Furr, A Keith Ph.D "NONCHEMICAL LABORATORIES" CRC Handbook of Laboratory Safety Edited by A Keith Furr, Ph.D Boca Raton: CRC Press LLC,2000 Chapter NONCHEMICAL LABORATORIES I INTRODUCTION The emphasis in the previous chapters has been on laboratories in which the primary concerns were due to the use of chemicals, although in order to not completely avoid a topic unnecessarily, some of the problems arising in other types of operations have been addressed For example, in the latter part of the last chapter some serious issues involving problems with specific contagious diseases were discussed due to the increasing importance these diseases have in our society and in our laboratorie s , as a continuation of the topic of health effects The problems of dealing with infectious waste were considered at some length as well as chemical wastes as part of the larger problem of dealing with hazardous wastes In this chapter laboratory operations which involve special problems in other classes of laboratories will be presented in greater detail However, in responding to these special problems, one should be careful not to neglect the safety measures associa t e d w i t h t h o s e hazards already covered II RADIOISOTOPE LABORATORIES Exposure of individuals to ionizing radiation is a major concern in laboratories using radiation as a research tool or in which radiation is a byproduct of the research Although there are many types of research facilities in which ionizing radiation is generated by the equipment, e.g., accelerator laboratories, X-ray facilities, and laboratories using electron microscopes, the most common research application in which ionizing radiation is a matter of concern is the u s e of unstable forms of the commo n elements which emit radiation A very brief discussion of some atomic and nuclear terms will be given next, with apologies for those not requiring this introduction to the subject A Brief Summary of Atomic and Nuclear Concepts An atom of an element can be simply described as consisting of a positively charged nucleus and a cloud of negatively charged electrons around it The electron cloud defines the chemical properties of the atom, which have been the subject up until now, while the processes primarily within the nucleus give rise to the nuclear concerns which will be addressed next Although the nucleus is very complex, for the present purposes an atom of a given element may be considered to have a fixed number of positive protons in the nucleus, equal in number to the numb er of electrons around the neutral atom, but can differ in the number of neutral neutrons, the different forms being called isotopes It is the property of the unstable forms, or radioisotopes, to emit radiation which makes them useful, since their chemical properties are essentially identical to the stable form of the element (where a stable form exists; for elements with atomic numbers greater than that of bismuth, there are no ©2000 CRC Press LLC completely stable forms) The radia tion which the radioisotopes emit allows them to be distinguished from the stable forms of the element in an experiment There are three types of radiation normally emitted by various radio i s o t o p e s , a l p h a (α) particles, electrons (β), and gamma rays (γ ) The properties of these radiations will be discussed later A fourth type of radiation, neutrons, may be emitted under special circumstances, by a small number of radioactive materials Most laboratories will not use materials emitting neutrons The properties of these radiations will be discussed in more detail later B Radiation Concerns The radiation which makes radioisotopes useful also makes their use a matter of concern to the users and the general public Exposure to high levels of radiation is known t o c a u s e health problems; at very high levels, death can follow rapidly A t lower, but still substantial levels, other health effects are known to occur, some of which, including cancer, can be delayed for many years A t very low levels, knowledge of the potential health effects is much more uncertain The generally accepted practice currently is to extrapolate statistically known effects on individuals exposed to higher levels to large groups of persons exposed to low levels of radiation in a linear fashion The concept is similar to the u s e of higher concentrations of chemicals using a limited number of animals in health studies of chemical effects, instead of more normal concentrations in a very large number of test animals There are some who question the validity of this assumption in both cases, but it is a conservative assumption and, in the absence of confirmed data, is a generally safe cours e of action to follow However, the practice may have led to a misleading impression of the risks of many materials When a scientist makes the statement that he does not know whether a given material is harmful or not, he is often simply stating in a very honest way that the data not clearly show whether, at low levels of use or exposure, a harmful effect will result It does not necessarily imply, as many assume, that there is a lack of research in discovering possible harmful effects In many cases, major efforts have been made to unambiguously resolve the issue, as in the case of radiation, and the data not support a definite answer There are levels of radiation below which no harmful effects can be detected directly In the case of radiation, there is even a substantial body of experimental data (to which proponents of a concept called “hormesis” call attention) that supports possibly positive effects of radiation at very low levels This position is, of course, very controversial However, in chemical areas there are many examples of chemicals essential to health in our diets in trace amounts that are poisonous at higher levels It is not the intent of this section to attempt to resolve the issue of the effects of low-level radiation, but to emphasize that there are concern s b y m a n y employees and the general public It may well be that, by being very careful not to go beyond known information, scientists have actually contributed to these concerns Another way of looking at the issue, and certainly a more comforting way, is that many unsuccessful attempts have been made to demonstrate negative effects at low levels Radiation levels which normally accompany the u s e of radioisotopes are deliberately kept low, and the perception of risk by untrained individuals may be overstated However, a linear dose-effect relation is the accepted basis for regulatory requirements at this time, and until better data are available scientists using radioactive substances must conform to the standards Users owe it to themselves and the public to use the materials in ways known to be safe However, as a general concept, it would be wise for scientists, when speaking to persons not trained in their field, to be sure that when they say they not know of possible harmful effects of any material, that this s tatement is understood to be an informed uncertainty where this is the case, as opposed to being based on a lack of effort It is unfortunate that there is so much concern about radiation sin ce there are many beneficial effects, but because of the dramatization of the concerns, many individuals fear radiation out of all proportion to any known risks In an opinion poll in which members of the ©2000 CRC Press LLC general public were asked to rank the relative risks of each of a number of hazards, nuclear radiation was ranked highest but in reality, was the least dangerous of all the other risks based on known data with all the others being much more likely to cause death or injury than radiation Individuals who urgently have needed X-rays, the application of diagnostic u s e of radioactive materials, or radiation therapy have declined to have them because of this heightened fear Used properly, radiation is an extremely valuable research tool and has many beneficial aspects Used improperly, it can be dangerous, but so can many other things in the laboratory, many very much more so C Natural Radioactivity A common misconception is that radiation is an artificial phenomenon Many of the most commonly used radioisoto pes have been created artificially, but there are abundant sources of natural radiation which emit radiation of exactly the same three types as artificially created radioisotopes Elements such as potassium and carbon, which are major constituents of our body have radioactive isotopes Many other elements, such as the rare earths, have radioactiv e versions Every isotope of elements with atomic numbers (i.e., the number of protons in the nucle us of the element, or the number of electrons around the nucleus in a neutral atom) above 83 is unstable and these elements are common in the soils and rocks which make up the outer crust of the earth There are areas in the world in which the natural levels of radiation could significantly exceed that permitted for the general public resulting from the operation of any licensed facility using radioisotopes Radiation constantly bombards us from space due to cosmic rays Pers ons who frequently take long airplane flights receive a significantly increased amount of radiation over a period of time compared to persons who fly rarely or not at all Arguments that these natural forms of radiation are acceptable because they are natural has absolutely no basis in fact As was mentioned earlier, there are only a modest number of varieties of radiation, and these are produced by both natural and artificially produced radioactive materials Similarly, there are only a few ways in which radiation may interact with matter, and they also are the same for all sources of radiation The acknowledgment of natural sources of radiation is not intended to minimize concerns about radiation, even the natural forms, but to point out that if there are concerns about low levels of radiation, then these natural levels must be considered as well as the artificial sources One of the naturally occurring radioactive materials, radon, has been receiving much attention and may be a significant hazard, perhaps contributing to an increase of to 5% of the number of lung cancer deaths each year This estimate, as in most cases dealing with attribution of specific effects of low levels of radiation, is supported by some and disputed by others Note, however, that even in this case at least 95 to 99% of the lung cancer deaths are attributable to other causes Radon as an issue will be discussed in a separate section later in this chapter An isotope of potassium, an essential element nutritionally and present in substantial amounts in citrus fruits and bananas, for example, emits significant amounts of very penetrating radiation There are various estimates of the average source of radiation exposure for most individuals An article by Komarov,1 who is associated with the World Health Organization, provides the following data about sources of radiation: 37% from cosmic r a y s a n d t h e terrestrial environment, 28% from building materials in the home, 16% from food and water, 12% from medical usage (primarily X-rays), perhaps 4% from daily color television viewing, 2% from long-distance airplane flights, and 0.6% (under normal operating conditions) from living near a nuclear power plant Note that the medical exposure to radiation is 20 times larger than from nuclear power plants even for those living near one The Komarov article was written before the Chernobyl incident, but even this outstanding example of poor ©2000 CRC Press LLC management is not sufficient to change the general picture Unlike the Chernobyl reactor, commercial nuclear power plants in the United States are protected by very strong confinement enclosures to prevent unscheduled releases In the case of the Three-Mile Island incident, in which the reactor core melted down, the confinement enclosure performed as designed and minimal amounts of radioactive material were released As the news media reported some time after the initial furor, “the biggest danger from Three-Mile Island was psychological fear,” to which the media contributed significantly by exaggerated news reports of the potential dangers In summary, radiation is a valuable research tool In order to prevent raising public concerns and perhaps lead to further restrictions on its use, scientists need to be scrupulously careful to conform to accepted standards governing releases or over exposures Fortunately, for common uses of radiation in research laboratories, this goal is easily achieved with reasonable care D Basic Concepts Each scientific discipline has its own special terms and basic concepts on which it is founded This section is, of course, not necessary for most scientists who routinely work with radiation, but it may be useful for establishing a framework within which to define some needed terms As scientists work with accelerators of higher and higher energies, the concept of matter is at once growing more complex and simpler; more complex in that more entities are known to make up matter, but simpler in that theorists working with the data generated by these gigantic machines are developing a coherent concept unifying all of the information For the purposes of this discussion, a relatively simple picture of the atom will suffice, as noted earlier The Atom and Types of Decay In the simple model of the atom employed here, as briefly described earlier in this chapter, the atom can be thought of as consisting of a very small dense nucleus, containing positively charged particles called protons and neutral particles called neutrons, surrounded by a cloud of negatively charged electrons The number of protons and the number of electrons are equal for a neutral atom, but the number of neutrons can vary substantially, resulting in different forms of an element called, as already noted, isotopes of the element Some elements have only one stable isotope, although tin has ten There are unstable isotopes, logically called radioisotopes, in which, over a statistically consistent time, a transition of some type occurs within the nucleus Different types of transitions lead to different types of emitted radiation Hydrogen, for example, has two stable forms and one unstable one, in which a transition occurs to allow an electron to be generated and emitted from the nucleus, producing a stable isotope of helium Prior to the transition, the electron did not exist independently in the nucleus A neutron is converted to a proton in the process, and the electron is created by a transformation of energy into matter This process is called beta decay No element with more than 83 protons in the nucleus has a completely stable nucleus, although some undergo transitions (including by processes other than beta decay) extremely slowly In some cases, the mass energy of the nucleus favors emission of a positive electron (positron) instead of a normal electron which has a negative charge This is called positive beta decay or positron decay Here a proton is converted into a neutron A competitive process to positive beta decay is electron capture (ε) in which an electron from the electron cloud around the nucleus is captured by the nucleus, a proton being converted into an neutron in the process In the latter process, X-rays are emitted as the electrons rearrange themselves to fill the vacancy in the electron cloud However, following positron emission, ©2000 CRC Press LLC the positive Table 5.1 Properties of Radioactive Emissions Type Mass (amu) Alpha (a) Beta (B) Gamma X-Rays 1/1 840 0 Charge (Electron Units) Range of Energy +2 4-6 MeV +1 0 eVs-4 MeV eVs-4 MeV eVs-100 KeV An amu is the mass of a single nucleon based on the 1/12th the mass of a carbon-12 nucleus electron eventually interacts with a normal electron in the surrounding medium, and the two vanish or annihilate each other in a flash of energy The amount of energy is equal to the energy of conversion of the two electron masses according to E = mc This amounts to, in electron volts, 1.02 million electron volts, or 1.02 MeV In order to conserve momentum, two photons or gamma rays of 0.511 MeV each are emitted 180 " apart in the process In many case, the internal transitions accompanying adjustments in the nucleus results in the emission of electromagnetic energy, or gamma rays These can be in the original or parent nucleus, in which case they are called internal transitions, and the semi-stable states leading to these transitions are called metastable state s More often, the gamma-emitting transitions occur in the daughter nucleus after another type of decay such as beta decay (metastable states can exist in the daughter nucleus also) The gamma emission distribution can be very complex In some instances, the internal transition energy is directly transferred to one of the electrons close to the nucleus in a process called internal conversion, and the electron is emitted from the atom In this last case, energy from transitions in the orbital electron cloud is also emitted as X-rays Finally, the most massive entity normally emitted as radiation is the alpha (a) particle which consists of a bare (no electrons), small nucleus having two protons and two neutrons The nucleons making up an alpha particle are very strongly bound together, and unlike electrons, the alpha particle appears to exist in the parent nucleus as a cohesive unit prior to the decay in our simple model This process is somewhat more rare than β or γ decay The processes briefly described above are the key decay processes in terms of safety in the u s e of radioisotopes There is another very important aspect of the decay processes, and that is the energy of the emitted radiation The electrons emitted in beta decay can have energies ranging from a few eV to between and MeV There is an unusual feature of the beta decay process in that the betas are not emitted monoenergetically from the nucleus as might be expected, and as does occur for alpha and gamma decay The most probable energy of the betas in a decay process is approximately one third of the maximum energy beta emitted in the process The reason is that, in addition to a beta being emitted, another particle, called a neutrino, of either zero mass or very close to it, is emitted simultaneously and shares the transitional energy, with varying amounts going to the two entities The neutrino does not play a role in radiation safety as it interacts virtually negligible with matter, although its existence is very important for many other reasons Gammas can have a similar range of energies to that of electrons, but the energies of the gammas are discrete instead of a distribution Alpha particles have a relatively high energy, normally ranging from to MeV The decay of alphas with lower energies is so slow that it occurs very rarely while with an energy just a little higher, the nucleus decays very rapidly The high energy, accompanying the high mass and the double positive charge, make the alpha particle a particularly dangerous type of radiation, if it is emitted in the proximity of tissue which can be inj u r e d T h i s l a s t i s a n important safety qualification as will be seen later Table 5.1 summarizes the properties of the ©2000 CRC Press LLC Figure 5.1 Schematic representation of the decay process types of radiation Graphically the decay process can be depicted as shown in Figure 5.1, where N = the neutron number and Z = the nuclear charge The box with N,Z is the parent nucleus and the others are the possible daughters for the processes shown The Fission Process A major omission deliberately not mentioned in the preceding Section is not involved in most laboratories using radioisotopes However, without this process many of the commonly used radioisotopes would not be available, since they are obtained from reprocessing spent fuel and recovery of the remnants left over after the fission process The process of fission describes the process by which a few very heavy atoms decay by splitting into two major components and a few neutrons, accompanied by the release of large amounts of energy, ~200 MeV The process can be spontaneous for some very heavy elements, e.g., Californium252 but also can be initiated by exposing specific heavy nuclei to neutrons There are no common radioisotopes that normally emit neutrons, but there are several interactions in which a neutron is generated Among these are several reactions in which a gamma ray interacts with beryllium to yield neutrons, so that a portable source of neutrons can be created There are many other ways to generate neutrons but there is no need to describe these in this book However, if a source of neutrons, n, is available and is used to bombard an isotope of uranium, 235U, the following reaction can occur ©2000 CRC Press LLC n+ 235 U -> X+Y + ~ ~2.5n + energy (1) Here, X and Y are two major atomic fragments or isotopes resulting from the fission process On average about 2.5 neutrons are emitted in the reaction plus energy The process is enhanced if the initiating neutrons are slowed down until they are in or near thermal equilibrium with their surroundings X and Y themselves will typically decay after the original fission event, a few by emitting additional neutrons, as well as betas and gammas A s noted earlier, about 200 MeV of energy are released in the process, much of it as kinetic energy shared by the particles Some of these fission fragments are long lived, and can be chemically separated to provide radioisotopes of u s e in the laboratory These fission fragment derived radioisotope s are the major source of the byproduct radioisotopes regulated by the Nuclear Regulatory Commission (NRC) The fission reaction can, under appropriate circumstances, be self-sustaining in a chain reactio n In some configurations, the chain reaction is extremely rapid, and an atomic bomb is the result However, by using the neutrons emitted by the fission fragments (called delayed neutrons), the process can be controlled safely in a reactor Over a period of time, the fission products build up in the uranium fuel eventually can be recovered when the fuel element is reprocessed Additional radioactive materials or radioisotopes are made by the following reaction: n + AX-> (A+1) y* + a (2) The asterisk indicates that the product nucleus, Y may be unstable and will undergo one (or more) of the modes of decay discussed previously The 'a’ indicates that there may be a particle directly resulting from the reaction In many cas es, the source of neutrons for radioisotopes created by this reaction is a nuclear reactor so these radioactive materials also are “byproduct materials, ” and are regulated by the Nuclear Regulatory Commission or State surrogates Plutonium is made in nuclear reactors by the above reaction where 238U is the target nucleus Although there are other reactions using different combinations of particles in Equation 2, in most cases these require energetic bombarding particles generated in accelerators Also, since there are no common radioisotopes that generate neutrons, there is essentially no probability that other materials in laboratories will be made radioactive by exposure to radiation from byproduct materials Materials which will undergo fission and can be used to sustain a chain reaction are, in the nomenclature of the NRC, “special” nuclear materials These inclu d e t h e i s o t o p e s o f uranium with mass numbers 233 and 235, materials enriched in these isotopes, or the artificially made element, plutonium Materials which have uranium or thorium, which also has a fissionable isotope, in them to the extent of 0.05% are called source materials Radioactive Decay An important relationship concerning the actual decay of a given nucleus is that it is purely statistical, dependent only upon the decay constant for a given material, i.e., the activity A, is directly proportional to the number, N, of unstable atoms present: Activity = A = dN/dt = C N (3) This can be reformulated to give the number of radioactive atoms N at a time t in terms of the number originally present N(t) = N0e 8t ©2000 CRC Press LLC (4) where λ = ln2/τ Table 5.2 Typical Decay of a Group of 1000 Radioactive Atoms Number 1000 502 249 125 63 31 Time (t) Number 14 1 Time (t) 10 11 Equation shows that during any interval, t = τ, theoretically half of the unstable nuclei at the beginning of the interval will decay In practice, approximately half will decay in a halflife, τ This is illustrated in Table 5.2 The data in this table illustrate clearly that when small numbers are involved, the statistical variations cause the decrease to fluctuate around a decay of about one half of the remaining atoms during each successive half-life, but obviously between and half-lives in this table, it would have been impossible to go down by precisely half The table also illustrates a fairly often used rule-of-thumb: after radioactive waste has been allowed to decay by 10 halflives, the activity has often decayed sufficiently to allow safe disposal This, of course, depends upon the initial activity The daughter nucleus formed after a decay can also decay as can the second daughter, and so forth However, eventually a nucleus will be reached which will be stable This is, in fact, what occurs starting with the most massive natural elements, uranium and thorium All of their isotopes are unstable, and each of their daughters decays until eventually s t a b l e i s o topes of lead are reached The existence of all of the elements above atomic number 83 owe their existence to the most massive members of these chains that have very long half-lives that are comparable to the age of the earth, so a significant fraction remains of that initially present Units of Activity The units of activity are dimensionally the number of decays or nuclear disintegr a t i o n s per unit time Until fairly recently, the standard unit to measure practical amounts of activity was the curie (Ci), which was defined to be 3.7 x 1010 disintegrations per second (dps) Other units derived from this were the millicurie (mCi) or 3.7 x 107 dps, the microcurie (µ Ci) or 3.7 x 104 dps, the nanocurie (nCi) or 37 dps and the picocurie (pCi) or 0.037 dps Many health physicists prefer to u s e disintegrations per minute (dpm), and the NRC also prefers the data logged in laboratory surveys to be expressed in dpm The curie was originally supposed to equal the amount of activity of g of radium This unit, and the derivative units, are still the ones most widely used daily in this country; however, an international system of units, or SI system, has been established (and is used in s cientific articles) In this system, one disintegration per second is defined as a becquerel (Bq) Larger units, which are multiples of 103, 106, 109, and 10 12, are indicated by the prefixes kilo, mega, giga, and tera, respectively In most laboratories that u s e radioisotopes as tracers, the quantities used are typically about 10 to 10 dps There are other uses of radioisotopes (e.g., therapeutic use of radiation) which use much larger amounts ©2000 CRC Press LLC Interaction of Radiation with Matter a Alphas A s an alpha particle passes through matter, its electric field interacts primarily with the electrons surrounding the atoms Because it is a massive particle, it moves comparatively slowly and spends a relatively significant amount of time passing each atom Therefore, the alpha particle has a good opportunity to transfer energy to the electrons by either removing them from the atom (ionizing them) or raising them or exciting them to higher energy states Because it is so much more massive than the electrons around the atoms, it moves in short, straight tracks through matter and causes a substantial amount of ionization per unit distance An alpha particle is said to have a high linear energy transfer (LET) A typical alpha particle has a range of only about 0.04 mm in tissue or about cm in air The thickness of the skin is about 0.07 mm so that a typical alpha particle will not penetrate the skin However, if a material that emits alphas is ingested, inhaled, or, in an accident, becomes imbedded in an open skin wound, so that it lodges in a sensitive area or organ, the alpha radiation can cause severe local damage Since many heavier radioactive materials emit alpha radiation, this often makes them more dangerous than materials that emit other types of radiation, especially if they are chemically likely to simulate an element retained by the body in a sensitive organ If they are not near a sensitive area, they may cause local damage to nearby tissue, but this may not cause appreciable damage to the organism as a whole b Betas Beta particles are energetic electrons They have a single negative or positive charge and are the same mass as the electrons around the atoms in the material through which they are moving Normally, they also are considerably less energetic than an alpha particle They typically may move about two orders of magnitude more rapidly than alpha particles They still interact with matter by ionization and excitation of the electrons in matter, but the rate of interaction per unit distance traveled in matter is much less Typically, beta radiation, on the order of MeV, can penetrate perhaps 0.5 cm deep into tissue, or about meters of air, although this is strongly dependent upon the energy of the beta Low-energy betas, such as from 14C, would penetrate only about 0.02 cm in tissue or about 16 cm in air Therefore, only those organs lying close to the surface of the body can be injured by external beta irradiation and then only by the more energetic beta emitters Radioactive materials emitting betas taken into the body can affect tissues further away than those that emit alphas, but the LET is much less There is a secondary source of radiation from beta emitters As the electrons pass through matter, they cause electromagnetic radiation called “bremstrahlung,” or braking radiation to be emitted as their paths are deflected by passing through matter The energy that appears as bremstrahlung is approximately ZE/3000 (where Z is the atomic charge number of the absorbing medium and E is the β energy in MeV.) This is not a problem with alpha particles since their paths through matter are essentially straight Bremstrahlung radiation can have important implications for certain energetic beta emitters such as 32P Protective shielding for energetic beta emitters should be made of plastic or other low-Z material instead of a high-Z material such as lead Because of the silicon in glass, even keeping 32P in a glass container can substantially increase the radiation d o s e to the hands while handling the material in the container as compared to the exposure that would result were bremstrahlung not a factor c Gammas Since gamma rays are electromagnetic waves, they are not charged and not have any mass, they interact differently with matter than alpha and beta particles, although the net effect is usually still ionization of an orbital electron They interact with the electrons in matter ©2000 CRC Press LLC ii Appendix K-II-B Written instructions and training of personnel shall be provided to assure that cultures of viable organisms containing recombinant DNA molecules are handled prudently and that the work place is kept clean and orderly iii Appendix K-II-C In the interest of good personal hygiene, facilities (e.g., hand washing sink, shower, changing room) and protective clothing (e.g., uniforms, laboratory coats) shall be provided that are appropriate for the risk of exposure to viable organisms containing recombinant DNA molecules Eating, drinking, smoking, applying cosmetics, and mouth pipetting shall be prohibited in the work area iv Appendix K-II-D Cultures of viable organisms containing recombinant DNA molecules shall be handled in facilities intended to safeguard health during work with microorganisms that not require containment v Appendix K-II-E Discharges containing viable recombinant organisms shall be handled in accordance with applicable governmental environmental regulations vi Appendix K-II-F Addition of materials to a system, sample collection, transfer of culture fluids within/between systems, and processing of culture fluids shall be conducted in a manner that maintains employee *s exposure to viable organisms containing recombinant DNA molecules at a level that does not adversely affect the health and safety of employees vii.AppendixK-II-G The facility's emergency response plan shall include provisions for handling spills 11 Appendix M Points to Consider in the Design andSubmission of Protocols for the Transfer of Recombinant DNA Molecules into One or More Human Subjects (Points to Consider) Appendix M applies to research conducted at or sponsored by an institution that receives any support for recombinant DNA research from NIH Researchers not covered by the NIH Guidelines are encouraged to use Appendix M This appendix will be abridged, as was Appendix K, because it applies to a much smaller group of laboratories than most recombinant DNA operations It is, however, an extremely sensitive area and anyone to whom it applies must adhere to all sections In addition to this introductory portion, Appendix M-3 and M-4 will be included which apply to informed consent on the part of the participants Any research involving human subjects must be cleared through the Institutions Institutional Review Board (IRB)according to the regulations found in Title 45 CFR 46 which provides for the protection of Human subjects Preparation of an adequate Informed Consent form often appears to provide difficulties to a researcher who is more comfortable with the technical aspects of the research The acceptability of human somatic cell gene therapy has been addressed in several public documents as well as in numerous academic studies In November 1982, the President *s Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Research published a report, Splicing Life, which resulted from a two-year process of public deliberation and hearings Up on release of that report, a U.S House of Representatives subcommittee held three days of public hearings with witnesses from a wide range of fields from the biomedical and social sciences to theology, philosophy, and law In December 1984, the Office of Technology Assessment released a background paper, Human Gene Therapy, which concluded that civic, religious, scientific, and medical groups have all accepted, in principle, the appropriateness of gene therapy of somatic cells in humans for specific genetic diseases Somatic cell gene therapy is seen as an extension of present methods of therapy that might be preferable ©2000 CRC Press LLC to other technologies In light of this public support, RAC is prepared to consider proposals for somatic cell gene transfer RAC will not, at present, entertain proposals for germ line alterations but will consider proposals involving somatic cell gene transfer The purpose of somatic cell gene therapy is to treat an individual patient, e.g., by inserting a properly functioning gene into the subject *s somatic cells Germ line alteration involves a specific attempt to introduce genetic changes into the germ (reproductive) cells of an individual, with the aim of changing the set of genes passed on to the individual's offspring Research proposals involving the deliberate transfer of recombinant DNA, or DNA or RNA derived from recombinant DNA, into human subjects (human gene transfer) will be considered through a review process involving both NIH/ORDA and RAC Investigators shall submit their relevant information on the proposed human gene transfer experiments to NIH/ORDA Submission of human gene transfer protocols to NIH will be in the format described in Appendix M-I Submission to NIH shall be for registration purposes and will ensure continued public access to relevant human gene transfer information conducted in compliance with the NIH Guidelines Investigational New Drug (IND) applications should be submitted to FDA in the format described in 21 CFR, Chapter I, Subchapter D, Part 312, Subpart B, Section 23 Institutional Biosafety Committee approval must be obtained from each institution at which recombinant DNA material will be administered to human subjects (as opposed to each institution involved in the production of vectors for human application and each institution at which there is ex vivo transduction of recombinant DNA material into target cells for human application) Factors that may contribute to public discussion of a human gene transfer experiment by RAC include: (i) new vectors/new gene delivery systems, (ii) new diseases, (iii) unique applications of gene transfer, and (iv) other issues considered to require further public discussion Among the experiments that may be considered exempt from RAC discussion are those determined not to represent possible risk to human health or the environment Full RAC review of an individual human gene transfer experiment can be initiated by the NIH Director or recommended to the NIH Director by: (i) three or more RAC members, or (ii) other Federal agencies An individual human gene transfer experiment that is recommended for full RAC review should represent novel characteristics deserving of public discussion If the Director, NIH, determines that an experiment will undergo full RAC discussion, NIH/ORDA will immediately notify the Principal Investigator RAC members may forward individual requests for additional information relevant to a specific protocol through NIH/ORDA to the Principal Investigator In making a determination whether an experiment is novel, and thus deserving of full RAC discussion, reviewers will examine the scientific rationale, scientific context (relative to other proposals reviewed by RAC), whether the preliminary in vitro and in vivo safety data were obtained in appropriate models and are sufficient, and whether questions related to relevant social and ethical issues have been resolved RAC recommendations on a specific human gene transfer experiment shall be forwarded to the NIH Director, the Principal Investigator, the sponsoring institution, and other DHHS components, as appropriate Relevant documentation will be included in the material for the RAC meeting at which the experiment is scheduled to be discussed RAC meetings will be open to the public except where trade secrets and proprietary information are reviewed RAC prefers that information provided in response to Appendix M contain no proprietary data or trade secrets, enabling all aspects of the review to be open to the public Any application submitted to NIH/ORDA shall not be designated as ‘confidential' in its entirety In the event that a sponsor determines that specific responses to one or more of the items described in Appendix M should be considered as proprietary or trade secret, each item should be clearly identified as such The cover letter (attached to the submitted material) shall: (1) clearly indicate that select portions of the application contain information considered as proprietary or trade secret, (2) a brief explanation as to the reason that each of these items is ©2000 CRC Press LLC determined proprietary or trade secret Public discussion of human gene transfer experiments (and access to relevant informa-tion) shall serve to inform the public about the technical as pects of the proposals, meaning and significance of the research, and significant safety, social, and ethical implications of the research RAC discussion is intended to ensure safe and ethical conduct of gene therapy experiments and facilitate public understanding of this novel area of biomedical research In its evaluation of human gene transfer proposals, RAC will consider whether the design of such experiments offers adequate assurance that their consequences will not go beyond their purpose, which is the same as the traditional purpose of clinical investigation, namely, to protect the health and well being of human subjects being treated while at the same time gathering generalizable knowledge Two possible undesirable consequences of the transfer of recombinant DNA would be unintentional: (i) vertical transmission of genetic changes from an individual to his/her offspring, or (ii) horizontal transmission of viral infection to other persons with whom the individual comes in contact Accordingly, Appendices M-l through M-V request information that will enable RAC and NIH/ORDA to assess the possibility that the proposed experiment(s) will inadvertently affect reproductive cells or lead to infection of other people (e.g., medical personnel or relatives) Appendix M will be considered for revisions as experience in evaluating proposals accumulates and as new scientific developments occur This review will be carried out periodically as needed 12 Appendix M-III Informed Consent In accordance with the Protection of Human Subjects (45 CFR Part 46), investigators should indicate how subjects will be informed about the proposed study and the manner in which their consent will be solicited They should indicate how the Informed Consent document makes clear the special requirements of gene transfer research If a proposal involves children, special attention should be paid to the Protection of Human Subjects (45 CFR Part 46), Subpart D, Additional Protections for Children Involved as Subjects in Research a Appendix M-III-A Communication About the Study to Potential Participants i Appendix M-III-A-1 Which members of the research group and/or institution will be responsible for contacting potential participants and for describing the study to them? What procedures will be used to avoid possible conflicts of interest if the investigator is also providing medical care to potential subjects? ii Appendix M-III-A-2 How will the major points covered in Appendix M-ll, Description of Proposal, be disclosed to potential participants and/or their parents or guardians in language that is understandable to them? iii Appendix M-III-A-3 What is the length of time that potential participants will have to make a decision about their participation in the study? iv Appendix M-III-A-4 If the study involves pediatric or mentally handicapped subjects, how will the assent of each person be obtained? ©2000 CRC Press LLC v Appendix M-III-B Informed Consent Investigators submitting human gene transfer proposals must include the Informed Consent document as approved by the local Institutional Review Board A separate Informed Consent document should be used for the gene transfer portion of a research project when gene transfer is used as an adjunct in the study of another technique, e.g., when a gene is used as a “marker” or to enhance the power of immunotherapy for cancer Because of the relative novelty of the procedures that are used, the potentially irreversible consequences of the procedures performed, and the fact that many of the potential risks remain undefined, the Informed Consent document should include the following specific information in addition to any requirements of the DHHS regulations for the Protection of Human Subjects (45 CFR 46): indicate if each of the specified items appears in the Informed Consent document or, if not included in the Informed Consent document, how those items will be presented to potential subjects; include an explanation if any of the following items are omitted from the consent process or the Informed Consent document b Appendix M-III-B-1 General Requirements of Human Subjects Research i Appendix M-III-B-1-a Description/Purpose of the Study The subjects should be provided with a detailed explanation in non-technical language of the purpose of the study and the procedures associated with the conduct of the proposed study, including a description of the gene transfer component ii Appendix M-III-B-1-b Alternatives The Informed Consent document should indicate the availability of therapies and the possibility of other investigational interventions and approaches iii Appendix M-III-B-1-c Voluntary Participation The subjects should be informed that participation in the study is voluntary and that failure to participate in the study or withdrawal of consent will not result in any penalty or loss of benefits to which the subjects are otherwise entitled iv Appendix M-III-B-1-d Benefits The subjects should be provided with an accurate description of the possible benefits (to themselves), if any, of participating in the proposed study For studies that are not reasonably expected to provide a therapeutic benefit to subjects, the Informed Consent document should clearly state that no direct clinical benefit to subjects is expected to occur as a result of participation in the study, although knowledge may be gained that may benefit others v Appendix M-III-B-e Possible Risks, Discomforts, and Side Effects There should be clear itemization in the Informed Consent document of types of adverse experiences, their relative severity, and their expected frequencies For consistency, the following definitions are suggested: side effects that are listed as mild should be those which not require a therapeutic intervention; moderate side effects require an intervention; and severe side effects are potentially fatal or life-threatening, disabling, or require prolonged hospitalization If verbal descriptors (e.g., “rare,” “uncommon,” or “frequent”) are used to express quantitative information regarding risk, these terms should be explained The Informed Consent document should provide information regarding the approximate number of people who have previously received the genetic material under study It is necessary to warn potential subjects that, for genetic materials previously used in relatively few or no humans, unforeseen risks are possible, including some that could be severe The Informed Consent document should indicate any possible adverse medical consequences that may occur if the subjects withdraw from the study once the study has started ©2000 CRC Press LLC vi Appendix M-III-B-1-f Costs The subjects should be provided with specific information about any financial costs associated with their participation in the protocol and in the long-term follow-up to the protocol that are not covered by the investigators or the institution involved Subjects should be provided an explanation about the extent to which they will be responsible for any costs for medical treatment required as a result of research-related injury c Appendix M-III-B-2 Specific Requirements of Gene Transfer Research i Appendix M-III-B-2-a Reproductive Considerations To avoid the possibility that any of the reagents employed in the gene transfer research could cause harm to a fetus/child, subjects should be given information concerning possible risks and the need for contraception by males and females during the active phase of the study The period of time for the use of contraception should be specified The inclusion of pregnant or lactating women should be addressed ii Appendix M-III-B-2-b Long-Term Follow-Up To permit evaluation of long-term safety and efficacy of gene transfer, the prospective subjects should be informed that they are expected to cooperate in long-term follow-up that extends beyond the active phase of the study The Informed Consent document should include a list of persons who can be contacted in the event that questions arise during the follow-up period The investigator should request that subjects continue to provide a current address and telephone number The subjects should be informed that any significant findings resulting from the study will be made known in a timely manner to them and/or their parent or guardian including new information about the experimental procedure, the harms and benefits experienced by other individuals involved in the study, and any long-term effects that have been observed iii Appendix M-III-B-2-c Request for Autopsy To obtain vital information about the safety and efficacy of gene transfer, subjects should be informed that at the time of death, no matter what the cause, permission for an autopsy will be requested of their families Subjects should be asked to advise their families of the request and of its scientific and medical importance iv Appendix M-III-B-2-d Interest of the Media and Others in the Research To alert subjects that others may have an interest in the innovative character of the protocol and in the status of the treated subjects, the subjects should be informed of the following: (i) that the institution and investigators will make efforts to provide protection from the media in an effort to protect the participants ' privacy, and (ii) that representatives of applicable Federal agencies (e.g., the National Institutes of Health and the Food and Drug Administration), representatives of collaborating institutions, vector suppliers, etc., will have access to the subjects ' medical records d Appendix M-IV Privacy and Confidentiality Indicate what measures will be taken to protect the privacy of patients and their families as well as to maintain the confidentiality of research data i Appendix M-IV-A What provisions will be made to honor the wishes of individual patients (and the parents or guardians of pediatric or mentally handicapped patients) as to whether, when, or how the identity of patients is publicly disclosed ©2000 CRC Press LLC ii Appendix M-IV-B What provisions will be made to maintain the confidentiality of research data, at least in cases where data could be linked to individual patients? 13 Appendix P Physical and Biological Containment for Recombinant DNAResearch Involving Plants Appendix P specifies physical and biological containment conditions and practices suitable to the greenhouse conduct of experiments involving recombinant DNA-containing plants, plantassociated microorganisms, and small animals All provisions of the NIH Guidelines apply to plant research activities with the following modifications: Appendix P shall supersede Appendix G when the research plants are of a size, number, or have growth requirements that preclude the u s e of containment conditions described in Appendix G The plants covered in Appendix P include but are not limited to mosses, liverworts, macroscopic algae, and vascular plants including terrestrial crops, forest, and ornamental species Plant-associated microorganisms include viroids, virusoids, viruses, bacteria, fungi, protozoans, certain small algae, and microorganisms that have a benign or beneficial association with plants, such as c e r t a i n Rhizobium species and microorganisms known to cause plant diseases The appendix applies to microorganisms which are being modified with the objective of fostering an association with plants Plant-associated small animals include those arthropods that: (i) are in obligate association with plants, (ii) are plant pests, (iii) are plant pollinators, or (iv) transmit plant disease agents, as well as other small animals such as nematodes for which tests of biological properties necessitate the u s e of plants Microorganisms associated with such small animals (e.g., pathogens or symbionts) are included The Institutional Biosafety Committee shall include at least one individual with expertise in plant, plant pathogen, or plant pest containment principles when experiments utilizing Appendix P require prior approval by the Institutional Biosafety Committee a Appendix P-I General Plant Biosafety Levels i Appendix P-I-A The principal purpose of plant containment is to avoid the unintentional transmission of a recombinant DNA-containing plant genome, including nuclear or organelle hereditary material or release of recombinant DNA-derived organisms associated with plants ii Appendix P-I-B The containment principles are based on the recognition that the organisms that are used p o s e no health threat to humans or higher animals (unless deliberately modified for that purpose), and that the containment conditions minimize the possibility of an unanticipated deleterious effect on organisms and ecosystems outside of the experimental facility, e.g., the inadvertent spread of a serious pathogen from a greenhouse to a local agricultural crop or the unintentional introduction and establishment of an organism in a new ecosystem iii Appendix P-I-C Four biosafety levels, referred to as Biosafety Level (BL) 1-Plants (P), BL2-P, BL3-P, and BL4P, are established in Appendix P-II, Physical Containment Levels The selection of containment levels required for research involving recombinant DNA molecules in plants or associated with plants is specified in Appendix P-III, Biological Containment Practices T hese biosafety levels are described in Appendix P-II, Physical Containment Levels This appendix describes greenhouse practices and special greenhouse facilities for physical containment ©2000 CRC Press LLC iv Appendix P-I-D BL1-P through BL4-P are designed to provide differential levels of biosafety for plants in the absence or presence of other experimental organis ms that contain recombinant DNA These biosafety levels, in conjunction with biological containment conditions described in Appendix P-III, Biological Containment Practices, provide flexible approaches to ensure the safe conduct of research v Appendix P-I-E For experiments in which plants are grown at the BL1 through BL4 laboratory settings, containment practices shall be followed as described in Appendix G These containment practices include the use of plant tissue culture rooms, growth chambers within laboratory facilities, or experiments performed on open benches Additional biological containment practices should be added by the Greenhouse Director or Institutional Biosafety Committee as necessary if botanical reproductive structures are produced that have the potential of being released b Biological Containment Practices Appropriate selection of the following biological containment practices may be used to meet the containment requirements for a given organism The present list is not exhaustive; there may be other ways of preventing effective dissemination that could possibly lead to the establishment of the organism or its genetic material in the environment resulting in deleterious consequences to managed or natural ecosystems i Appendix P-III-A Biological Containment Practices (Plants) ii Appendix P-III-A-1 Effective dissemination of plants by pollen or seed can be prevented by one or more of the following procedures: (i) cover the reproductive structures to prevent pollen dissemination at flowering and seed dissemination at maturity; (ii) remove reproductive structures by employing male sterile strains, or harvest the plant material prior to the reproductive stage; (iii) ensure that experimental plants flower at a time of year when cross-fertile plants are not flowering within the normal pollen dispersal range of the experimental plant; or (iv) ensure that cross-fertile plants are not growing within the known pollen dispersal range of the experimental plant iii Appendix P-III-B Biological Containment Practices (Microorganisms) iv Appendix P-III-B-1 Effective dissemination of microorganisms beyond the confines of the greenhouse can be prevented by one or more of the following procedures: (i) confine all operations to injections of microorganisms or other biological procedures (including genetic manipulation) that limit replication or reproduction of viruses and microorganisms or sequences derived from microorganisms, and confine these injections to internal plant parts or adherent plant surfaces; (ii) ensure that organisms, which can serve as hosts or promote the transmission of the virus or microorganism, are not present within the farthest distance that the airborne virus or microorganism may be expected to be effectively disseminated; (iii) conduct experiments at a time of year when plants that can serve as hosts are either not growing or are not susceptible to productive infection; (iv) use viruses and other microorganisms or their genomes that have known arthropod or animal vectors, in the absence of such vectors ; ( v ) u s e microorganisms that have an obligate association with the plant; or (vi) u s e microorganisms that are genetically disabled to minimize survival outside of the research facility and whose natural mode of transmission requires injury of the target organism, or assures that inadvertent release is unlikely to initiate productive infection of organisms outside of the experimental facility ©2000 CRC Press LLC v Appendix P-III-C Biological Containment Practices (Macroorganisms) vi Appendix P-III-C-1 Effective dissemination of arthropods and other small animals can be prevented by using one or more of the following procedures: (i) use non-flying, flight-impaired, or sterile arthropods; (ii) use nonmotile or sterile strains of small animals; (iii) conduct experiments at a time of year that precludes the survival of escaping organisms; (iv) u s e animals that have an obligate association with a plant that is not present within the dispersal range of the organism; or (v) prevent the escape of organisms present in run-off water by chemical treatment or evaporation of run-off water 14 Appendix Q Physical and Biological Containment for Recombinant DNA Research Involving Animals Appendix Q specifies containment and confinement practices for research involving whole animals, both those in which the animal's genome has been altered by stable introduction of recombinant DNA, or DNA derived therefrom, into the germ-line (transgenic animals ) and experiments involving viable recombinant DNA-modified microorganisms tested on whole animals The appendix applies to animal research activities with the following modifications Appendix Q shall supersede Appendix G when research animals are of a size o r h a v e growth requirements that preclude the use of containment for laboratory animals Some animals may require other types of containment The animals covered in Appendix Q are those species normally categorized as animals including but not limited to cattle, swine, sheep, goats, horses, and poultry The Institutional Biosafety Committee shall include at least one scientist with expertise in animal containment principles when experiments utilizing Appendix Q require Institutional Biosafety Committee prior approval The institution shall establish and maintain a health surveillance program for personnel engaged in animal research involving viable recombinant DNA-containing microorganisms that require Biosafety Level (BL) or greater containment in the laboratory a Appendix Q-l General Considerations b Appendix Q-I-A Containment Levels The containment levels required for research involving recombinant DNA associated with or in animals is based on classification of experiments in Section III For the purpose of animal research, four levels of containment are established These are referred to as BL1-Animals (N), BL2-N, BL3-N, and BL4-N and are described in the appendices of Appendix Q The descriptions include: (i) standard practices for physical and biological containment, and (ii) animal facilities c Appendix Q-l-B Disposal of Animals (BL1 -N through BL4-N) i Appendix Q-I-B-1 When an animal covered by Appendix Q containing recombinant DNA or a recombinant DNA-derived organism is euthanized or dies, the carcass shall be disposed of to avoid its u s e as food for human beings or animals unless food use is specifically authorized by an appropriate Federal agency ii Appendix Q-l-B-2 A permanent record shall be maintained of the experimental use and disposal of each animal or group of animals Since animals are mobile and could conceivably escape or intermingle with other animals, the provisions of Appendix Q contain many provisions to make sure the animals involved with ©2000 CRC Press LLC recombinant DNA not have opportunitie s to spread and transfer any modified genetic materials either directly or via experimental procedures Those intending to u s e recombinant DNA procedures in work with animals should be thoroughly familiar with the provisions of appendix Q of the NIH Guidelines REFERENCES National Institutes of Health guidelines for research involving recombinant DNA molecules, Fed Reg., 51, May 7, 1986, 16958 Classification of Etiologic Agents on the Basis of Hazard, 4th Edition, U.S Department of Health 10 in 11 12 13 14 15 16 17 18 19 20 Education and Welfare, Center for Disease Control, Office of Biosafety, Atlanta, GA, July, 1974 Laboratory Safety at the Center for Disease Control, Publ No CD.C 75-8118, U.S Department of Health, Education and Welfare, 1974 National Cancer Institute Safety Standards for Research Involving Oncogenic Viruses, Pub No (NIH) 75790, U.S Department of Health, Education and Welfare, 1974 National Institutes of Health Biohazards Safety Guide, Stock No 1740-003 83, Public Health Service, National Institutes of Health, U.S Department of Health, Education and Welfare, Washington, D.C., 1974 Hellman, A., Oxman, M.N., and Pollack, R., Eds., Biohazards in Biological Research, Cold Spring Harbor Laboratory, New York, 1974 Bodily, J.L., General administration of the laboratory, in Diagnostic Procedures for Bacterial, Mycotic and Parasitic Infections, Bodily, H.L., Updyke, E.L., and Mason, J.O., Eds., American Public Health Association, New York, 1970, 11 Darlow, H.M., Safety in the microbiological laboratory, in Methods in Microbiology, Norris, J.R and Robbins, D.W, Eds., Academic Press, New York, 1969, 169 Collins, C.H., Hartley, E.G., and Pilsworth, R., The Prevention of Laboratory Acquired Infection,, Monogr Ser No 6, Public Health Laboratory Service, 1974 Chatigny, M.A., Protection against infection in the microbiological laboratory: devices and procedures, Advances in Applied Microbiology, Vol 3, Umbreit, W.W., Ed., Academic Press, New York, 1961, 131 Design Criteria for Viral Oncology Research Facilities, Publ No (NIH) 75-891, U.S Department of Health, Education and Welfare, Washington, D.C., 1975 Kuehne, R.W., Biological containment facility for studying infectious disease, Appl Microbiol., 26, 239, 1973 Runkle, R.S and Phillips, G.E., Microbial Containment Control Facilities, Van Nostrand Reinhold, New York, 1969 Chatigny, M.A and Clinger, D.I., Contamination control in aerobiology, in An Introduction to Experimental Aerobiology, Dimmick, R.L and Akers, A.B., Eds., John Wiley & Sons, New York, 1969, 194 Matthews, R.E.E, Ed., Third report of the international committee on taxonomy of viruses: classification and nomenclature of viruses, Intervirology, 12, 129, 1979 B uchanan, R.E and Gibbons, N.E., Eds., Bergey’s Manual of Determinative Bacteriology t h e d , Williams and Wilkins, Baltimore, MD, 1974 Richmond, J.Y and McKinney, R.W., Eds., Biosafety in Microbiological and Biomedical Laboratories, 3rd ed., Publ No (CD.C.) 84-8395, U.S Department of Health, and Human Services, National Institutes of Health, Centers for Disease Control, Washington, D.C., 1993 Laboratory Safety Monograph — A Supplement to the NIH Guidelines for Recombinant Office of Recombinant DNA Activities, National Institutes of Health, Bethesda, MD Hershfield, V., Boyer H.W., Yanofsky, C., Lovett, M.A., and Heliaski, D.R., Plasmid molecular vehicle for cloning and amplification of DNA, Proc.Natl Acad Sci., U.S.A , Wensink, P.E., Finnegan, D.J., Donelson, J.E., and Hogness, D.S., A system for sequences in the chromosomes of Drosophila melanogaster, Cell, 3, 315, 1974 ©2000 CRC Press LLC DNA Research, Col E l a s a 71, 3455, 1974 mapping DNA 21 22 23 Tanaka, T and Weisbium, B., Construction of a coli in E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid, J Bacteriol., 121,354, 1975 Armstrong, K.A., Hershfield, V., and Helsinki, D.R., Gene cloning and containment properties of plasmid Col El and its derivatives, Science, 196, 172, 1977 Bolivar, F., Rodriguez, R.L., Batlach, M.C., and Boyer, H.W., Construction and characterization 25 of new cloning vehicles I Ampicillin-resistant derivative of pMB9, Gene, 2, 75, 1977 Cohen, S.N., Chang, A.C.W., Boyer, H., and Helling, R., Construction of biologically functional plasmids in vitro, Proc Nati Acad Sci U.S.A., 70, 3240, 1973 Bolivar, F., Rodriguez, R.L., Greene, R.J., Batlach, M.C., Reynekei H.L., Boyer, W., Crosa, J.H., 26 and Falkow, S., Construction and characterization of new cloning vehicles II A multi-purpose cloning system, Gene, 2, 95, 1977 Thomas, M., Cameron, I.R., and Davis, R.W, Viable molecular hybrids of bacteriophage lambda and 24 27 28 29 eukaryotic DNA, Proc Natl Acad Sci U.S.A., 71, 4579, 1974 Murray, N.E and Murray, K., Manipulation of restriction targets in phage lambda to form receptor chromosomes for DNA fragments, Nature, 251, 476, 1974 Rambach, A and Tiolais, P., Bacteriophage having ecor1 endonuclease sites only in the non-essential region of the genome, Proc Natl Acad Sci U.S.A., 71, 3927, 1974 Blattner, E.R., Williams, G.G., Bleche, A.E., Denniston-Thompson, K., Faber, H.E., Furlong, L.A., Gunwald, D.J., Kiefer, D.O., Moore, D.D., Shumm, J.W., Sheldon, E.L., and Smithies,O, 31 Charon phages: safer derivatives of bacteriophage lambda for DNA cloning, Science, 196, 163, 1977 Donoghue, D.J and Sharp, P.A., An improved lambda vector: construction of model recombinants coding for kanamycin resistance, Gene, 1, 209, 1977 Leder, P., Tiemeier, D., and Enquist, L., EK2 derivatives of bacteriophage lambda useful in the cloning 32 33 of DNA from higher organisms: the gt WES System, Science, 196, 175, 1977 Skalka, A., Current Status of Coliphage EK2 Vectors, Gene, 3, 29, 1978 Szybalski, W., Skalka, A., Gottesman, S., Campbell, A., and Botstein, D., Standardized laboratory 30 tests for EK2 certification, Gene, 3, 36, 1978 INTERNET REFERENCE Guidelines for Research Involving Recombinant DNA Molecules, May 1998, http://www.nih.gov/od/orda/guidelines.pdf/toc.htm VIII RESEARCH ANIMAL CARE AND HANDLING12 A Introduction Animal use in research, teaching, and testing has provided advances in health care and preventive medicine for both animals and humans Experimental results are greatly dependent upon the humane care and treatment of animals used in research There is a large body of laws, regulations, and guidelines governing the use of animals in research to assure humane animal care and use These regulations should not be feared as inhibitory to scientific freedom Rather, as Aristotle said, “Shall we not like the archer who has a mark to aim at, be more likely to hit upon that which is right?” Compliance with these laws and guidelines assures healthy, high-quality animal models for u s e in research, assuring consistency from laboratory to laboratory throughout the nation, thus enhancing experimental reliability The use of high-quality healthy animals and experimental methodologies which seek to minimize or eliminate pain or discomfort to the animals should be incorporated because not only is it the law, but because it makes scientific s e n s e and is the most humane and ethical thing to The following sections briefly describe the laws and * This Section was written by David M Moore, D.V.M current versions of any regulatory references ©2000 CRC Press LLC It was written in 1994 so the reader should refer to regulations governing animal care and use, and programs for health maintenance of animals and research personnel who come into contact with those animals B Laws and Regulations Relating to Animal Care and Use Two major types of regulatory activities impacting the use of animals at a research facility involve voluntary and involuntary regulations Involuntary regulations are statutory in nature, are uncompromising, and include federal and state laws which dictate minimum standards for the acquisitions of animals, provision of veterinary and husbandry care, use, and disposition of laboratory animals, and personal and institutional compliance is mandatory Voluntary regulations are those which a research facility imposes on itself above and beyond the minimum standards set forth by the government Knowledge of and compliance with applicable institutional, state, and federal policies, regulations, and laws will assure humane care of animals, improve scientific reliability, and deflect criticism from the small segment of society which questions whether animals used in research are humanely treated Animal Welfare Act The major federal law affecting and regulating u s e of animals in research is the Federal Animal Welfare Act (PL 89-544, and its amendments, PL 1-579, PL 94-279, and PL 99-198) Full text copies of the Act are published in the Code of Federal Regulations CFR Title 9, Animals and Animal Products, Subchapter A, Animal Welfare, Parts 1, 2, and 3, and copies of these laws and regulations can be obtained from the Director, Regulatory Enforcement and Animal Care, USDA, APHIS, Room 207, Federal Building, 6505 Belcrest Road, Hyattsville, M D 20782, or the U.S Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, Import-Export Products Staff, Room 756, Federal Building, 6505 Belcrest Road, Hyattsville, M D 20782 They can also be obtained via the Internet The Act is administered and enforced by the United States Department of Agriculture Animal, Plant Health Inspection Service (USDA/APHIS) All research institutions using animals defined by the Act (dog, cat, nonhuman primate, guinea pig, hamster, or rabbit) must complete and submit VS Form 18-11 “Applications for Registration of a Research Facility” to the USDA-APHIS-VS Veterinarian-in-Charge for the state in which the facility is located (contact the national office listed above for the local address) The form asks for the location of animal holding facilities, and the species and numbers of animals to be used Failure to register as a research facility using covered animals may result in fines or sanctions prohibiting future u s e of animals at that institution Currently, rodent species, birds, farm animals, and exotic species are not considered “Animals” under the definition cont ained in the Act These may be included in subsequent amendments, so it would be advisable to contact the veterinarian-in-charge for your state to determine if you must register The Act addresses and sets minimum standards for the care and use of animals in research in the areas of facilities, construction, caging, and operations of the facility; animal health and husbandry, standards covering feeding, watering, sanitation, employee qualifications, separation of species, record keeping, and provision of adequate veterinary care; and transportation standards of animals to and from the facility Please refer to a copy of the Act for detailed specifications The size of a facility and its research program may determine whether a full-time veterinarian is required on the staff, or if the animal health care needs can be met with a part-time or consulting veterinarian with laboratory animal training or experience The institution must file a “Program of Veterinary Care” with the USDA/APHIS-VS Veterinarian-in-Charge for that state, detailing programs of disease control and prevention, euthanasia methods, and use of appropriate anesthetic, analgesic, or tranquilizing drugs when necessary as determined by the institutional veterinarian Each registered research facility must submit VA 18-23 “Annual Report of Research Facility” with USDA/APHIS each year, listing the numbers of animals by species and by category of potential pain or discomfort used during that year, with the signatures of the attending ©2000 CRC Press LLC institutional veterinarian and a designated senior administrative official at the institution Federal penalties may be invoked against the signatories for falsification of information in the body of the report The Improved Standards for Laboratory Animals Act (P.L 99-198), the most recent amendment to the Animal Welfare Act, sets even more important requirements Each research facility must have an institutional animal care committee of not fewer than three members to be appointed by the chief executive officer of the facility, to include a doctor of veterinary medicine (usually the institutional veterinarian), another facility employee, and a nonemployee, community member who has no family member affiliated with the facility The committee must inspect all animal study areas and animal facilities twice annually keeping all inspection reports on file for years, and notifying the administrative representative of the facility of any deficiencies or deviations from the Act Additionally, each research facility must establish a program for the training of scientists, animal technicians, and other personnel involved with animal care and treatment to include instruction on: Humane practice of animal maintenance and experimentation Research or testing methods that minimize or eliminate animal pain or distress Utilization of the information service at the National Agricultural Library Methods whereby deficiencies in animal care and treatment should be reported This amendment also calls for establishing institutional standards for exercise of dogs, and provision for environmental enrichment for nonhuman primates The Good Laboratory Practices Act The Good Laboratory Practices Act (22 December 1978 issue of the Federal Register 43 FR 59986-60025) regulates “nonclinical laboratory studies that support applications for research or marketing permits for products regulated by the Food and Drug Administration, including food and color additives, animal food additives, human and animal drugs, medical devices for humane use, biological products, and electronic products.” Standards addressed by GLP regulations include: (i) compliance with the “NIH Guide for the Care and Use of Laboratory Animals” (to be discussed in the following pragraph); (ii) establishment of standard operating procedures (SOPs) for animal husbandry and experimental treatment; (iii) meticulous record keeping and documentation of activities; and the establishment of a functional quality assurance unit reporting to the highest administrative levels of the facility Please consult this document for further details and applicability to your studies The Guide for the Care and Use of Laboratory Animals The “Guide” (NIH Publications 85-23) was prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council The Guide presents recommendations and basic guidelines for: (i) appropriate cage and enclosure sizes for a variety of commonly used laboratory species; (ii) social environmental enrichment; (iii) appropriate environmental temperature and humidity ranges; (iv) ventilation of animal facilities,levels of illumination,noise; (v) separation of species, sanitation of caging and facilities; (vi) provision for quarantine, and provision of adequate veterinary care Whereas the standards in the Guide are more stringent than those in the Animal Welfare Act, they are simply recommendations and not by themselves carry legal penalties However, other government agencies use the Guide as a measure for animal care, and will terminate funding support for an Institution for noncompliance with the Guide Single copies of the Guide can be obtained from the Animal Resources Program, Division of Research Resources, National Institutes of Health, Bethesda, Maryland 20205 Public Health Service Policies Institutions receiving federal grant support for animal research activities from the Public ©2000 CRC Press LLC Health Service (including the National Institutes of Health) must file an Animal Welfare Assurance statement with the Office for Protectio n from Research Risks (OPRR), National Institutes of Health, 9000 Rockville Pike,Building 31, Room 4B09, Bethesda, Maryland 20892 The components of the Assurance are listed in the publications “Public Health Service Policy on Humane Care and Use of Laboratory Animals” which can be obtained from OPRR The intent of this policy is to “require Institutions to establish and maintain proper measures to ensure the appropriate care and use of all animal involved in research, research training, and biological lasting activities.” The PHS “requires that institutions, in their Assurance Statement, u s e the Guide for the Care and Use of Laboratory Animals as the basis for developing and implementing an institutional program for activities involving animals.” In contrast to the Animal Welfare Act, PHS mandates an institutional animal care and u s e committee with a minimum of five members (the membership credentials mirror those specified in the Act) Institutions with approved assurance statements must file an annual report with OPRR detailing any changes in the animal program, and listing the dates of the twice-yearly facility inspections by the committee Failure to comply with the provision of this policy will result in non-funding or withdrawal of funding for ongoing activities As with the Animal Welfare Act, investigators submitting proposals to PHS agencies must submit a research protocol detailing animal use for review of and approval by the institutional animal care and u s e committee This committee must approve the project before funding is released, and may require modification of the project if it is not in compliance with Animal Welfare Act standards or PHS policies The institutional animal care and u s e committee may halt or terminate an ongoing project for noncompliance with federal laws and policies Voluntary Regulations Institutions may develop their own internal policies regarding animal care and use, providing that they are equal to or more stringent than those contained in the Guide or the Animal Welfare Act Internal policies might include SOPs for animal care and use, mechanisms for selection of and purchase from commercial animal vendors, quarantine policies, and human health monitoring programs C Personnel “The Guide for the Care and Use of Lab6ratory Animals” promotes Institutional personnel policies requiring the use of technicians qualified to provide proper, humane animal care and husbandry, and recommends that these individuals apply for and receive certification from the American Association for Laboratory Animal Science (AALAS, 70 Timbercreek Drive, Suite 5, Cordova, TN 38018) There are three levels of certification based upon educational background and training, and on-the-job experience dealing with laboratory animals: Assistant Technician, Technician, and Laboratory Animal Technologist M o s t facilities require facility supervisors or managers to have the AALAS Technologist certification In-house training of technicians using AALAS course materials will satisfy the training requirement set forth in the Improved Standards for Laboratory Animals Act Training of the scientific staff in humane animal care would be best accomplished by the lab animal veterinarian or an AALAS technologist The qualifications for full-time or consulting veterinarians to the research facility should include either specialty board certification by the American College of Laboratory Animal Medicine (ACLAM) or indications of postdoctoral training or experience with laboratory animals The role of the veterinarian in assuring the provision of “adequate, veterinary care” (as referred to in the laws and policies section of this chapter) is described in a report by ACLAM on “Adequate Veterinary Care” issued in October 1966 D Animal Holding Facilities Animal holding facilities should be designed and constructed following the recommendations ©2000 CRC Press LLC of “The Guide for the Care and Use of Laboratory Animals,” which also assures compliance with the Animal Welfare Act Consult the references at the end of this section for further information on animal facility design and management Facilities should be designed and operated for the comfort of the animals and the convenience of the investigator Another critical factor in facility design operations involves the prevention of transmission of latent diseases from animal to animal, or animals to humans The first step involves purchase of animals from “clean” sources who have a documented animal health quality assurance program Newly arrived animals should be held in a quarantine area in the facility to prevent potential contamination of existing research animal populations The quarantine facility should be located in an area adjacent to the main colony, but with separate access to prevent cross-contamination of the colony as would be the case with common traffic flows The second step for maintaining clean animals involves control of the micro-environment through the u s e of appropriate housing units (i.e., micro-isolator caging), laminar airflow housing racks, or mass air displacement “clean” rooms The items suggested above may be cost prohibitive for some facilities, and adequate care could involve simply following sanitation and hygiene recommendations in the “Guide.” Air pressure in the room can be changed to protect either the animals or humans working in the facility Making the room air pressure slightly positive with respect to the hallways will minimize the chances of entry of airborne disease agents into an animal room However, if the research involves animals infected with animal or human pathogens, or they are treated with toxic or carcinogenic agents, then the room air pressure should be made slightly negative as compared to the adjacent hallways, to prevent contamination of animals in other nearby rooms or humans who u s e that hallway Hessler and Moreland discuss the use of HEPA filtration in rooms using nonvolatile carcinogens E Animal Care and Handling The Improved Standards for Laboratory Animals Act requires that experimental procedures “ensure that animal pain and distress are minimized.” A stressed animal is not a good experimental model, since its biochemical and physiological attributes are altered during stress Minimizing animal stress can be easily accomplished by: Purchasing animals free of latent and overt clinical diseases, and providing adequate veterinary care to maintain their health Familiarizing animals with experimental devices or rooms prior to the start of the experiment Limiting restraint to that which is necessary to accomplish the experimental goals, preconditioning the animals to the restraint apparatus, or using other nonrestraint alternatives Controlling or eliminating environmental stressors (i.e., inappropriate temperature, humidity, light, noise, aggressive cage mates, cage size) Providing environmental enrichment programs and/or exercise for animals, especially dogs, cats, and nonhuman primates Selecting and using appropriate anesthetics, tranquilizers, sedatives, and analgesic drugs for procedures in which pain or distress are likely Allowing only skilled, trained individuals to perform surgery Providing training for technicians and investigators in humane animal care and use techniques F Human Health Monitoring It is imperative that a human health monitoring program be established for those individuals having limited or full-time contact with research animals Caretakers and investigators can be exposed to hazardous aerosols, bites, scratches, bodily wastes and discharges, and fomites ©2000 CRC Press LLC contaminated with zoonotic agents A preemployment physical should be conducted to obtain baseline physical and historical data, and a serum sample should be drawn and frozen for future reference Additional examinations should be scheduled periodically depending on the nature and risks in the work environment Training programs should be established to acquaint personnel with biologic (zoonotic), chemical, and physical hazards within the animal facility.Appropriate hygiene should be stressed, and protective clothing and equipment (gloves, protective outer garments, masks, respirators, face shields or eye protectors) should be made available, and their use made mandatory where appropriate in SOPs Technicians should be aware of clinical signs of disease, notifying the facility veterinarian for confirmation, treatment, isolation of the animal(s), or euthanasia of the affected animal REFERENCES Clark, J.D., Regulation of animal use: voluntary and involuntary, I Vet Med Educ., 6(2), 86, 1979 McPherson, C.W., Legislation regulations pertaining to laboratory animals - United States, in Handbook of Laboratory Animal Science, Vol I, Melby, E.C and Altman, N.H., Eds., CRC Press, OH, Cl eveland, 1974, McPherson, C.W., Laws, regulations, and policies affecting the use of laboratory animals, in Laboratory Animal Medicine, Fox, J.G et al., Eds., Academic Press, Orlando, FL, 1984, 19 Poiley, S.M., Housing requirements - general considerations, in Handbook of Laboratory Animal Science, Vol I., Melby, E.C and Altman, N.H., Eds., CRC Press, Cleveland, OH, 1974, 21 Hessler, J.R and Moreland, A.F., Design and management of animal faci l i t i e s i n Laboratory Animal Medicine, Fox, J.G et al., Eds., Academic Press, Orlando, FL, 1984, 505 Simmonds, R.C., The design of laboratory animal homes, in Aeromedical Review, Vol 2, USAF School of Aerospace Medicine, Brooks Air Force Base, San Antonio, TX, 1973 Runkle, R.S., Laboratory animal housing - part II, Am Inst Architect J., 41, 77, 1964 Comfortable Quarters for Laboratory Animals, Animal Welfare Institute, Washington, D.C., 1979 Richmond, J,Y and McKinney, R.W., Biosafety in Microbiological and Biomedical Laboratories, 3rd ed., U.S Department of Health and Human Services, HITS Publ 93-8395, 1993 INTERNET REFERENCE Internet URL for laws cited: http://www.access.gpo.gov/nara/cfr/waisidx/9cfrv1_99.html ©2000 CRC Press LLC ... Gonads 0. 25 x 1 0 -5 in 25, 000 Breast 0. 15 2 .5 x 1 0 -5 in 40,000 Red bone marrow 0.12 x 1 0 -5 in 50 ,000 Lung 0.12 x 1 0 -5 in 50 ,000 Thyroid 0.03 x 1 0-6 in 200,000 Bone surfaces 0.03 x 1 0-6 in 200,000... days 0.212 0.279 1 25 59.7 days , 0.0 355 131 8.04 days 0.606, others Mn 22 Na 312 .5 days 2.6 years , ,, $+ 0 .54 5 59 203 I I 54 0.6616 1.099, 1.292 0.364, others 0.8 35 1.27, 0 .51 1 (annihilation... Gamma X-Rays 1/1 840 0 Charge (Electron Units) Range of Energy +2 4-6 MeV +1 0 eVs-4 MeV eVs-4 MeV eVs-100 KeV An amu is the mass of a single nucleon based on the 1/12th the mass of a carbon-12