortuatelp. the more active the decontaminant the more likely it will posse55 undesirable char- acterbtics. For example, peracetic acid 15 a fast-acting, universal decontaminant. How- ever, in the concentratted state it 1s a hazard- ous compound that can readily decompose with explosive violence, When diluted for use, it has a short half-life, produce5 strong, pungent, irritating cdors. and is extremely corrosive to metals. Nevertheless, it is such an outstanding decontaminant that it is commonly used in germ-free animal studies despite these under&able characteristics. The halogen5 are probably the second most active group al decontamlnants. Chlorine, iodine, bromine, and fluorine will rapidly kill bacterial spores, viruses. rickettsiae. and fungi. These decontaminants are effective over a wide range of temperatures. In fact, chlorine has been shown to be effective at -40 F. (On the other hand, phenols and formaldehyde have hlgh temperature coefa- cients). The halogens have several undesir- able features. They readily combine with protein, 50 that an excess of the halogen must be used if proteins are present. Also. the halogem are relatively unstable so that fresh solutions must be prepared at frequent fntervals. Finally, the halogens corrode metals. A number of manufacturera of de- contaminant5 have treated the halogen5 to remove some of the undesirable features. For example, sodium hyp+chlorite reacts with p- toluenesulfonamlde to form Chloramine T, and iodine reacts with certain SurfaCe-active agents to form the popclar iodophors. These "tamed" halogens are stable, non-toxic, odorless, and relatively ncncorrosive to metals. However, the halogen5 are highly reactive elements, and, because they are reactive they are good germicides. When a haolgen acts a5 a decontamlnant, fret halo- gen ls the effect,lve agent. Raking the pH or combining the halogen with other com- pounds to decrease the corrosive effect will also decrease the germicidal power. -4 trade- off situation occurs. Ineffectiveness of a decontaminant LY due primarily to the failure of the de- contaminant to contact the microorga- nisms rather than failure of the decon- taminnnt to act:If one places an item in FEDERAL REGISTER, VOL. 41, NO. 176-THURSDAY, SEPTEMBER 9, 1976 NOTICES 38471 a liquid decontaminant, one can see that the item is covered with tiny bubbles. of course, the area under the bubbles ls dry, and microorganisms in these drY areas will not be tiect&by the decon- taminant. Also, if there are spots Of grease, rust or dirt on the object, micro- organisms under these protective coat- lngs will not be contacted by the decon- taminant. Scrubbing an item when im- mersed ln a decontaminant is helpful, und a decontaminant should have, and most do have, incorporated surface- active agents. F. Propertfes of some common decon- taminants-1. Alcohol. Ethyl or iSO- propyl alcohol in a concentration of 70- 80 percent by weight is often used. Al- cohols denature proteins and are some- what slow in their germicidal action. However, they are effective decontami- nants against lipid-containing viruses. 2. Ether and Chloroform These com- pounds are not ordinarily used as decon- taminants, but they do demonstrate the fact that lipid-containing viruses are inactivated by these organic solvents, whereas non-lipid-containing viruses are quite resistant. 3. Formaldehyde. Formaldehyde for use as a decontaminant is usually mar- keted as a solution of about 3'7 percent concentration referred to as formalin or as a solid polymerized compound called paraformaldehyde. Formaldehyde in a concentration of 5 percent active ingredient is an effective liquid decon- taminant. It loses considerable activity at refrigeration temperatures and the pungent, irritating odors make formal- dehyde solutions difficult to use in the laboratory. Formaldehyde vapor gener- ated from formaldehyde solution is an effective space decontaminant for decon- taminatlng rooms or buildings, but in the vapor state with water it tends to polymerize out on surfaces to form para- formaldehyde, which ls persistent and unpleasant Formaldehyde gas can be liberated by heating paraformaldehyde to depolylplerize it. In the absence of high moisture content in the air, formal- dehyde released in the gaseous stats forms less polymer&d residues on sur- faces and less time is required to clear treated areas of fumes than formalde- hyde released in the vapor state. 4. P7~enoZ. Phenol Itself is not often used BY n decontaminant. The odor la somewhat unpleasant and s sticky. gummy residue remnine on treated surfnces. This is espb clally true during steam sterilization Al- though phenol itself may not be in wlde- spread use., phenol homologs and phenollc compounds are basic to a number 6f popular decontamlnant.8. The phenollc compounds nre effective decontarnlnanta against some viruses. rlckettslae, fungl and vegetative bat- terla. The phenollca are not effective in ordl- nary usage against bacterial spores. 5. Quaternary Ammonium Compound8 or Quats. After 30 years of testing and use, there ls still 8 considerable controversy about the efficacy of the Quats as decontamlnants. These catlonlc detergents are strongly sur- face-active and BPB effective against llpld- containing viruses. The Quata will attach to protein so thet dilute solutlone of Quata will quickly lose effectiveness in the presence of protelm The Quatd tend to clump micro- orgsnlsms and are neutralized by anlonlc detereents. such 88 8080. The Qusts have the advantageS of being n&toxic, odorless, non- staining, noncorrosive to metals, stable, and inexpensive. 6. Chlorine. Thb halogen ls a universal decontamlnant active against all microor- ganisms. including bacterial spores. Chlorine combines wlth protein and rapidly decreasea in concentration in its presence. Free, avsll- able chlorlue ls an active element. It is a strong oxldizlng agent, corrosive to metals. Chlorine solutions will gradually lose strength so that fresh solutions must be pre- pared frequently. Sodium hypochlorlde ls usually used as a base for chlorine decon- tamlnants. An excellent decontamlnant can be prepared from household or laundry bleach. These bleaches usually contain 5.25 percent available chlorine or 52,500 ppm. If one dilutes them 1 to 100, the solution will contain 525 ppm of available chlorine. and, lf a nonlonic detergent such 88 Naccanol la added in a concentration of about 0.7 uer- cent, a very good decontamlnant ls crer;ted 7. Iodine. The characteristics of chlorine and iodine are similar. One of the most popular groups of decontamlnanta used ln the laboratory ls the iodophors, and Wes- wdyne is perhaps the most popular. The range of dllutlon of Wescodyne recommend@ by the manufacturer ls 1 oz. ln 5 gal. of water glvlng 25 ppm of available lodlne to 3 oz. in 5 gnl. giving 75 ppm. At 75 ppm, the con- centratlon of free iodine ls .0075 percent. This em&l amount can be rapidly taken up by any extraneous protein present. Clean surfaces or clear water can be effectively treated by 75 ppm available iodine. but dlfficultles may be experienced lf any appreciable amount of protein ls present. For bacterial spores. a dilution of 1 to 40 ~lvlne 750 Darn is recum- mended by the m&uf&turer-. -For washing the hands, It ls recommend@ that Wescodyne be diluted 1 to 10 or 10 percent in 50 percent ethyl alcohol (a reasonably good -d-n- tnmhnnt itself I which wlll elve 1.500 rmm of avsllable iodine: at which c¢&i& reb- tlvely rapid lnaotivatlon of any and .sll mlcro- orgnnisme will occur. 0. Vapor8 and gases. The use of fcumalde- hyde as a vapor or ges hss already been dls- cussed. Other chemical decontamlnanta which have been used thla way Included ethylene oxide, peracetlc acid. beta-proplolac- tone (BPL), methyl bromide, and ethylene amine. When these oan be used in closed systems and under controlled conditions of temperature and humidity. excellent deam- tamln&.lon can be otbalned. Residues from ethylene oxlde must be removed by aeration; but otherwise it ls convenient to use. versatile, and noncorrosive. Paracetic acid h corrosive for metals and rubber. BPL ln the vapor form ncte rapidly against bacter& rlckettslae. and viruses. It has a half-life of 3.5 hours &hen mlxed with water, la easily neutralized wlth water, and lends itself to removal by aeration. The National Institutes of Health does not_ recommend BPL as a deoontamlnant because it has been identified s a suspect carcinogen. H. Residual action of &contaminants. Ae noted ln the preceding discussion of decon- tamlnant DroDertles. manv of the chemical decontamihadts often have residual proper- ties that may be considered a desirable fea- ture in terms of aiding ln the control of background contamlnatlon. One ls cautioned, however, to consider residual properties care fully. Ethylene 6xlde used to sterlll%.e labora- tiry shoes can leave residues which cause skin irritation. Animal cell cultures, as well 88 viruses of interest, nra also lnhiblted or lnactlvated by decontamjnants persisting af- ter routie cleaning procedures. Therefore, reusable Items that are routinely held ln liquid decontamlnant prior to sutoclavlng and cleaning should receive particular atten- tion in rinse cycles. Slmliarly. during gen- eral area decontamlnatlon wlth gases or va- pors, it may be necessary to protect new and used clean items by removing them from the area or by enclosing them in gastlght bags or by insuring adequate aeration following decontamination. I. Selecting chemic&l decontaminanta fM research on reclombinant DNA molecules. No single chemical decontamlnant or method will be effective or practical for all situations in which decontamination ls required. Selec- tion of chemical decontamlnants and proce- dures must be preceded by practical consld- eration of the purposes for the decontamlna- tlon and the lnteractlne factors that will ul- timately determine ho& that purpose ls to be achieved. Selection of any glven procedure wlU be lnliuenced by the information derived from answers-to the following questions: 1. What is the tareet mlcrooreanlsm~s~ ? 2. What decont&lnanta in ihat form are known to. or can be expected to, lnnctivate the target microorganism(s) 7 3. What degree of inactivation ls required? 4. In what menstruum ls the mlcroorga- nlsm suspended; i.e., simple or complex, on solid or porous surfacea, and/or airborne? 5. mat ls the highest concentration o! cells anticipated to be encountered? 6. Can the decontamlnant either aa an squeous solution. a vapor, or * gas reason- ably be expected to contact the mlcroorga- nlsms, and can effective duration of contact be maintained? `7. What restrictions apply with respect to compatibility of materials? 8. Does the anticipated use situation re- quire immediate avallablllty of an effective concentration of the decontamlnant or wlll auf&lent time be available for preparation of the worklng concentration shortly before its anticipated use? The primary target of decontamination ln the lnfectlous disease laboratory is the microorganism under active lnvestlgatlon. Laboratory preparations or infectious agents usually have titers grossly ln excess of those normally observed ln nature. The decontamlnatlon of theea h&h-titer ma- terlals presents cer%ln problems. Malnte- nnnce systems for bacteria or vlrusea rue speclflcally selected to preserve viability of the agent Agsr, protelnacwue nutrients, and cellular materials can be extremely ef- fective lm physically retarding or chemically blndlng active moletles of chemical decon- tamlnants. Such lnterferencea with the de- sired action of decontamlnanta may requlre the use of decontamlnant concentrations and co&act tlmea in excess 02 those shown to be effective in the teat tube. SlmiMulv. a major portion of d-tamlnan t wnkct time required to achieve a given level of agent inactivation may be expended ln in- actlvatlng a relatively small number of the more resistant members of the population. The current state of the fut provides llttle information on wblch to predict the prob- able virulence of these survivors. These problems are, however, common to all po- tentially psthogenlc agents and must alwtrya be considered ln selecting decontamlnanta and procedures for thelr use. Microorganisms exhibit a range of reslst- ante to chemical decontamlnants. In terms of practical decontamlnatlon. most vegeta- tive bacteria, fungi and llpld-containing vl- ruses, are relatively susceptible to chemical decontamination. The non-lipid-containing viruses and bacteria with a waxy coating such sa tubercle bacillus occupy a mld-range of resistance. Spore forms are the most re- slstnnt. A dewntamlnant selected on the basis of its effectiveness against mlcroorgnnlsms on" any range of the reslstanoe scale will be ef- FEDERAL REGISTER, VOL 41, NO. 17&THURSDAY, SEPTEMBER 9, 1976 38472 NOTICES fective 8gainst microorganisms lower on the scale. Therefore, lf decontamlnants that ef- fectively control spore forms 8re selected for routine laboratory decontamiaation, lt c8n be assumed that any other microorganisms generated by laboratory operations, even in high concentrations, would also be lnacti- vated. An additional area that must be considcrcd and for which there is little definitive infor- mation available is the "inactivation" of nucleic acids. Nucleic acids often have better survival characteristics under adverse con- ditions than do the intact virions and cells from which they were derived. Strong oxi- dlzers. strong acids and bases, and either gaseous or aqueous formaldehyde should re- act readily with nucleic acids. Their ability -. to destroy the nucleic acid being studied, however, should be confirmed in the experl- menter's laboratory. Because of inns& dlf- ferences in the chemlstrv of RNA end DNA the effectiveness of 8 decbntamlnant for one cannot be extrapolated to the other. For ex- ample, RNA molecules are susceptible to mild alkaline hydrolysis by virtue of the free hy- droxvl erouo in the 2' nositlon. whereas DNA mole&yes dre not susceptible.to mild alks- line hydrolysis. Table II summrtrizes pertinent characteris- tics and potential applications for several categories of chemical decontaminants most likely to be used in the biological laboratory. Practical concentrations and contact times that may differ markedly from the recom- mend&ions of manufacturers of proprietary VII. HOUSEKEEPniG A. Introduction. Well-defined housekeep- ing procedures and schedule8 are essential in reducing the risks of working with etio- logic agents and in protecting the integrity of the research program. This is p8rticul8rly true in the biological Laboratory operating under less than totsl containment concepts mui in all are&s used for the housing of 8ni- m8ls. whether or not they have been inten- tiormlly infected. A well-conceived 8nd well- executed housekeeping program 1imlt.a physi- Cal clutter that could distract the attention 8nd interfere with the activities of labora- tory personnel at 8 critical moment in 8 po- tentlslly h8z8rdous procedure, provide8 a work are8 th8t will not in itself be 8 source of physical ln]ury or contamination, and pro- vides 8n 8re8 that promotes the efllclent use of decontamlnants-in the event of the In- sdvertent release of 8 harmful egent. Less immediately evident are the benefits of es- tablishing. among personnel of widely vary- ing levels of education. an appreciation of the nature 8nd sources of biological con- tamination. Housekeeping is an omnibus term that can be interpreted 8s broadly or 8s narrowly 88 one choose8 It c.8n be seen that many of the procedures found under special headings, such ss decont8minatlon, disposal, and ani- mal care. are. in reality. specific instructions for safely accomplishing otherwise routine housekeeping chores. In these safety sug- gestions for resesrch on recombinant DNA molecules, It has been elected Ito address 8pecl5c8lly only t88ks of 8 janitorl8l nature under the subject of housekeeping. products are suggested. It has been assumed that micoorganlsms will be afforded 8 high degree of potential protection by organic menstruums. It has not been assumed that 8 sterile state will result from application of the indicated concentrations and contact times. It should be emphasized that these data 8re only indicative of efficacy under artificial test conditions. The efficacy of any of the decontaminants should be conclu- sively determined by individual investigators. It is readily evident that each of the de- contaminants has a range of advantages and d&advantages 8s well 8s 8 range bf potentla! for inactivation of 8 diverse mlcroflor. Eausl- ly evident is the need for compromise &. an 8lternative to maintaining 8 veritable "drug store" of decontaminants. - - -. -.-.-am The objectives of*housekeeping In the blo- logical 18bor8tory 8re to: 1. Provide 84 orderly work 8re8 conducive to the 8ccompllshment of the research pmgrsm. 2. Provide work 8re8s devoid of physical hazsrds. 3. Provide 8 clean work are8 with back- ground contamln8tlon ideally held to a xem level but more reallstlcslly to 8 level such that extr8ordin8ry measures in sterile t&n- nlques 8re not required to nalnterln lnteg- rlty of the biological systems belng researched. 4. Prevent the 8ccumul8tion of materhIs from current and pest experiments that con- stitute 8 hazard to laboratory personnel. 6. Prevent the cm&ion of aerosols of h8.z- 8rdous materials as a result of the housekeep- ing prwedures used. Procedures developed in the 8re8 of house- keeping should be based on the highest level of risk to which the personnel and integrity of the experiments will be subject. Such 8n appm8c.h avoids the confusion of multiple praoticee and retmining of personnel. The prlm8ry function, then. of routine honse- keeping procedures is to prevent the ~cumu- lation of organic debris th8t (1) may harbor microorganisms that are 8 potential threat to the integrity of the biologlaal systems un- der investlg8tion, (ii) may enhance the 8ur- vlval of microo~ inadvertently re- leased In experimental procedures, (iii) m8y retard penetration of deeont8mlnants, (iv) may be tm.ns.fer8ble from one 8re8 to 8n- other on clothing and shoes, (v) may, with sufliclent buildup. become 8 bloh8z8rd 88 8 consequence of secondary aerosolization by personnel and 8ir movement. and (vi) m8y cause allergenic sensitlz8tlon of personnel, e.g., to anlm81 dander8 Housekeeping ln animal c8re unit&i has the same primary function a9 that stated for the laboratory and 8hould, $ addition, be as meticulously carried out in quarsntine and conditioning areas 8s in area.5 used t.o house experlment8lly infected animals. No other are&s in the laboratory have the con- stant potential for creation of significant quantities of contaminated organic debris than do animal 08re facilities. In 8ll laboratories. efforts to achieve f&&l decontamination and to conduct 8 major cleanup of the biological complex are nor- mally undertaken at relatively long time ln- tervals. Routine housekeeping must be relied on to provide a work 8rea free of slgnlfioant sources of background contamination. The provision of such 8 work area is not simply 8 nmtter of indicating in 8 general way what has to be done, who will do it, and how often. The supervisor must view each task critically in terms of the potential ,bioh8rard involved. decide on 8 detailed procedure for its accomplishment, and provide lnstructlons to 18boratory personnel in 8 manner that minimizes the opportunity for misunder- stsnding. The following check&t outlines a portion of the items requiring critic& review by the laboratory supervlsor. It is not intended to be complete but is presented es 8n example of the detailed manner ln which housekeep- tng in the blologicsl laboratory complex must be viewed. FEDERAL REGISTER, VOL. 41, NO. 176THURSDAY, SEPTEMBER 9, 1976 Administrat~ion Areas Aisles Animal Food Storage Animal Bedding Storage Biological Safety cabin&3 Bench Tops and Other Wc& Surfaces Ceilings Change Rooms Cleaning Solution Disposal Cages and Oage Racks Dry Ice Chests Deep Freeze Chests Entry and Exit Ways Equipment Storage FlOOIX Glassware General Laboratory Equipmetit Cleanup Hallways Incubators Instruments Insect and Rodent, Control Light Fixtures Mechanical Equipment Areas MOPS Pipes-Wall and Ceiling Hung Refrigerators Showers supply storage uv Lamps Vacuum Cleaners Waste Accumulations Waste Water Disposal Others Housekeeping in the laboratory is One Of the avenues that leads to accomplishing the research program safely. It is important that housekeeping tasks be asSigned DAY personnel who are knowledgeable of the research pro- gram and special hazards of the research en- vironment. The recommended approach to housekeeping iii the assignment of house- keeping tasks to the research teams on an ln- dividual basis for their immediate work areaa and on a cooperative basis for areas of com- mon usage. Similarly, animal caretaker per- sonnel should be responsible for houeekeep- lng in animal care areas. The laboratory su- pervisor must determine the frequency with which the individual and cooperative house- keeping chores need be accomplished. He should provide schedules and perform fre- quent inspection to aesure compliance. This approach assures that research work flow patterns will not be interrupted by an alien cleanup crew, delicate laboratory equipment will be handled only by those most knowl- edgeable of its particular requirements. and the location of concentrated biological prep- arations and contaminated equipment used in their preparation and application will be kIlOWL B. Floor care. Avoidance of dry sweeping and dusting will reduce the formation of nonspecific environmental aerosols. Wet mop- ping or vacuum cleaning with a hlgh-effi- ciency particulate air (HEPA) filter on the exhaust is recommended. Careful consideration must be given to de- sign and quality in the selection of cleaning equipment and materials and in their use to prevent the substitution of one hazard for another. In the absence of overt hazardous spills, the cleaning process commonly will consist of an initial vacuuming to remove all gro.ss particulate matter, and a follow-up wet mopping with a solution of chemical de- contaminant containing a detergent. De- pending on the nature of the surfaces to be cleaned and availability of floor drains. re- moval of residual cleaning selutlons can be accomplished by a nu&ber of methods. Among them are: Pickup with a partially dry mop, pickup with a wet vacuum that has an adequately filtered exhaust, or remov- al to a convenient floor drain by use of a floe squeegee. After cleaning up a spill of infected mate- rial. the residual solution should not be NOTICES discharged io a sanitary sewer until it has been autoclaved or given further chemical treatment, such as by the addition of sodi- um hypochlorite suiiicient to pro\-ide a fitlal concentration of 500 ppm chlorine. Most household bleaches are marketed with a chlorine content of 5.25%. These in a final dilution of 1 :lOO, yield 5i5 ppm of a\,ailable chlorine. After allowing a contact time of 15 minutes, these solutions may be flushed down any available drain. Chlorine solutions in these high concentrations may be too corrosive for general application to floors and equipment. In any event, if solutions are used in this way, after the contact time the area should be rinsed with water. C. Dry sweeping.-While it is recommended that dry sweeping be minimized, this may be the only method available or practicable under certain circumstances. In such cases, sweeping compounds used with push brooms and dry-dust mop heads treated to suppress aerosollzation of dust should be used. Sweeping compounds available from the usual janitorial supply firms fall in three categories: Wax-baaed compounds used 011 vinyl floors and waxed floor ooverings. oil-based compounds for concrete floors. Oil-based compounds with abrasives (such as eand) to achieve a dry scouring action where much soil is present. Dry-dust mop heads can be purchased as treated disposable units or as reusable, wash- able heads that must be treated with appro- priate sprays or by other means to improve their dust-capturing property. D. Vacuum cleaning. In the absence of a HEPA filter on the exhaust, the usual wet and dry industrial-type vacuum cleaner is a potent aerosol generator. The HEPA-filtered exhaust used in conjunction with a well- sealed vacuum unlt. however, can negate this factor because of its ability to pass huge volumes of exhaust air while retaining par- ticles with a mlnhnum efeciency of 99.9'7 per- cent. Wet and dry unlta ticorporatLng a HEPA filter on the exhaust are available from a number of manufadurers. There are no particular requirements with respect to the manner in which the dry vacuuming is accomplished other than to emphasize that the objective is to remove alI debris and particulate matter. The manufac- turer's directions adequately detail the fre- quency of bag changes, fflter changes, and mechanical adjustments. Drv material vacuum-collected during these floor-cleaning ectivlties is potentially con- taminated, but the nature of the risk in probably greater to the experiment than to the experimenter. It Is wise to effect bag and fflter changes and to clean out Eollection tanks in a m&nner that will avoid or mini- mize aerosolizlng the contents of the vacuum cleaner. A vacuum machine that collects debris in a disposable bag Is preferable to machines that collect the major debris in a tank and on an exposed primary fllter. Even though it may serve as a primary fflter. the disposable bag must be removed with caution A bel- lows effect may pump dust out of the bag if its intake onenina is not sealed before moving it to a &tic-bag for transfer out of the area. In any event, the outer Surface of the disposable bag will probably bear some dust contamination. which also may occur on inner surfaces of the machine. To avoid contaminating experimental ma- terials, the emptying of vacuum collection tanks and chaneina of baas and fllters are best done away from the immediate labora- tory area, for example, in a small area that can be easily cleaned afterwards. The use of heavy rubber gloves is recommended when removing wastes from tanks in case broken glwd k present. After making the fllter changes, all external surfaces of the imme- 3&73 diate work area and the equipment should be wiped with a cloth moistened In decon- taminant. The operator might plan for a change of laboratory clothing afterwards so as to minimize carrying contamination into other areas of the laboratory. Avoid use of dry vacuum cleaning equip- ment in work with high risk agents in the open laboratory. Should it be necessary to use it, it Is recommended that gaseous ster- ilization may be used to minimize aerosoll- zation of microorganisms before waste is emptied from the vacuum container. Be- cause complete penetration of sterill+g gases into the collected dry dust may be B problem, all wastes should be Dlaced in a plastic bag. which then is tightly closed and incinerated or disposed of in an approved manner. When dry vacuum cleaning equipment has been used within a gastight safety cab- inet system, it can be treated ln an attached double-door carboxyclave (an autoclave equipped with an ethylene oxide gas sterili- zation system) to allow for removal and emptying of the collectton tank. If a wet vacuum is to be used for pickup of the detergent-germicide solution from the floor, the manufacturer's reconunenda- tions on filter life should be followed. In addition, the operation of the vacuum should be closely observed for evidence of operating changes indicating restricted air- flow or. conversely, increased flow indicating filter fsllure. Liquids collected in the vac- uum cleaner after floor mopping will con- tain decontamlnant materials. These liquids may be poured down a convenient floor drain, except in the case of cleanup wastes from an overt spill. The collected liquid should then be autoclaved or treated with chlorine solution before disposal. Provisions should be made for regular de- contamination of the entire vacuum clean- er with formaldehyde gas or vapor, or ethyl- ene oxide. This should be done after use if the vacuum is used In any manner for cleanup of overt spills of infectious material. E. Selection of 0 cleaning solutton. The selection of a detergent-&contaminant corn- binatlon for routine cleaning of the labora- tory complex should be based on the require- ments of the area of greatest potential for contamination by the widest spectrum of microorganisms. Wlth rare exception. this will be identl6ed as the animal holding area and the expected microorganisms may WI1 include fungi. virwes. and the vegetative and spore forms of bacteria A decontamlnat- Ing solution for such a range of mlcroorga- nkms would, however, be expensive and ex- cessively wrroslve for routine use. Except in those rare instances where it can be as- sumed that pathogenic sporea are being shed by laboratory animals. the risks from the spores are more likely to affect the experi- ments than the personnel The spores tend to be associated with organic debils from bedding and food. thus offering potential for removal or at least a large initial reduc- tlon in their numbers by vacuum cleaning. A wide range of cleaning solutions that am mildly sporicidal, reasonably residual. and are not destructive to the physical plant am available. Phenol derlvatlves In combination with a detergent have these characteristics and have been selected for routine use In a number of research facilities. There are num- erous detergent-phenolic combinations avail- able on the market. The phenols are one type of a broad spectrum of blocidal substan- that include the mercuriale, quaternary ammonium compounds, chlorine compound& lodophores. alcohols, formaldehyde, glutaral- dehyde, and combinations of alcohol with either iodine or formaldehyde. These havm been discussed in Section VI. The laboratiry supervisor should make a selection from those types most readily wuU- FEOERAL REGISTER, VDC 41, NB. 17~THURSOAY, SEPTEMBER 9, 1976 3YP74 NOTKES able which meet the gerleral criteria of effec- tiveness, residual propert,&, and low corro- siveness. F. Wet mopping-&Go-bucket ?nen:od. Wet mopping of floors in laboratory and animal care areas is, from a safety standpoint, most conveniently and etaclently accomplhhed using a two-bucket system. The principal feature of such a system is that fresh detor- gent-decontaminant solution is always ap- plied to the fl@X from one bucket, while all spent cleaning solution wrung from the mop is collected in the second bucket. Compact dolly-mounted double-bucket units with foot-operated wringers are available from most janitorial supply houses. A freshly laundered mop head of the cotton String type should be used daily. This requires that a mop with removable head be provided as opposed to a fixed-head type. In practice, t.he mop is saturated with fresh solution, very lightly wrung into the second bucket and applied to the floor using a figure eight mo- tion of the mop head. After every four or five strokes, the mop head is turned over and the process continued until an area of approxi- matelv 100 ft* has been covered. After allow- ing a contact time of five minutes, the solu- tion is removed with either a wet vacuum cleaner with HEPA-filtered exhaust or with the wrung-out mop. The mopping is con- t.inued in 100 it? increments untfl the total floor area has been covered. Floor-cleaning procedures are most effectively completed after the majority of the work force has de- parted and should progress from areas of lea& potential contamination to those of greatest potential. Before a mop head is sent lo a laundry, it should be autoclaved. Spent cleaning fluids are disposed of by flushing down tl?e drain. If the cleanun follows an overt sl)ill of in- fe&ous materlrll, the spent cleaning solution, titer removal from the floor, should be auto- claved or treated with chlorine solution. Chlorine (as household bleach) should be added to give 600 ppm and held for a contact time of 16 minutes before dumping in the sanitary sewer. 0. Alternatizie floor cleaning ?nethod for animal care areas and areas wills monolithtc floors. The absence of permanently placed laboratory benches and fixed equipment, coupled with the mobility of modern cage rs&ks, makes possible alternate floor-cleaning procedures in animal care facilities. As in all considerations of methodologies in blomedi- ti laboram facilities. M, is necessary to aseeea the compattbillty of procedures and facilities from the hazard point of view. The alternative floor-cleaning procedure to be d&cussed requires that floom are completely sealed or of monolithic con.struction so that liquid leakage to adjaent arlreas does not oemr and that, floor drains or Wet vacuum cleaners are available. Subsequent to the removal of all debris by dry vacuum, move the cage racks to one sfde of tme room. Cover the floor of the remaiting cleared portion of the - wim detergent- decontamlnant solution applied at a rate of approximately one gallon per 144 ft? from a one-gallon tank sprayer, using a setting on the nozzle which will cause the solution to flow on and not create a spray, The nozzle is pl,ac%d close t.o the floor. Allow a fifteen- minute contact period; then push the clean- ing solution to the floor drain with a large floor squeegee or pick 1% up with a wet vat- uum. Nlow the floor to air dry; move the csge racks into the cleaned area, and repeat the process for the remaining floor area. Floor drains in these areas should be rim-fish, at lea& stx inches in diameter, and fitted with a screen or poreus trap bucket ta catch large debris that es&spas the initial dry cleaning. such screens and mket.6 should be emptied after treatment with a decontaminant. If qaee ut.ilWion doea MA. require frWUent Iloo? WaShdoWn, pour I) half-gallon of deter- gellt-dccollt;tnlll~al~t solution into the drain each week to keep the t.rap in the waste line filled against backup of sewer gases. A. Biokacardo&s spill in a biological sa,@Cy cabzrLet. Chemical decontamination pme- dures should be initiated at once while the cabinet continues to operate to prevent escape of contaminants from the cabinet. 1. Spray or wipe walls, work surfaces, and equipment with a 2 percent solution of an ivdophor-decont.aminant ( Wescvd yne or equivalent). A decontaminsnt detergent has tile advantage of detergent activity, which is import.ant because extraneous organic sub- stances frequently interfere with the reaction between the microorganisms and the active agent of the decontaminnnt. Operator should wear gloves during this procedure. 2. Flood the ton work surface tray. and. if a Class II cabined, the drain pans aiid catch barilns below the work surface, with a decon- t.aminant and allow to stand IO-15 minutes. 3. Remove excess decontaminant from t.he tray by wiping with a sponge or cloth soaked in a decontaminant. For Class II cabinets, drain the tray into the cabinet base, lift out tray and removable exhaust grille work, and wipe off top and bottom (underside) surfaces wit.h a sponge or cloth soaked in a decon- taminant. Then replace in position and drain recontaminant from cabinet base into ap- propriate coutainer and autoclave according to standard procedures. Gloves, cloth or sponge should be discarded in en autoclave pan and autoclaved. B. Biohazard spill outside a biological sajety cabinet. 1. Hold your breath, leave the room immediately, and close the door. 2. Warn others not to enter the contami- nated area. 3. Remove and put into a container con- taminated garments for autoclavlng and thoroughly wash hands and face. 4. Wait 30 minutes to allow disslpatioil of aerosols created by the spill. 5. Put on a long-sleeve gown, mask, and rubber gloves before reentering the room. (For a high risk agent, a jumpsuit with tight-flttlng wrists and use of a respirator should be considered). 6. Pour a decontaminant solution (670 lodophor or 5% hypochlorite are recom- mended) around the spill and allow to flow Into the spill. Paper towels soaked with the decontaminant may be used to cover the area. To minimize aerosolization, avoid pour- ing the decontaminant solution directly OntO the spill. 7. Let stand 20 minutes to allow an ade- quate contact time. 8. Using an auticlavable dust pan and squeege, transfer all contaminated materials @aper towels, glass, liquid, gloves, etc.) into a deep autoclave pan. Cover the pan with aluminum foil or other suitable cover and sutoclave according to st.andard direc- tions. s. The dust pan and squeegee should be placed in an autoclavable bag and auto- claved according to standard directions. Con- tact of reusable items with non autoclavable plastic bags should be avoided-separation of the plastic after autoclaving can be very dim- cult. C. Radioactive biohazard spill outside a biologfcal safety cabinet. In the event that a biohazardous spill also involves a radiation hazard, the clean-up procedure may have to be modifled, depending on an evaluation of the risk assessment of relative biological and radiological hazard. Laboratories handling radioactive sub- stances must have the services of a deslgnat%d radiation protection ofacer available for con- sultatlon. The following procedure indicates suggest- ed variations from the bloheuard spill pro- cx?dure (above) that should be considered when a radioactive biohazard spill occurs out- side a Biological Safety Ca13lnet.l 1. Holding your breath, leave the r@zm im- mediately and close the door. 2. Warn others not to enter the con&ml- nated area. 3. flemove and put In a container con- tanilnated garments for autoclaving ad thoroughly wash hands and face. 4. Wait thirty minutes to allow &&pa- tion of aerosols created by the spill. *Before clean-up proceaures begin, (I radiu- tion protection o#irer sho?rld survey the spill for External radiation ilacard to determine the relaiive degree of risk. 5. Put on a long-sleeve gown, mask, and rubber gloves before reentering the room (For a high risk agent, a jumpsuit with tight- fit,ting slce~;?~ nlld a respirator should be ccn- sidered). 6. Pour a decontanlinant solutjon (5 `> lodc- pho: or 5',;) hypochlorite are recommended) around the soil1 and allow to flow Into the spill. Paper tawels soaked with the decon- tam~uant. may be used to cover the area. Tc minmize aerosolization, avoid pouring the deaont~millnnt solution directly onto t?i? :,p111. 7 l.et .-lxnd 30 lninutes to allow adequaie dlsinfectwlt contact tinle. 8. *In niost cases, the spill will Znuolve `,C or :H, whtch present tu) external haard Hou:ei:er, if more energetic beta or gamma emitters are inrolved, care must be taken to prevent hand and body radiation exposure. The radiation ,protection officer must make ihis determinalfon before the clean-up opera- tion is begun. If the radiation protection officer approvee. the bio-hazard-handling procedure may be- gin: Using an autoclavable dust pan and Yqueegee, transfer all contaminated materials (paper towels, glass, liquid, gloves, etc.) into a deeg autoclave pan. Cover the pan with aluminum foil or other suitable cover and autoclave according to standard directions. *If the radiation protection officer deter- mines tha&radioactice vapors may be re- leased and thereby contaminate the auto- clut'e, the material must not be autoclaved In that case, suflcfent deccmtaminant solu- tion to immerse the contents should be added to the wastes contafner. The cover should be seaZed wiih waterprooj tape, and the con- tainer stored and handled /or disposal a.* radioarttive taaste. Radioactive and bioha.zard warn&g symbols should be affixed to the waste container. As a general rule', autoclar- fngf should be avoided. 9. If autoclavlng has been approved, ths dust pan and squeegee should be placed in an autoclavable bag and autoclaved according to standard directions. Contact of reusable Items with plastic bags should b% avoided- separation of the plastic after autoclaving can be verl difficult. *A final radioactive survey should be made ot the spell area, dust pan and squeegee wfth a Geiger counter, or a smear should be taken and counted in a liquid scintdllation counter. The sspiratlon of tissue culture medih from monolayer cultures and of superna- tants from centrifuged samples into collec- tion vessels or reservoirs is a common pro- cedure in many laboratories. To prevent the accidental contamination by aerosola or fluids of house vacuum systems or labora- tory pumps, some investigators have in- stalled side arm flasks containing cottos sulfuric acid or decontamunt between abbe reservoir and the vaculim line. Cotton 1% not completely effective 85 a Altering sg%nt, 1 wp in procedures have been starred and itallciz&. FEDERAL REGISTER, VOL. 41, NO. 176lHURSDAY, SEPTEMBER 9. 1976 NOTICES 3847& sulfuric acid will corrode pipes, and con- taminants may lose their inactivating abil- ltv unon standing. The introduction of a c&ridge-type filter that is moisture resist- ant and has a rated capacity to remove particles 350 nm (0.35u) or larger in size provides an &ective barrier to ilrus aerosols. The secondary reservoir and filtration ap- paratus can be assembled from readily avail- able units as shown in Figure 1. A length of plastic tubing `/ inch 1.D x I:16 inch wall is attached at one end of the reservoir and at the other end to the lowrr arm of a filtration and media storage flask. These flasks vary in capacity from 250 to 4000 ml. the choice of flask depending on available space and amount of fluid that could be accident.ally aspirated. A second tube `of the same dimensions is attached from the upper arm of the flask to the inlet port of the disposable filter assembly. The third tube is attached from the filter assembly to a vacuum source. The t.uhes are securely held to the filter by fittings supplied with the filter and the other tubing connections can be secured by worm drive hose clamps. Ideally the flask should be placed higher than the reservoir of collection vessel. If fluid is accidentally drawn into the flask, the liquid can drain back into the reservoir by gravity if the connection at the vacuum line is broken. This prevents t,he loss of fluid which the investigator needs to retain. Should the flask be used onlv for the re- covery and storage of waste fluids, then the addition of a few grams of Dow Cornlng Antifoam A to the flask will reduce violent foaming of fluids aspirated into it. Such fluids can be decontaminated by introducing into the reservoir a flnal 5~~ concentration of an iodophor or other appropriate decon- taminant. holding for 30 minutes and drain- lng as above. If the filter becomes contamlnat,ed or re- quires changing, the Alter and flask can be safely removed by clamping the line between filter and vacuum source. The filter and flask should be autoclaved before the filter Is discarded. A new fllter can t.hen be Installed and the assembly replaced. logical materials. The NIH.Guidelines specify that all DNA recombinant materials will be packaged and shipped in containers that meet the requirements of these regulatiOnS and carrier tariffs. In addition when any por- tion of the recombinant DNA material is derived from an etiologtc agent l&ted in paragraph (c) of 42 CFR 72.25 (which is included at the end of this section, page D-85) the labeling requirements in these reg- ulations and carrier tariffs shall apply. B. Packaging of recombinant DNA mnt~,- rials. 1. Volume less than 50 ml. Material shall be placed in a securely closed, water- tight container [primary container ( test tube, vial, etc.) ] which shall be enclosed in a second, durable watertight container (secondary container). Several primary con- tainers may be enclosed in a single secondary container, if the total volume of all the primary containers so enclosed does not er- teed 50 ml. The space at the top, bottom and sides between the primary and secondary containers shall contain sufficient nonpar- ticulate absorbent material to absorb the entire contents of the primary container(s) in case of breakage or leakage. Each set of primary and secondary containers shall then be enclosed in an outer shipping container constructed of corrueated fiberboard. card- board, wood, or other-material of equivalent strength. If dry ice is used as a refrigerant, it must be placed outside the secondary container(s), W(S). Descriptions of thls packaging method a?e giren in Table III. 2. Volumes of 50 ml or Greater. Material shall be placed in a securely closed, waier- tight container (primary container) which shall be enclosed in a second, durable wat,er- tight container (secondary container). Single prhrn~ry coniainers shall not contain more i!~r\n 500 ml. of material. However, two or more primary containers whose combined voilunes do not exceed 500 ml. may be placed in a single secondary container. The space at the top, bottom, and sides between the primary and secondary containers shall con- tain sufficient non-particulate absorbent ma- terial to absorb the entire contents of the primary container(s) in case of breakage or leakage. Each set of primary and secondary containers shall then be enclosed in an outer shipping container constructed of corrugated fiberboard, cardboard, wood, or other ma- terial of equivalent strength. A shock absorb- ent material. in volume at least equal to that of the absorbent material between the pri- mary and secondary containers, shall be placed at the top, bottom, and sides between the secondary container and the outer ship- plng container. Not more than eight sec- ondary shipping containers may be enclosed in a single outer shipping container. (The maximum amount of materials which may be enclosed within a single outer shipping container should not exceed 4,000 ml.). If dry ice is used as a refrigerant, it must be placed out&de the secondary container(s). If dry ice is used between the secondary con- tainer and the outer shipping container. the shock absorbent material shall be placed so that the secondary container does not become loose inside the outer shipping container as the dry ice sublimates. Dcscrintions of nackagee which comnlv with the regulntiot;? of Ithe Department- of Transportation (DOT) are given in Table IV. C. Labeling of packages containing re- combinatat DNA n~nterials. 1. nlaterlals which do not cont.ain any portion of an etiologic agent listed in paragraph (c) of 42 CFR 72.25. Material data forms. letters, and other information identifying or describing the material should be placed around the out- side of the secondary container. Place only the addre-s label on the outer shipping con- tainer. DO NOT USE THE LABEL FOR ETIOLOGIC AGENTS,`BIOMEDICAL MATERIAL. 2. Materials which contain any portion of an et.iologic agent listed in paragraph (c) of 42 CFR 72.25. Material data forms, letters, and other information identifvina or describing the material should be piaced around the outside of the secondary container. In addition to the address label, the label for Etiologic Agents:Biomedical hZateria.1 must be affixed to t.he outer shipping container. This label l.9 described in paragraph (c) (4) of 42 CFR 72.25. 3. Materials which contain any portion of a plant pest (plant pathogens) which are so defined by the Department of Agriculture (USDA). Material data forms, letters, and other in- formation identifying or describing the ma- terial should be placed around the outside of the secondary-container. In addition to the address label, the shipping labels fur- nished bv the USDA as oart of the General. Courtesy or Special Permits required for re: search with and shipment of such agents shall be affixed to the outer shipping container. D. Additional shipipng requirements and limitations for recombinant DNA mate- rials.-1. Ddmestic Transportation. CiVil Aeronautics Board Rule No. 83 (Air Trans- port Association Restrlcted Articles TarifY 6-D) requires that a Shipper's Certificate, depicted below, be completed and arllxed to all shipments which bear the E'I'IOLOGIC AGENT;BIOMEDICAL MATERIALS label re- quired under the provisions of the Inter- state Quarantine regulations (42 CFR 72.25(c) 1. The Certiilcate must be com- pleted in duplicate and affixed to the outer shipping container. .A. Introduction. Federal regulations and carrier tariifa have been promulgated to ensure the safe transport of hazardous blo- Tbls L to cettifr that the contents ol this cansidnment are Ptonertv classified. described bp P~OFEC shipDing name end are Dncked. marked and Isbelled and are ia proocr condition for car&v+? by +Ic according to 811 eo~licabk carrier and Rovrrnmeat regulations. (For intcrnationnl rhirmenrr add "and to the TATA Restricted Articla Reauhtion.s'*J This consxnment 1s within the limitutio~ prescribed for: PASSENGER AIRC&WTICARGO ONLY Icross out nonapolicnblel. . Number d Specifr Each Article Senaretely Pnchkxa fl'roncr 8hlDDinP Name) Cleuificrtion ETIOLOGIC AGENT. r1.0~ . I I NG Ch~ct!$' ETIO. AC. FEDERAL REGISTER, VOL 41, NO. I76THURSDAY, SEPTEMBER 9, 1975 .38476 NOTICES Bhlpments of recombinant DNA Materials exceeding 60 nd in volume and containin.g any portion of an etiologic agent listed in paragraph (c) of 42 CFR 72.25 an3 restricted, by DOT regulations, to transport by $argo only aircraft. When the volume of a single primary container exceeds the 60 ml limita- tion, &Is restriction must be indicated on the Bhlpper's Certificate by crossing out "Passen- ger AlrcrW. When dry ice it3 used as a refrigerant an "OR&-Group A-DRY ICE LABEL" should be affIxed to the outer shipping container. The amount of dry ice used and the dale (2) Parcel Post Customs Declarat.ion (Pe packed should be designated on the label. 2. International Transportation.-In addi- 296&A) label. tion to the packaging and labeling require- (3) International Parcel PosLInstructions ments of the regulations previously cited, in- Given by Sender (poD asaa) 1abe'. ternational shipments of recombinant DNA (4) Dispatch note (POD 2972) tsrg materials in which any portion of the mate- (6) "Violet Label" rial is derived from an etiologic agent listed in paragraph (c) of 42 CFR 72.25 must have (6) Shipper's Certificate specifKed hx the one or more of the following documents- current International Air Transport Associa- depending on the country of destination: tion Tariff. Individual country requirementa (1) Parcel POst Customs Declaxation (PS are listed in "International Postage Ftatea and m30) tag. _ Fees" (USPO Publication 51). A.P?ENDIX D, PACE D-83 FEDERAL REGISTER, VOL. 41, NO. 17kTHURSDAY, SEPTEMBER 9, X976 Vlth rlclring WIthout Rcfripcrant Rcfrlnermlt m J 3 J .d d Refrinermt Plbrrbiwd box closely f:ttJnS the atyroform box, taped aha No. 3 erlnp eta1 t1n f14 404 x 700 or . l-6Sll.m frletion-seal tin cm, 610 x 70.3, top aolderad or clipped ,f 4 polnte u Styroloan box shuck- absorbent inmulatlon Plbcrbb.rd box closrly fitting the styrofom box, taped rhut V3C cardboard bar PSI type, 9-l/16- x 9-3116" x 11-l/&~ hIth O.D. taped ahuc with 3" typo * Pi: :ipe Styrofoam box shock- rbaorbtnt In.ul.tlon Ho, 3 crimp Beal t1n cm 404 I 700 or . I-nalIan friction-se*1 tGc._n, 610 x 708. top soldered or cltpped at 4 points L/ VIC cardboard box PS3 type. V-3/16* x Y-3/16" I 11-114" high O.D. toped *hut vlth 3" typ. PS3 tmpc VIC cardboard box 5 PSI type. 9-l/16" x g-1116" x 11-114' a hi6h O.D. taped -, #hut with 3" typ, PSI tape f P VX crrdborrd b&x g 12-114" I 12-l/4" I IO-3/16" hinh D.D. taped ahut with 1" wide PSI tape. No. 3 crlny se*1 tin esa 404 I 700 or . I-gsllon Jrictian-seal tin cm, 610 x 708, top mldcred DI Clipped at 4 pulnf# hl I-pallon rrlctlon-8fal t1n cm, 804 x 908, top o oldercd c.t clipped at 4 pohrl b/ ?Ibarborrd box &rely fittIn& the atyrofou box, iapcd #hut 500 ml Pyr.x gloss bottla, rubber-sLlrc .toppcr, taped, 01 so0 ml phtlc* bottle. iuwrow,oy wlda mouth. ,erw cap*, taped 603 x 510 2-S*110,, frtctfo~ ,bwt`wnt inbUt6ttM maa1 t1n can, SO4 x 901, top mldered or clipped at 4 polntl a, box lo ok taped shut .- mN'E& voi 4t, NO. 176THURSDAY, SEPTEMBER 9, 1976 APPENDIX D, Page D-85 ATTACHMENT I OEPARTMENf OF HEALTH, EDUCATJC;N, AND WELFARE PlJELlC HEALTH SERVICE CENTER FOR OISEASE CONTROL ATLANTA, GEORGIA 30333 IrIsphone: (404) 633.331 I, Ext. lfi8J TITLE 42-PUBLIC HEALTH Chaiter I-Fublic Health Service, t)epartrnent of Health, Education, and Welfare BUBCHAPrER F-QlJARANTtNE. INSPECTlC&;ilCENSlNG &&on 72.25 of Pwt 72, TN@ 42, Code a Federal Regulations, is amended to &ad as follows: Q 72.25 Etiologic rg~:en~sf